CN101444525A - RNAi-mediated inhibition of ocular targets - Google Patents
RNAi-mediated inhibition of ocular targets Download PDFInfo
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- CN101444525A CN101444525A CNA2008102131399A CN200810213139A CN101444525A CN 101444525 A CN101444525 A CN 101444525A CN A2008102131399 A CNA2008102131399 A CN A2008102131399A CN 200810213139 A CN200810213139 A CN 200810213139A CN 101444525 A CN101444525 A CN 101444525A
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Abstract
The present invention provides a RNA interference for inhibition of ocular hypertension target mRNA expression for lowering elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Ocular hypertension targets include carbonic anhydrase II, IV, and XII; beta1- and beta2 adrenergic receptors; acetylcholinesterase; Na<+>/K<+>-ATPase; and Na-K-2Cl cotransporter. Ocular hypertension is treated by administering interfering RNAs of the present invention.
Description
The application is to be that on February 1st, 2006, denomination of invention are applied for dividing an application of 200680007300.6 (PCT/US2006/003515) for the China of " inhibition of the ocular hypertension targets of RNAi mediation " applying date.
Invention field
The present invention relates to the field of RNA interfering compositions, said composition is used to suppress the particularly expression of primary open angle glaucoma intraocular pressure target of glaucoma.
Background of invention
Glaucoma is heterogeneousization of the optic nerve colony with certain Clinical symptoms.The forfeiture of glaucoma vision is because the retinal ganglial cells selectivity death in the neural retina causes, and the characteristic that clinical diagnosis shows as the visual field changes, nerve fibre layer is damaged and the carrying out property depression of optic disc (ONH).One of main hazard factor that glaucoma forms be exist high intraocular pressure (intraocular pressure of rising, IOP).Suitable intraocular pressure is necessary for keeping eye shape and providing barometric gradient that aqueous humor is flowed to depletion of blood limb film and crystalline lens.The pathogeny of common intraocular pressure glaucoma (NTG) may also relate to the IOP level, benefits from the patient who falls the IOP medicine and can prove this point.When NTG patient is carried out tonometry,, may will find that many people have high intraocular pressure among these patients in case adjust central corneal thickness.
The elevated IOP relevant with glaucoma is because due to the discharge of the aqueous humor of rising was obstructed in the girder (TM), girder was the small special tissue in iris-cornea angle that is arranged in the anterior chamber of eyeball.The glaucoma of TM change comprise the minimizing of TM cell and comprise protein speckle sample material the extracellular residue deposition and gather.In addition, also change among the glaucomatous ONH.In glaucoma, there is morphologic and ambulant variation in the ONH neurogliocyte.Reply the damage of elevated IOP and/or transient ischemic attack, the ONH extracellular matrix components changes and the form of glial cell and retinal ganglial cells aixs cylinder changes.
It is moving disorderly that primary glaucoma results from the ophthalmic liquid stream with anatomy or physiological Foundations.Secondary glaucoma is because due to ocular injury or ocular injury or the original disease.Primary open angle glaucoma (POAG) is also referred to as chronic or donders' glaucoma, accounts for 90% of all primary glaucomas.POAG is characterised in that the degeneration of girder, causes drain in the eye is produced unusual high resistance.The result of this resistance causes IOP to raise, and it is in order to drive by the normal fluid that produces of eyes to overcome the resistance of increase that IOP raises.
At present, antiglaucomatous treatment comprises by inhibitor that uses aqueous humor formation or reagent reduction IOP, laser trabeculoplasty or the trabeculectomy (it is a filtration surgery of improving drain) that increases uvea sclera external flux.Glaucoma method on the materia medica shows multiple adverse side effect.For example, miotic such as pilocarpine can cause blurred vision and other negative vision side effect.The carbonic anhydrase inhibitors that general is used (CAI) also can cause nausea, dyspepsia, fatigue and metabolic acidosis.In addition, it is relevant with serious pulmonary's side effect that some beta-blocker becomes day by day, and this is owing to they influences to β in the lung tissue-2 receptor.The amine thing of imitative sympathetic nerve effect causes tachycardia, arrhythmia and hypertension.The termination that this negative side effect may cause patient's compliance to reduce or treat.In addition, it is shorter relatively that present IOP reduces the curative effect of therapy, need repeat administration every day, and in some cases, drug effect weakens in time.
In view of high intraocular pressure in glaucoma importance and before the deficiency of Therapeutic Method, need the basic reason that a kind of method for the treatment of ocular hypertensive improvement solves its progress.
Summary of the invention
The present invention be directed to RNA interfering, the reticent ocular hypertension targets mRNA of this RNA interfering expresses, thereby reduces open angle glaucoma and high intraocular pressure patient's intraocular pressure.Ocular hypertension targets comprises: carbonic anhydrase II, IV and XII; β 1 and beta 2-adrenergic receptor; Acetylcholinesterase; Na
+/ K
+-ATP enzyme and Na-K-2Cl cotransporter.RNA interfering of the present invention helps to treat open angle glaucoma and high intraocular pressure patient.
One embodiment of the invention provide and weakened the method that ocular hypertension targets mRNA expresses in the experimenters, and this ocular hypertension targets mRNA is as carbonic anhydrase II, IV or XII; β 1 or beta 2-adrenergic receptor; Acetylcholinesterase; Na
+/ K
+-ATP enzyme or Na-K-2Cl cotransporter mRNA.This method comprises to the experimenter uses compositions, and said composition comprises the RNA interfering with 19-49 length of nucleotides and the drug acceptable carrier of effective dose.To experimenter's dosing eyes, to weaken the expression of ocular hypertension targets in the human body.
In one embodiment of the invention, RNA interfering includes the intimate at least continuous complementary zone fully of adopted nucleotide chain, antisensenucleic acids chain and at least 19 nucleotide.In addition, antisense strand is hybridized with the part of mRNA under physiological condition, described mRNA is corresponding to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ IDNO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ IDNO:133 or SEQ ID NO:134, these are that adopted cDNA sequence is arranged, and carbonic anhydrase II and IV encode respectively, β 1 and beta 2-adrenergic receptor, the variant E4-E5 of acetylcholinesterase (ACHE), Na
+/ K
+-ATP enzyme α 2 polypeptide, Na-K-2Cl cotransporter NKCC2 (SLC12A1), carbonic anhydrase XII variant 1, acetylcholinesterase variant E4-E6, Na
+/ K
+-ATP enzyme α 1 polypeptide variants 1 and 2, Na
+/ K
+-ATP enzyme α 3 polypeptide, Na
+/ K
+-ATP enzyme α 4 polypeptide variants 1 and 2, Na
+/ K
+-ATP enzyme β 1 polypeptide variants 1 and 2, Na
+/ K
+-ATP enzyme β 2 polypeptide, Na
+/ K
+-ATP enzyme β 3 polypeptide, Na-K-2Cl cotransporter NKCC1 (SLC12A2) and carbonic anhydrase XII variant 2.Antisense strand has the continuous complementary zone fully that is close to of at least 19 nucleotide, the hybridization portion of mRNA is contained in this zone, and described mRNA corresponds respectively to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ IDNO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ IDNO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ IDNO:133 or SEQ ID NO:134.The expression of using the ocular hypertension targets mRNA that has weakened the experimenter of this compositions.
In one embodiment, ocular hypertension targets mRNA coding carbonic anhydrase II, IV or XII, and antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:101 or SEQ ID NO:134, and comprise with the hybridization portion that corresponds respectively to the mRNA of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:101 or SEQ ID NO:134 and have at least 19 nucleotide to be close to complete continuous complementary zone.
In another embodiment, ocular hypertension targets mRNA coding β 1 or beta 2-adrenergic receptor, and antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:3 or SEQ ID NO:4, and comprise with the hybridization portion that corresponds respectively to the mRNA of SEQ ID NO:3 or SEQ IDNO:4 and have at least 19 nucleotide to be close to complete continuous complementary zone.
In the another one embodiment, the ocular hypertension targets mRNA acetylcholinesterase of encoding, and antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:5 or SEQ ID NO:123, and comprise with the hybridization portion that corresponds respectively to the mRNA of SEQ ID NO:5 or SEQ ID NO:123 and have at least 19 nucleotide to be close to complete continuous complementary zone.
In another embodiment, ocular hypertension targets mRNA coding Na
+/ K
+The subunit of-ATP enzyme, and antisense strand (this mRNA is corresponding to SEQ IDNO:6 with the hybridization of the part of mRNA under physiological condition, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131 or SEQ ID NO:132), and comprise hybridization portion with mRNA have at least 19 nucleotide to be close to fully continuous complementary zone (this mRNA corresponds respectively to SEQ ID NO:6, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ IDNO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, or SEQ IDNO:132).
In another embodiment, ocular hypertension targets mRNA coding Na-K-2Cl cotransporter, and antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:7 or SEQ ID NO:133, and comprise with the hybridization portion that corresponds respectively to the mRNA of SEQ ID NO:7 or SEQ ID NO:133 and have at least 19 nucleotide to be close to complete continuous complementary zone.
In one embodiment of the invention, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 232 corresponding to SEQ ID NO:1,527,721,728,809,810,855,856,921,1139,506,547,548,740,911,1009,1140,1149,1150,1151,1188,1194,1195,1223,1239,1456,1457,1458,100,158,166,247,286,318,322,328,371,412,482,504,505,541,734,772,777,814,972,998,1232,317 or 401.
In another embodiment of the invention, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 213,252,258,266,399,457,463,490,595,1064,109,112,125,126,150,261,265,280,398,453,459,462,467,492,534,785,801,825,827,876,1003 or 1012 corresponding to SEQ ID NO:2.
In another embodiment of the invention, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 191,239,274,275,341,389,412,413,423,687,689,695,710,791,792,794,983,993,994,995,691,1039,1568,2326,2332,2425,2433,2844,2845,2880,2884,2891,2954,2955,2956,2957,2964,2965,3006,3007,3012 or 3026 corresponding to SEQ ID NO:101.
In another embodiment, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 687,1535,2293,2299,2392,2400,2811,2812,2847,2851,2858,2921,2922,2923,2924,2931,2932,2973,2974,2979 or 2993 corresponding to SEQ ID NO:134.
Another embodiment of the invention provides and is designed to mRNA is the RNA interfering of target, this mRNA comprises nucleotide 468,523,799,1563,1565,1569,1593,1613,1614,1626,310,322,726,769,772,801,802,1501,1576,1577,1579,1580,1581,1586,1590,1592,1594,1615,1616,1632,1633 or 1654 corresponding to SEQ ID NO:3.
Another embodiment of the invention provides and is designed to mRNA is the RNA interfering of target, this mRNA comprises nucleotide 329 corresponding to SEQ ID NO:4,375,1031,1046,1149,1163,1371,1401,1426,1880,283,607,608,609,619,623,722,857,1037,1091,1115,1124,1136,1137,1151,1164,1393,1394,1395,1406,1407,1427,1428,1429,1442,1725,1726,1756,1757,1758,1767,1790,1791,1792,1793,1803,1861,1869,1971,1972 or 1979.
In another embodiment of the invention, it is target that RNA interfering is designed to mRNA, and this mRNA comprises nucleotide 1875,1890,1891,2011,2012,2133 or 2134 corresponding to SEQ ID NO:123.
Another embodiment of the invention provides and is designed to mRNA is the RNA interfering of target, this mRNA comprises nucleotide 366 corresponding to SEQ ID NO:5,370,384,385,525,588,768,1045,1046,1061,1090,1232,1314,1316,1460,1461,1462,1528,1607,1705,1713,382,393,397,622,1131,1459,1530,2251,2885,2886,386,1231,1315,2047,2049,2053,2055,2057,2125,2126,2127,2250,2253,2258,2260,2318,2395,2397,2404,2405,2643,2645 or 2887.
In another embodiment, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 2208,2275,2307,2526,2538,2592,2628,2979,2985,3093,3474,3504,3505,3506,3518,343,442,700,707,811,907,1059,1363,1594,1662,1758,1760,1896,2037 or 2147 corresponding to SEQ ID NO:124.
In another embodiment, it is target that RNA interfering is designed to mRNA, and this mRNA comprises nucleotide 436,441,443,552,617,701,702,832,2204,2291 or 2495 corresponding to SEQ ID NO:125.
Another embodiment of the invention provides and is designed to mRNA is the RNA interfering of target, this mRNA comprises nucleotide 471 corresponding to SEQ ID NO:6,1990,3080,3797,4037,4093,4225,4323,5213,5285,214,467,470,472,473,632,825,946,1693,1767,1768,2157,2263,2589,2590,2765,2988,3094,3144,3145,3344,3345,3418,3666,3828,3850,4040,4041,4061,4882,4894,4900,5040,5114,5115,5128,5129,5253,5296,5375,5384 or 5385.
In another embodiment of the invention, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 240,272,362,1836,1851,2103,2137,2138,2139,2157,2158,2160,2425,2580,2601,2646,2650,2794,2803,3116,3124,3126,3129 or 3377 corresponding to SEQ ID NO:126.
In another embodiment of the invention, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 113,612,702,833,1101,1732,1733,1836,2070,2071,2143,2328,2475,2861,2862,2952,3203,3281,3377,3379,3470,3471,3554,3614,3615,3616,3617,3625,3626,3642,3646,3647,3653,3655,3797,3801,3803,3809 or 3810 corresponding to SEQ ID NO:127.
In another embodiment, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 126,251,252,253,331,427,429,520,521,530,601,602,603,604,664,665,666,667,675,676,692,696,697,702,703,705,707,847,851,853,859 or 860 corresponding to SEQ ID NO:128.
In another embodiment, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 1096,1099,1130,1131,1167,1299,1441,1450,1451,1452,1564,1746,1750,1751,1752,1795,203,204,214,222,224,225,226,380,525,591,612,613,615,635,636,663,664,669,699,765,790,839,840,841,900,909,933 or 947 corresponding to SEQ ID NO:129.
In another embodiment, it is target that RNA interfering is designed to mRNA, and this mRNA comprises nucleotide 1063,1102,1106,1107,1108,1109,1111 or 1151 corresponding to SEQ ID NO:130.
In another embodiment, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 653,654,771,773,841,849,853,917,918,926,927,931,981,983,984,996,998,1022,1023,1160,1214,1355,1356,1381,1394,1425,1474,1550,1620,1707,1740,1753,1825,1956,1965,2598,2599,2608,2828,2829,2888,3012 or 3251 corresponding to SEQ ID NO:131.
In another embodiment of the invention, it is target that RNA interfering is designed to mRNA, this mRNA comprises nucleotide 292,434,438,457,459,488,490,498,499,592,639,723,774,775,788,857,858,910,911,930,931,932,1009,1010,1023,1024,1111,1146,1147,1220,1246,1321,1325,1326,1327,1331,1437,1548,1571,1785,1786 or 1787 corresponding to SEQ ID NO:132.
Another embodiment of the invention provides and is designed to mRNA is the RNA interfering of target, this mRNA comprises nucleotide 675,974,1373,1780,2102,2151,2315,2542,2609,3197,67,71,73,353,405,864,911,912,913,1409,1748,1811,1935,1937,1993,2012,2346,2388,2437,2586,3007,3008,3022,3130,3210,3237 or 3271 corresponding to SEQ ID NO:7.
Another embodiment of the invention provides and is designed to mRNA is the RNA interfering of target, this mRNA comprises nucleotide 748,749,753,1119,1169,1499,1509,1820,2081,2118,2147,2615,2644,2659,2663,2671,2672,2793,2812,2914,2948,3044,3334,3391,3480,3520,3549,3639,3840,3941,3944,4001,4995,4997,5141,5143,5249,5375,5834,5852,5981 or 6678 corresponding to SEQ ID NO:133.
Except that first kind of RNA interfering, the present invention also provides to the experimenter and uses second kind of RNA interfering.This method comprises to the experimenter uses second kind of RNA interfering that length is 19-49 nucleotide, and described second kind of RNA interfering also includes the continuous complementary zone fully that is close to of adopted nucleotide chain, antisensenucleic acids chain and at least 19 nucleotide; Wherein the antisense strand of second kind of RNA interfering is hybridized with the part of mRNA under physiological condition, this mRNA is corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133 or SEQ ID NO:134, and second hybridization portion of antisense strand and mRNA has the continuous complementary zone fully that is close to of at least 19 nucleotide, and this mRNA corresponds respectively to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133 or SEQ ID NO:134.Second RNA interfering may be target with identical mRNA with first RNA interfering or be target with different mRNA.In addition, also can use in a similar manner the third, RNA interfering such as the 4th kind or the 5th kind.
Another embodiment of the invention is to answer the experimenter need treat ocular hypertensive method.This method comprises that to experimenter's ocular administration compositions the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide.This RNA interfering contains the continuous complementary zone fully that is close to that adopted nucleotide chain, antisense nucleotide chain and at least 19 nucleotide are arranged.This antisense strand is hybridized with the part of mRNA under physiological condition, this mRNA is corresponding to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ IDNO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ IDNO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ TD NO:133 or SEQ ID NO:134, and the hybridization portion that comprises with mRNA has at least 19 nucleotide to be close to continuous complementary zone fully, and this mRNA corresponds respectively to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ IDNO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ IDNO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ IDNO:133 or SEQ ID NO:134.Treat high intraocular pressure thus.
Another embodiment of the invention is to weaken the method that ocular hypertension targets mRNA expresses in the experimenter, comprises to the experimenter and use compositions that the length that said composition comprises effective dose is the strand RNA interfering and the drug acceptable carrier of 19-49 nucleotide.For weakening the expression of ocular hypertension targets, this strand RNA interfering under physiological condition with part hybridization corresponding to the mRNA of the above-mentioned nucleotide site of sequence identifier and antisense strand.
Another embodiment of the invention is to weaken the method that ocular hypertension targets mRNA expresses in the experimenter, comprise to the experimenter and use compositions, the length that said composition comprises effective dose is the strand RNA interfering and the drug acceptable carrier of 19-49 nucleotide, wherein, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:8, SEQ ID NO:14-SEQ ID NO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ ID NO:135-SEQ ID NO:717, SEQ ID NO:720 and SEQ ID NO:721.
When ocular hypertension targets mRNA coding carbonic anhydrase, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:8, SEQ ID NO:14-SEQ ID NO:32, SEQ IDNO:83-SEQ IDNO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ ID NO:135-SEQ IDNO:219, SEQ ID NO:720 and SEQ ID NO:721.
When ocular hypertension targets mRNA coding B-adrenergic receptor, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:33-SEQ ID NO:52 and SEQ ID NO:220-SEQ IDNO:282.
When ocular hypertension targets mRNA coding ACHE, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQID NO:53-SEQ ID NO:62 and SEQ ID NO:283-333.
When ocular hypertension targets mRNA coding ATP1A1, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:334-SEQ ID NO:374.
When ocular hypertension targets mRNA coding ATP1A2, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:63-SEQ ID NO:72 and SEQ ID NO:375-SEQ ID NO:416.
When ocular hypertension targets mRNA coding ATP1A3, RNA interfering comprises 3 of this continuous nucleotides of zone of at least 13 continuous nucleotides and following arbitrary sequence ' penultimate 13 nucleotide of end and has complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQIDNO:417-SEQIDNO:440.
When ocular hypertension targets mRNA coding ATP1A4, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:441-SEQ ID NO:511.
When ocular hypertension targets mRNA coding ATP1B1, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:512-SEQ ID NO:563.
When ocular hypertension targets mRNA coding ATP1B2, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:564-SEQ ID NO:606.
When ocular hypertension targets mRNA coding ATP1B3, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:607-SEQ ID NO:648.
When ocular hypertension targets mRNA coding SLC12A1, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:73-SEQ ID NO:82 and SEQ ED NO:649-SEQ ID NO:675.
When ocular hypertension targets mRNA coding SLC12A2, RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:676-SEQ ID NO:717.
In another embodiment of the invention, at least 14 continuous nucleotides are contained in the continuous nucleotide zone, and penultimate 14 nucleotide of 3 of the sequence of this continuous nucleotide and sequence identifier ' end have complementary or at least 85% the sequence homogeneity of at least 85% sequence.And in another embodiment of the invention, at least 15,16,17 or 18 continuous nucleotides are contained in the continuous nucleotide zone, have complementary or at least 80% the sequence homogeneity of at least 80% sequence with 3 of the sequence of sequence identifier ' penultimate 15,16,17 or 18 nucleotide of end respectively.
One embodiment of the invention are compositions and the drug acceptable carriers that comprise RNA interfering, and this RNA interfering length is 19-49 nucleotide and has any one nucleotide sequence or its complementary series among SEQ ID NO:8, SEQ IDNO:14-SEQ ID NO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ IDNO:135-SEQ ID NO:717, SEQ ED NO:720 and the SEQ ID NO:721.In one embodiment, RNA interfering is isolating.It is free from its total natural surroundings that term " isolating " means RNA interfering.
Another embodiment of the invention is to answer the experimenter need treat ocular hypertensive method.This method comprises to experimenter's ocular administration compositions, the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide, this RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, described sequence is SEQIDNO:8, SEQ ID NO:14-SEQ ID NO:100, SEQ ID NO:102-SEQ IDNO:122, SEQ ID NO:135-SEQ ID NO:717, SEQ ID NO:720 and SEQ IDNO:721 treat high intraocular pressure thus.
Another embodiment of the invention is to weaken the expression of first kind of variant of experimenter's ocular hypertension targets mRNA and the method that do not weaken the expression of second kind of variant of ocular hypertension targets mRNA.This method comprises to the experimenter uses compositions, the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide, this RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of described continuous nucleotide and first kind of variant ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, wherein the expression of first kind of variant mRNA is weakened, and do not weaken the expression of second variant mRNA, and wherein first kind of variant target mRNA is SEQ ID NO:101, SEQ ID NO:5, SEQ ID NO:124, SEQ IDNO:127 or SEQ ID NO:129 and second kind of variant target mRNA are SEQ ID NO:134, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128 or SEQ ID NO:130.
In another embodiment of the above method, first kind of variant target mRNA is SEQID NO:134, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128 or SEQID NO:130, and second kind of variant target mRNA is SEQ ID NO:101, SEQ IDNO:5, SEQ ID NO:124, SEQ ID NO:127 or SEQ ID NO:129.
Any embodiment described herein also is embodiment of the present invention in the purposes that preparation is used for weakening the medicine that high intraocular pressure mRNA expresses.
The accompanying drawing summary
Fig. 1 provides western trace, detects with CA2siRNA# 1, #3, #4 and #5, no targeting contrast siRNA and buffer contrast (siRNA) the HeLa cell of transfection with anti-CA2 and actin antibody.SiRNA concentration is 100nM or 1nM.The position of arrow indication～30-kDa CA2 albumen and 42-kDa actin band.
Detailed Description Of The Invention
It is the process of using double-stranded RNA (dsRNA) to come cryptiogene to express that RNA disturbs (RNAi). Although do not expect to be bound by theory, RNAi at first cuts into little RNA interfering (siRNA) by class RNaseIII enzyme-dicer with long dsRNA. SiRNA is that length is generally about 19-28 nucleotides or 20-25 nucleotides or 21-22 nucleotides and usually contains 3 ' jag of 2-nucleotides and the dsRNA of 5 ' phosphoric acid and 3 ' C-terminal. It is in the RNA silencing complex (RISC) of inducing that the chain of siRNA is impregnated in ribonucleoprotein complex. RISC utilizes this siRNA chain to identify mRNA molecule with at least part of complementation of siRNA chain of incorporating into, and then cuts these targets mRNA or suppress their translation. Therefore, the siRNA chain that mixes RISC is called as guiding chain or antisense strand. Another siRNA chain (being called transfer chain or sense strand) is removed from siRNA, and with target RNA Homoeology is arranged at least. Those skilled in the art generally acknowledge that arbitrary the chain of siRNA can both be impregnated among the RISC and as guiding chain and work in principle. Yet siRNA design (the siRNA double helix stability that for example, reduces at 5 of antisense strand ' end) can be conducive to antisense strand and mix among the RISC.
The mRNA cutting with the sequence of at least part of complementation of guiding chain of having of RISC mediation causes this mRNA and is reduced by the steady-state level of the related protein of this mRNA coding. Alternatively, RISC also can reduce the expression of related protein by suppressing translation, and does not cut target mRNA. Other RNA molecules and class RNA molecule also can interact with RISC and cryptiogene is expressed. Can comprise with the example of interactional other RNA molecules of RISC short hairpin RNA (shRNA), strand siRNA, Microrna (miRNA) and dicer-substrate 27 aggressiveness double helixs. Except as otherwise noted, term ＂ siRNA ＂ refers to double-chain interference RNA. Can comprise with the example of the interactional class RNA molecule of RISC and contain the RNA molecule that one or more nucleotides through chemical modification, one or more deoxyribonucleotide and/or one or more non-phosphodiester bond are connected. For the purpose of this discussion, all RNA or class RNA molecules that can interact with RISC and participate in the change of gene expression of RISC mediation are called " RNA interfering ". Therefore, SiRNA, shRNA, miRNA and dicer-substrate 27 aggressiveness double helixs all are the subgroups of " RNA interfering ".
As if with catalytic way cutting target mRNA, namely RNA interfering can play inhibitory action to target mRNA to RNA interfering in the embodiment of the present invention on substoichiometric. Compare with antisense therapy, under such cutting condition, provide the needed RNA interfering of curative effect obviously to reduce.
The present invention relates to the expression that application of interference RNA suppresses ocular hypertension targets mRNA, thereby reduce open-angle glaucoma or high intraocular pressure patient's intraocular pressure. Ocular hypertension targets comprises carbonic anhydrase II, IV and XII; β 1 and beta 2-adrenergic receptor; Acetylcholinesterase; Na+/K
+-ATP enzyme subunit and Na-K-2Cl cotransporter. According to the present invention, the RNA interfering of expression that external source provides or endogenous makes ocular hypertension targets mRNA silence in the part tissue of eye.
The reversible hydration of carbonic anhydrase catalysis carbon dioxide and as if secretion of aqueous humor is played regulating action. Carbonic anhydrase inhibitor reduces intraocular pressure by reducing the Fluid Volume that produces. Carbonic anhydrase inhibitor can be used as eye drops (Dorzolamide (dorzolamide), brinzolamide (brinzolamide)) or tablets/capsules (acetazolamide (acetazolamide), methazolamide (methazolamide)) is used. The side effect of eye drops is lacked than tablet or capsule, can be accepted by a lot of patients better.The ophthalmic suspension of (brinzolamide) 1% is typical carbonic anhydrase inhibitor (Alcon Laboratories, Inc., Fort Worth, TX).
The eye beta-blocker reduces intraocular pressure by the Fluid Volume that reduces the intraocular generation. These medicines are divided into two classes: non-selective beta-blocker (Timolol (timolol), levobunolol (levobunolol), metipranolol (metipranolol), carteolol (carteolol)) and β-1 selective receptor retarding agent (betaxolol (betaxolol)). Usually dosage is each one every, one day one to twice, depends on employed medicine. The example of this product is
The ophthalmic suspension (Alcon Laboratories, Inc., Fort Worth, TX) of (betaxolol hydrochloride (betaxolol HCl)) 0.25%.
The inhibitor of acetylcholinesterase causes the enzyme (acetylcholinesterase) of acetylcholine hydrolyzation to make acetylcholine be retained in acceptor site by blocking-up. Acetylcholine accumulates in acceptor, and is by the contraction reduction intraocular pressure of ciliary muscle, similar to direct acting cholinergic agonist effect.
Na
+/K
+-atpase inhibitor for example unabain, nitric oxide donors and Endothelin reduces Na+/K
+The activity of-ATP enzyme reduces the driving force by ciliary process secretion aqueous humor.
The chloride transport inhibitors, for example ethacrynic acid changes the trabecular meshwork cell capacity to increase easily degree of outflow.
Unless otherwise noted, the nucleotide sequence of quoting here all by 5 ' write to 3 ' direction. Term used herein " nucleic acid ", refer to DNA or RNA or its modified forms, comprise the purine or the pyrimidine bases that are present among DNA (adenine " A ", cytimidine " C ", guanine " G ", thymidine " T ") or the RNA (adenine " A ", cytimidine " C ", guanine " G ", uracil " U "). Here the RNA interfering that provides may comprise " T " base, particularly at 3 ' end, although " T " base is not present among the RNA usually. " nucleic acid " comprises term " oligonucleotides " and " polynucleotides " and can refer to single chain molecule or duplex molecule. Duplex molecule forms by the Watson-Crick base pairing between A and T base, C and G base, A and the U base. Can be complementary partly, substantially or completely between the two strands of duplex molecule, and form a double helix crossbred, its bond strength depends on the nature and extent of base sequence complementation.
Be easy to infer the mRNA sequence from corresponding dna sequence dna. For example, SEQ ID NO:1 provides the sense strand sequence corresponding to the DNA of coding carbonic anhydrase II mRNA. The mRNA sequence is the same with DNA sense strand sequence, and wherein, " U " base replaces " T " base.
Therefore, from the SEQ ID NO:1 mRNA sequence of carbonic anhydrase II as can be known; From the SEQ ID NO:2 mRNA sequence of carbonic anhydrase IV as can be known; From the SEQ ID NO:3 mRNA sequence of β 1-adrenergic receptor as can be known; From the SEQID NO:4 mRNA sequence of beta 2-adrenergic receptor as can be known; From the SEQ ID NO:5 mRNA sequence of acetylcholinesterase splice variant E4-E5 as can be known; From SEQ ID NO:6 Na as can be known+/K
+The mRNA sequence of-ATP enzyme α 2; From the SEQ ID NO:7 mRNA sequence of Na-K-2Cl cotransporter Al as can be known; From the SEQ ID NO:101 mRNA sequence of carbonic anhydrase XII variant 1 as can be known; From the SEQ ID NO:123 mRNA sequence of acetylcholinesterase splice variant E4-E6 as can be known; From SEQ ID NO:124 Na as can be known+/K
+-ATP enzyme α 1, the mRNA sequence of variant 1; From SEQ ID NO:125 Na as can be known+/K
+-ATP enzyme α 1, the mRNA sequence of variant 2; From SEQ ID NO:126 Na as can be known+/K
+The mRNA sequence of-ATP enzyme α 3; From SEQ ID NO:127 Na as can be known+/K
+-ATP enzyme α 4, the mRNA sequence of variant 1; From SEQ ID NO:128 Na as can be known+/K
+-ATP enzyme α 4, the mRNA sequence of variant 2; From SEQ ID NO:129 Na as can be known+/K
+The mRNA sequence of-ATP enzyme β 1 variant 1; From SEQ ID NO:130 Na as can be known+/K
+-ATP enzyme β 1, the mRNA sequence of variant 2; From SEQ ID NO:131 Na as can be known+/K
+The mRNA sequence of-ATP enzyme β 2; From SEQ ID NO:132 Na as can be known+/K
+The mRNA sequence of-ATP enzyme β 3; From the SEQ ID NO:133 mRNA sequence of Na-K-2Cl cotransporter A2 as can be known; From SEQ ID NO:134 carbonic anhydrase XII as can be known, the mRNA sequence of variant 2.
MRNA (the CA2 of carbonic anhydrase II, IV and XII, CA4 and CA12): the GenBank database as the state-run biotechnology information centre of the U.S. on ncbi.nlm.nih.gov is described, and carbonic anhydrase (CA) II, IV and XII are the zinc metalloenzyme extended familys members of the reversible hydration of catalysis carbon dioxide. Carbonic anhydrase relates to for example CO between metabolizing tissue and the lung of important physiology course2The breathing of/bicarbonate and transportation, pH and CO2Homeostasis, the electrolyte secretion in Various Tissues and organ, biosynthesis reaction (generating and urea generates such as gluconeogenesis, fat), bone absorption, calcification and tumour occur.
Identify 14 kinds of different carbonic anhydrase isodynamic enzymes and had different Subcellular Localization and Tissue distribution. Carbonic anhydrase II is the kytoplasm isoenzymes, and carbonic anhydrase IV and XII are membrane-bound. Two of different subtype that identified coding CA-XII gene transcribe variant; The hypotype that variant 1 coding is long, and variant 2 is compared the extron that lacks coding in variant 1 transcript has lacked 11 amino acid whose sections thereby compare with hypotype 1. Systemic administration carbonic anhydrase inhibitor (CAI) is conducive to reduce the intraocular pressure (IOP) of the rising of glaucoma feature. The isodynamic enzyme inhibitory action (sulfanilamide (SN) susceptible isodynamic enzyme CA II and CA IV) that is present in ciliary process reduces the speed of bicarbonate and secretion of aqueous humor, thereby causes IOP to reduce 25-30%. Yet, an inhibitory action that is present in the multiple CA isoenzymes of the outer tissue of eye has side effects, and comprises that the paralysis of four limbs and tingle, metallic taste, depression, tired, uncomfortable, weight saving, sexual desire minimizing, intestines and stomach stimulation, metabolic acidosis, kidney stone and transience are near-sighted.
The GenBank database provides the dna sequence dna of CA2 with registration number NM_000067, and it is provided as SEQ ID NO:1 in " sequence table ". SEQ ID NO:1 provides the sense strand sequence of DNA, corresponding to the mRNA (except ＂ T ＂ base substitutes ＂ U ＂ base) of coding CAII. The coded sequence of CAII is from nucleotides 66-848.
Suitable with above-described CA2mRNA sequence is Alternative spliceo some, equipotential form, isodynamic enzyme or its homologue. Homologue refer to derive from other mammalian species and with the CA2mRNA (being the ortholog thing) of SEQ ID NO:1 homology. The CA2 nucleotide sequence relevant with SEQ ID NO:1 comprises those sequences of GenBank registration number M77181, X03251, BC011949, BC035424, CR536526, CR541875, J03037, M36532, S69526 and Y00339.
The GenBank database provides with SEQ ID NO:2 in " sequence table " with the dna sequence dna that registration number NM_000717 provides CA4. SEQ ID NO:2 provides the sense strand sequence of DNA, corresponding to the mRNA (except ＂ T ＂ base has substituted ＂ U ＂ base) of coding CAIV. The coded sequence of CAIV is from nucleotides 47-985.
Suitable with above-described CA2mRNA sequence is Alternative spliceo some, equipotential form, isodynamic enzyme or its homologue. Homologue refer to derive from other mammalian species and with the CA2mRNA (being the ortholog thing) of SEQ ID NO:1 homology. The CA4 nucleotide sequence relevant with SEQ ID NO:2 comprises those sequences of GenBank registration number L10955, BC057792, BC069649, BC074768, CR541766 and M83670.
The GenBank database provides with SEQ ID NO:101 in " sequence table " with the dna sequence dna that registration number NM_001218 provides CA12. SEQ ID NO:101 provides the sense strand sequence of DNA, corresponding to the mRNA (except ＂ T ＂ base has substituted ＂ U ＂ base) of coding CAXII variant 1. The coded sequence of CAXII variant 1 is from nucleotides 157-1221.
Suitable with the mRNA sequence of above-described CA12 variant 1 is Alternative spliceo some, equipotential form, isodynamic enzyme or its homologue. Homologue refer to derive from other mammalian species and with the CA12mRNA (being the ortholog thing) of SEQ ID NO:101 homology.
The GenBank database provides with SEQ ID NO:134 in " sequence table " for the DNA sequence of CA12 variant 2 is provided with registration number NM_206925. SEQ ID NO:134 provides the sense strand sequence of DNA, corresponding to the mRNA of coding CAXII variant 2 (except " T " base substituted ＂ U ＂ base). The coded sequence of CAXII variant 2 is from nucleotides 157-1188. Compare with variant 1, variant 2 lacks the extron of interior coding.
Suitable with above-described CA12 variant 2mRNA sequence is Alternative spliceo some, equipotential form, isodynamic enzyme or its homologue. Homologue refer to derive from other mammalian species and with the CA12mRNA (being the ortholog thing) of SEQ ID NO:134 homology.
Adrenergic receptor-β 1 and-β 2mRNA (ADRB1 and ADRB2): the GenBank database as the state-run biotechnology information centre of the U.S. on ncbi.nlm.nih.gov is described, and adrenergic receptor (subtype alpha 1, α 2, β 1 and β 2) is the prototype family of g protein coupled receptor of the physiological effect of mediation hormone adrenaline and neurotransmitter norepinephrine.
The GenBank database provides with SEQ ID NO:3 in " sequence table " with the dna sequence dna that registration number NM_000684 provides ADRB1. SEQ ID NO:3 provides the sense strand sequence of DNA, corresponding to the mRNA of coding β 1-adrenergic receptor (except " T " base substituted ＂ U ＂ base). The coded sequence of β 1-adrenergic receptor is nucleotides 87-1520.
Suitable with above-described ADRB1mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ADRB1mRNA (being the ortholog thing) of SEQ ID NO:3 homology. The ADRB1 nucleotide sequence relevant with SEQ ID NO:3 comprises those sequences of GenBank registration number AF169006, AF169007, AY567837 and J03019.
The GenBank database provides with SEQ ID NO:4 in " sequence table " with the dna sequence dna that registration number NM_000024 provides ADRB2. SEQ ID NO:4 provides the sense strand sequence of DNA, corresponding to the mRNA of coding beta 2-adrenergic receptor (except " T " base substituted ＂ U ＂ base). The coded sequence of beta 2-adrenergic receptor is nucleotides 220-1461.
Suitable with above-described ADRB2mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ADRB2mRNA (being the ortholog thing) of SEQ ID NO:4 homology. The ADRB2 nucleotide sequence relevant with SEQ ID NO:4 comprises those sequences of GenBank registration number AF022953, AF022954, AF022955, AF022956, AF169225, AF202305, AF203386, AY011291, J02960, Y00106, AY136741, BC012481, BC063486, BC073856, M15169 and X04827.
Acetylcholinesterase splice variant E4-E6 and E4-E5 (ACHE): the GenBank database as the state-run biotechnology information centre of the U.S. on ncbi.nlm.nih.gov is described, acetylcholinesterase is in neuromuscular junction and brain cholinergic synapse hydrolysis neurotransmitter acetylcholine, thus the termination signal transmission. Simultaneously find that also on erythrocyte membrane, it has consisted of the Yt blood group antigens. Acetylcholinesterase exists with the different kinds of molecules form, and it has similar catalytic performance, but different on the assembling of its oligomer and cell adhesion pattern to cell surface. It is by single ACHE gene code, and associates after the translation of the structure diversity of gene outcome from optionally mRNA montage and catalytic subunit and structure subunit. The principal mode of the acetylcholinesterase of finding in brain, muscle and other tissue is hydrophilic, this hydrophilic and collagen subunit or contain the oligomer that the structure subunit formation disulfide bond of fat is connected. On the other hand, the Alternative spliceo some that mainly is expressed in erythrocytic tissue is terminal different at C-, and contains the hydrophobic peptide that cuts with the GPI-anchored site. It associates with film by the part of the rear phosphoinositide (PI) that adds of translation. Splice variant E4-E6 is main transcript and results from extron 4 to the montage of exon 6. Splice variant E4-E5 results from extron 4 to the alternative splicing of extron 5.
The GenBank database provides with SEQ ID NO:5 in " sequence table " with the dna sequence dna that registration number NM_015831 provides ACHE splice variant E4-E5. SEQ ID NO:5 provides the sense strand sequence of DNA, corresponding to the mRNA of coding acetylcholinesterase E4-E5 (except " T " base substituted ＂ U ＂ base). The coded sequence of acetylcholinesterase E4-E5 is nucleotides 95-1948.
Suitable with above-described ACHE mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ACHE mRNA (being the ortholog thing) of SEQ ID NO:5 homology. The ACHE nucleotide sequence relevant with SEQ ID NO:5 comprises GenBank registration number AC011895, AF002993, AF312032, AY750146, CH236956, L06484, L42812, S71129, AF334270, BC026315, BC036813, M55040 and those sequences of NM_000665.
The GenBank database provides with SEQ ID NO:123 in " sequence table " with the dna sequence dna that registration number NM_000665 provides ACHE splice variant E4-E6. SEQ ID NO:123 provides the sense strand sequence of DNA, corresponding to the mRNA of coding acetylcholinesterase E4-E6 variant (except " T " base substituted ＂ U ＂ base). The coded sequence of acetylcholinesterase E4-E6 is nucleotides 95-1939.
Suitable with above-described ACHE mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ACHE mRNA (being the ortholog thing) of SEQ ID NO:123 homology. The ACHE nucleotide sequence relevant with SEQ ID NO:123 comprises GenBank registration number NM_015831, AC011895, AF002993, AF312032, AY750146, CH236956, L06484, L42812, S71129, AF334270, BC026315, BC036813 and those sequences of M55040.
Na
+/K
+-ATP enzyme α and β mRNA (ATP1-A1 variant 1 ,-A1 variant 2 ,-A2 ,-A3 ,-A4 variant 1 ,-A4 variant 2 ,-B1 variant 1 ,-B1 variant 2 ,-B2 and-B3): as describing at the GenBank database, protein by described gene code belongs to P-type cation transfer ATP enzyme family, and belongs to Na+/K
+-ATP enzyme subfamily. Na+/K
+-ATP enzyme is the memebrane protein of integrating, the responsible electrochemical gradient of setting up and keeping for sodium ion and potassium ion leap plasma membrane. These gradients are absolutely necessary for the transhipment of the sodium coupling of osmotic adjustment, various organic and inorganic molecule and the electrical excitation of N﹠M. This enzyme is comprised of two subunits, i.e. large catalytic subunit (α or A) and less glycoprotein subunit (β or B). Na+/K
+The catalytic subunit of-ATP enzyme is encoded by polygenes.
The GenBank database provides with SEQ ID NO:124 in " sequence table " with the DNA sequence that registration number NM_000701 provides ATP1A1 variant 1. SEQ ID NO:124 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme subunit A1 variant 1 (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme subunit A1 variant 1 is nucleotides 299-3370.
Suitable with above-described ATP1A1 variant 1mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1A1 variant 1mRNA (being the ortholog thing) of SEQ ID NO:124 homology.
The GenBank database provides with SEQ ID NO:125 in " sequence table " with the dna sequence dna that registration number NM_001001586 provides ATP1A1 variant 2. SEQ ID NO:125 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme subunit A1 variant 2 (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme subunit A1 variant 2 is nucleotides 299-2344.
Suitable with above-described ATP1A1 variant 2mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1A1 variant 2mRNA (being the ortholog thing) of SEQ ID NO:125 homology.
The GenBank database provides with SEQ ID NO:6 in " sequence table " with the dna sequence dna that registration number NM_000702 provides ATPl A2. SEQ ID NO:6 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme A2 subunit (except " T " base substituted ＂ U ＂ base). The coded sequence of Na+/K+-ATP enzyme A2 subunit is nucleotides 105-3167.
Suitable with above-described ATP1A2mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1A2mRNA (being the ortholog thing) of SEQ ID NO:6 homology. The ATP1A2 nucleotide sequence relevant with SEQ ID NO:6 comprises those sequences of GenBank registration number J05096, M27578, AB018321, AK091617, AK124581, AK126573, AL831991, AL831997, BC013680, BC047533, BC052271, M16795 and Y07494.
The GenBank database provides with SEQ ID NO:126 in " sequence table " with the dna sequence dna that registration number NM_152296 provides ATP1A3. SEQ ID NO:126 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme A3 subunit (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme A3 subunit is nucleotides 155-3196.
Suitable with above-described ATP1A3mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1A3mRNA (being the ortholog thing) of SEQ ID NO:126 homology.
The GenBank database provides with SEQ ID NO:127 in " sequence table " with the DNA sequence that registration number NM_144699 provides ATP1A4 variant 1. SEQ ID NO:127 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme A4 subunit variant 1 (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme A4 subunit variant 1 is nucleotides 469-3558.
Suitable with above-described ATP1A4 variant 1mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1A4 variant 1mRNA (being the ortholog thing) of SEQ ID NO:127 homology.
The GenBank database provides with SEQ ID NO:128 in " sequence table " with the dna sequence dna that registration number NM_01001734 provides ATP1A4 variant 2. SEQ ID NO:128 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme A4 subunit variant 2 (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme A4 subunit variant 2 is nucleotides 111-608.
Suitable with above-described ATP1A4 variant 2mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1A4 variant 2mRNA (being the ortholog thing) of SEQ ID NO:128 homology.
The GenBank database provides with SEQ ID NO:129 in " sequence table " with the DNA sequence that registration number NM_001677 provides ATP1B1 variant 1. SEQ ID NO:129 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme B1 subunit variant 1 (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme B1 subunit variant 1 is nucleotides 122-1033.
Suitable with above-described ATP1B1 variant 1mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1B1 variant 1mRNA (being the ortholog thing) of SEQ ID NO:129 homology.
The GenBank database provides with SEQ ID NO:130 in " sequence table " with the dna sequence dna that registration number NM_001001787 provides ATP1B1 variant 2. SEQ ID NO:130 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme B1 subunit variant 2 (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme B1 subunit variant 2 is nucleotides 122-1027.
Suitable with above-described ATP1B1 variant 2mRNA sequence is Alternative spliceo some, equipotential form or its homologue. Homologue refer to derive from other mammalian species and with the ATP1B1 variant 2mRNA (being the ortholog thing) of SEQ ID NO:130 homology.
The GenBank database provides with SEQ ID NO:131 in " sequence table " with the dna sequence dna that registration number NM_001678 provides ATP1B2. SEQ ID NO:131 provides the sense strand sequence of DNA, corresponding to coding Na+/K
+The mRNA of-ATP enzyme B2 subunit (except " T " base substituted ＂ U ＂ base). Na+/K
+The coded sequence of-ATP enzyme B2 subunit is nucleotides 584-1456.
Suitable with above-described ATP1B2mRNA sequence is alternative splicing body, equipotential form or its congener.Congener be meant derive from other mammalian species and with the homologous ATP1B2mRNA of SEQ ID NO:131 (being lineal congener).
The GenBank data base provides with SEQ ID NO:132 in " sequence table " with the DNA sequence that registration number NM_001679 provides ATP1B3.SEQ ID NO:132 provides the sense strand sequence of DNA, corresponding to coding Na
+/ K
+The mRNA of-ATP enzyme B3 subunit (except " T " base substituted " U " base).Na
+/ K
+The coded sequence of-ATP enzyme B3 subunit is nucleotide 175-1014.
Suitable with above-described ATP1B3mRNA sequence is alternative splicing body, equipotential form or its congener.Congener be meant derive from other mammalian species and with the homologous ATP1B3mRNA of SEQ ID NO:132 (being lineal congener).
Na-K-2Cl cotransporter (SLC12A1 and SLC12A2): sodium-potassium-chloride cotransporter (Na-K-2Cl cotransporter or NKCC) is convenient to Na
+, K
+Cross over plasma membrane with the ionic coupling co-transport of Cl-.Two kinds of hypotype: NKCC1 and NKCC2 are arranged.NKCC1 expresses in comprising the great majority tissue of eyes.By contrast, NKCC2 mainly expresses in kidney, yet, prove that on evidence this hypotype also has the expression of reduced levels in eyes.NKCC1 by SLC12A2 gene (solute carrier family 12, the member 2) coding and NKCC2 by the SLC12A1 gene code.Trabecular meshwork cell has strong Na-K-2Cl cotransporter.The activity of this cotransporter reduces the co-transport activity by neurotransmitter and hormonal regulation as norepinephrine, or vasopressin strengthens the co-transport activity.
The GenBank data base provides with SEQ ID NO:7 in " sequence table " with the DNA sequence that registration number NM_000338 provides SLC12A1.SEQ ID NO:7 provides the sense strand sequence of DNA, corresponding to the mRNA of coding Na-K-2Cl cotransporter NKCC2 (except " T " base substituted " U " base).The coded sequence of Na-K-2Cl cotransporter NKCC2 is nucleotide 20-3319.
Suitable with above-described Na-K-2Cl NKCC2 cotransporter mRNA sequence is alternative splicing body, equipotential form or its congener.Congener refer to derive from other mammalian species and with the homologous Na-K-2Cl cotransporter of SEQ ID NO:7 NKCC2mRNA (being lineal congener).The SLC12A1 nucleotide sequence relevant with SEQ ID NO:7 comprises those sequences of GenBank registration number AJ005332, AJ005333, AB032525, AB032527, BC040138, BX647067, BX647484 and U58130.
The GenBank data base provides with SEQ ID NO:133 in " sequence table " with the DNA sequence that registration number NM_001046 provides SLC12A2.SEQ ID NO:133 provides the sense strand sequence of DNA, corresponding to the mRNA of coding Na-K-2Cl cotransporter NKCC1 (except " T " base substituted " U " base).The coded sequence of Na-K-2Cl cotransporter NKCC1 is nucleotide 165-3803.
Suitable with above-described Na-K-2Cl NKCC1 cotransporter mRNA sequence is alternative splicing body, equipotential form or its congener.Congener refer to derive from other mammalian species and with the homologous Na-K-2Cl cotransporter of SEQ ID NO:133 NKCC1mRNA (being lineal congener).
MRNA weakens expression: phrase used herein " mRNA weaken expression " means and uses or express a certain amount of RNA interfering (as siRNA), by cutting mRNA or directly suppress translation and reduce target mRNA and translate into protein.The reduction of the expression of target mRNA or respective egg white matter is commonly referred to " knocking out ", and is with respect to using of being reported or the level after expressing no target contrast RNA (contrasting siRNA as no target).The embodiment is here considered and is comprised and the knocking out of the expression between 50%-100%.Yet, for purpose of the present invention, unnecessaryly reach such level that knocks out.In one embodiment, use the independent RNA interfering of one of targeting ocular hypertension targets to reduce IOP.In other embodiments, use two or more RNA interfering of the identical ocular hypertension targets of targeting (as CA2) to reduce IOP.Also have in some embodiments, two or more RNA interfering of using a plurality of ocular hypertension targets of targeting (as CA2 and ADRB2) are to reduce IOP.
Usually knock out by use quantitative polyase chain reaction (qPCR) amplification assay mRNA level, or by utilizing western trace or elisa (ELISA) mensuration protein level to assess.The analysis of protein level provides assessment for mRNA cutting and translation suppress both.Other technology that mensuration knocks out comprises RNA solution hybridization, nuclease protection, northern hybridization, uses the cell analysis of gene expression monitoring, antibodies, radioimmunoassay and the fluorescence-activation of gene chip.
By observing the improvement of glaucoma symptom, for example the inhibitory action that also has target described here in human body or mammal is inferred in the improvement of the improvement of the improvement of intraocular pressure, visual field loss or optic disc variation.
As if with catalytic way cutting target mRNA, promptly RNA interfering can play inhibitory action to target mRNA to RNA interfering in the embodiment of the present invention on substoichiometric.Compare with antisense therapy, under such cutting condition, provide the needed RNA interfering of curative effect obviously to reduce.
RNA interfering: in one embodiment of the invention, RNA interfering (as siRNA) has sense strand and antisense strand, and sense strand and antisense strand comprise at least 19 nucleotide be close to continuous complementary zone fully at least.In another embodiment of the invention, RNA interfering comprises the zone of at least 13,14,15,16,17 or 18 continuous nucleotides, respectively with mRNA in penultimate 13,14,15,16,17 or 18 nucleotide of 3 ' end of corresponding target sequence the sequence complementarity or the sequence homogeneity of certain percentage are arranged.
The length of every chain of RNA interfering comprises 19-49 nucleotide, also may comprise the length of 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48 or 49 nucleotide.
The antisense strand of siRNA is the active directed agents of siRNA, because antisense strand is impregnated among the RISC, thereby makes RISC identification and antisense siRNA chain have the complementary target mRNA of part at least to be used for cutting or to suppress translation.
In the present invention, by using the RNA interfering target sequence (as the siRNA target sequence) in the obtainable design tool selection target mRNA sequence.Check RNA interfering by the cell of transfection expression target mRNA, assess by knocking out as mentioned above subsequently corresponding to these target sequence.Select to produce the RNA interfering that knocks out of 50%-100% expression as further analyzing.
The technology of target sequence of selecting siRNA is by Tuschl, people such as T., " The siRNA UserGuide ", being revised on May 6th, 2004 provides, and can obtain in Rockefeller university website; Technical Bulletin #506 on also can the Ambion website by Ambion company in " siRNA Design Guidelines " provides; Also can pass through other based on network design tool, for example, Invitrogen, Dharmacon, Integrated DNA Technologies, Genscript or Proligo website are known.Initial search parameter can comprise the siRNA length between the G/C content between the 35%-55% and 19-27 nucleotide.Target sequence can be positioned the coding region of mRNA or be positioned 5 of mRNA ' or 3 ' end untranslated region.
In one embodiment, the DNA target sequence of 19 of carbonic anhydrase II nucleotide is present among the nucleotide 232-250 of SEQ ID NO:1:
5′-CCCTGAGGATCCTCAACAA-3′SEQ ID NO：8。
As the corresponding mRNA sequence of targeting SEQ ID NO:8 and have 21 nucleotide chains and the siRNA of the present invention of 2-nucleotide 3 ' jag is:
5′-CCCUGAGGAUCCUCAACAANN-3′SEQ ID NO：9
3′-NNGGGACUCCUAGGAGUUGUU-5′SEQ ID NO：10。
Each " N " residue can be the nucleotide of any nucleotide (A, C, G, U, T) or modification." N " residue number that 3 ' end can have is between 1,2,3,4,5 and 6 and comprise them." N " residue on arbitrary chain can identical (as UU, AA, CC, GG or TT) also can different (as AC, AG, AU, CA, CG, CU, GA, GC, GU, UA, UC or UG).3 ' jag can be the same or different.In one embodiment, two chains all have 3 ＇ UU jags.
The corresponding mRNA sequence of targeting SEQ ID NO:8 and have 21 nucleotide chains and on every chain, have the siRNA of the present invention of 3 ' UU jag to be:
5′-CCCUGAGGAUCCUCAACAAUU-3′SEQ ID NO：11
3′-UUGGGACUCCUAGGAGUUGUU-5′SEQ ID NO：12。
RNA interfering also has the jag of 5 ' nucleotide or it has flat terminal.The corresponding mRNA sequence of targeting SEQID NO:8 and have the terminal siRNA of the present invention of 19 nucleotide chains peace and be:
5′-CCCUGAGGAUCCUCAACAA-3′SEQ ID NO：722
3′-GGGACUCCUAGGAGUUGUU-5′SEQ ID NO：723。
The chain of double-chain interference RNA (as siRNA) can link hair clip of formation or loop-stem structure (as shRNA).The corresponding mRNA sequence of targeting SEQ ID NO:8 and have the double-stranded stem zone of 19 base pairs and the shRNA of the present invention of 3 ' UU jag is:
The known N of those of ordinary skills is nucleotide A, T, C, G, U or its modified forms.The number of nucleotide N is between 3-23 or 5-15 or 7-13 or 4-9 or 9-11 and comprise them in the ring, and perhaps the number of nucleotide N is 9.Some nucleotide in the ring may relate to ring in the base pair of other nucleotide interact.The example that can be used to form the oligonucleotide sequence of ring comprises 5 '-UUCAAGAGA-3 ' (Brummelkamp, people such as T.R., (2002) Science296:550) and 5 '-UUUGUGUAG-3 ' (Castanotto, people such as D., (2002) RNA8:1454).The single stranded oligonucleotide that those skilled in the art generally acknowledge to produce forms stem ring or hairpin structure, this structure comprise can with the double-stranded region of RNAi system interaction.
The siRNA target sequence of Que Dinging can be beneficial to the double-helical design of dicer substrate 27 aggressiveness 3 ' terminal the extension above.The extension of the DNA target sequence (SEQ ID NO:8) of 19 nucleotide in carbonic anhydrase II DNA sequence (SEQ ID NO:1), discerning by 6 nucleotide, obtain the DNA target sequence of 25 nucleotide, it is present in the nucleotide 232-256 among the SEQ ID NO:1:
5′-CCCTGAGGATCCTCAACAATGGTCA-3′SEQ ID NO：724。The dicer substrate of the present invention 27 aggressiveness Double helixs of the corresponding mRNA sequence of targeting SEQ ID NO:724 are:
5′-CCCUGAGGAUCCUAACAAUGGUCA-3′SEQ ID NO：718
3′-UUGGGACUCCUAGGAGUUGUUACCAGU-5′SEQ ID NO：719。
Two nucleotide of sense strand 3 ' end (being the CA nucleotide of SEQ ID NO:718) may be the Deoxydization nucleotides of the processing that is used to improve.As what here provide, from the double-helical design of dicer substrate 27 aggressiveness of 19-21 nucleotide target sequence at dna integration technology (IDT) website and Kim, people such as D.-H. (February, 2005) Nature Biotechnology 23:2; Further discussion is arranged among the 222-226.
When producing RNA interfering by chemosynthesis, phosphorylation in 5 ' terminal nucleotide, the 5 ' position of or two chains (if existence) can strengthen the siRNA effectiveness and the specificity of bonded RISC complex, but because phosphorylation can take place in cell, so do not need to carry out phosphorylation.
Table 1 is listed the example of the siRNA target sequence in the DNA sequence (being respectively SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:101 and SEQ ID NO:134) of CA2, CA4 and CA12 variant 1 and variant 2, designs siRNA of the present invention in the mode of as above illustrating from it.CA2, CA4 and CA12 variant 1 and variant 2 encode respectively carbonic anhydrase II, IV and XII variant 1 and 2.
The siRNA target sequence of table 1.CA2, CA4 and CA12
| The CA2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:1 | SEQ ID NO： |
| CCCTGAGGATCCTCAACAA | 232 | 8 |
| GGGCCTTCAGAAAGTTGTT | 527 | 14 |
| GCGAGCAGGTGTTGAAATT | 721 | 15 |
| GGTGTTGAAATTCCGTAAA | 728 | 16 |
| GCCACTGAAGAACAGGCAA | 809 | 17 |
| CCACTGAAGAACAGGCAAA | 810 | 18 |
| CCCATAGTCTGTATCCAAA | 855 | 19 |
| CCATAGTCTGTATCCAAAT | 856 | 20 |
| GGTGATTTGGACCCTGGTT | 921 | 21 |
| GGGTGATGAGCACTCACAA | 1139 | 22 |
| GAAGGTTGGCAGCGCTAAA | 506 | 83 |
| ATGTGCTGGATTCCATTAA | 547 | 84 |
| TGTGCTGGATTCCATTAAA | 548 | 85 |
| CCGTAAACTTAACTTCAAT | 740 | 86 |
| GATCTACCTTGGTGATTTG | 911 | 87 |
| GACCAATTGTCATGCTTGA | 1009 | 88 |
| GGTGATGAGCACTCACAAT | 1140 | 89 |
| CACTCACAATTGTTGACTA | 1149 | 90 |
| ACTCACAATTGTTGACTAA | 1150 | 91 |
| CTCACAATTGTTGACTAAA | 1151 | 92 |
| AGGAAAGTAGAATGGTTGA | 1188 | 93 |
| GTAGAATGGTTGAGTGCAA | 1194 | 94 |
| TAGAATGGTTGAGTGCAAA | 1195 | 95 |
| CAAGATAAATTGAGCTAGT | 1223 | 96 |
| AGTTAAGGCAAATCAGGTA | 1239 | 97 |
| GAGTTGTGATACAGAGTAT | 1456 | 98 |
| AGTTGTGATACAGAGTATA | 1457 | 99 |
| GTTGTGATACAGAGTATAT | 1458 | 100 |
| |
100 | 135 |
| TGACATCGACACTCATACA | 158 | 136 |
| ACACTCATACAGCCAAGTA | 166 | 137 |
| ACAATGGTCATGCTTTCAA | 247 | 138 |
| AGGACAAAGCAGTGCTCAA | 286 | 139 |
| GATGGCACTTACAGATTGA | 318 | 140 |
| GCACTTACAGATTGATTCA | 322 | 141 |
| ACAGATTGATTCAGTTTCA | 328 | 142 |
| ACAAGGTTCAGAGCATACT | 371 | 143 |
| CAGAACTTCACTTGGTTCA | 412 | 144 |
| ACTGGCCGTTCTAGGTATT | 482 | 145 |
| TTGAAGGTTGGCAGCGCTA | 504 | 146 |
| TGAAGGTTGGCAGCGCTAA | 505 | 147 |
| TTGTTGATGTGCTGGATTC | 541 | 148 |
| GAAATTCCGTAAACTTAAC | 734 | 149 |
| CCGAAGAACTGATGGTGGA | 772 | 150 |
| GAACTGATGGTGGACAACT | 777 | 151 |
| TGAAGAACAGGCAAATCAA | 814 | 152 |
| CTTACTTGATAGACTTACT | 972 | 153 |
| TGTGAAGACTAGACCAATT | 998 | 154 |
| TTGAGCTAGTTAAGGCAAA | 1232 | 155 |
| GGATGGCACTTACAGATTG | 317 | 720 |
| GAAATATGCTGCAGAACTT | 401 | 721 |
| The CA4 target sequence | Initial nucleotide position is with reference to SEQ ID NO:2 | SEQ ID NO： |
| TCGTCACCACCAAGGCAAA | 213 | 23 |
| GCTTCTTCTTCTCTGGCTA | 252 | 24 |
| TCTTCTCTGGCTACGATAA | 258 | 25 |
| GGCTACGATAAGAAGCAAA | 266 | 26 |
| GGTCCGACTTGCCATATAA | 399 | 27 |
| GGAGATGCACATAGTACAT | 457 | 28 |
| GCACATAGTACATGAGAAA | 463 | 29 |
| GACATCGAGGAATGTGAAA | 490 | 30 |
| GGTGGAGGCACTGTCTAAT | 595 | 31 |
| GGGACTTTAGGCATGATTA | 1064 | 32 |
| ACACTGGTGCTACGAGGTT | 109 | 156 |
| CTGGTGCTACGAGGTTCAA | 112 | 157 |
| GTTCAAGCCGAGTCCTCCA | 125 | 158 |
| TTCAAGCCGAGTCCTCCAA | 126 | 159 |
| CCTGCTTGGTGCCAGTCAA | 150 | 160 |
| TCTCTGGCTACGATAAGAA | 261 | 161 |
| TGGCTACGATAAGAAGCAA | 265 | 162 |
| GCAAACGTGGACTGTCCAA | 280 | 163 |
| TGGTCCGACTTGCCATATA | 398 | 164 |
| CCATGGAGATGCACATAGT | 453 | 165 |
| AGATGCACATAGTACATGA | 459 | 166 |
| TGCACATAGTACATGAGAA | 462 | 167 |
| ATAGTACATGAGAAAGAGA | 467 | 168 |
| CATCGAGGAATGTGAAAGA | 492 | 169 |
| TTGCGGTGCTGGCCTTTCT | 534 | 170 |
| GAACAGATCCTGGCATTCT | 785 | 171 |
| TCTCTCAGAAGCTGTACTA | 801 | 172 |
| AGGAACAGACAGTGAGCAT | 825 | 173 |
| GAACAGACAGTGAGCATGA | 827 | 174 |
| GGCAGCGCACGGTGATAAA | 876 | 175 |
| CAGCCTCTCTGTTGCCTCA | 1003 | 176 |
| TGTTGCCTCAGCTCTCCAA | 1012 | 177 |
| CA12, |
Initial nucleotide position is with reference to SEQ ID NO:101 | SEQ ID NO： |
| TCCTGCTGGTGATCTTAAA | 191 | 102 |
| ACGGTTCCAAGTGGACTTA | 239 | 103 |
| GAGAATAGCTGGTCCAAGA | 274 | 104 |
| AGAATAGCTGGTCCAAGAA | 275 | 105 |
| GTGACATCCTCCAGTATGA | 341 | 106 |
| GCTACAATCTGTCTGCCAA | 389 | 107 |
| CAGTTTCTCCTGACCAACA | 412 | 108 |
| AGTTTCTCCTGACCAACAA | 413 | 109 |
| GACCAACAATGGCCATTCA | 423 | 110 |
| CTCCTTCAATCCGTCCTAT | 687 | 111 |
| CCTTCAATCCGTCCTATGA | 689 | 112 |
| ATCCGTCCTATGACAAGAT | 695 | 113 |
| AGATCTTCAGTCACCTTCA | 710 | 114 |
| CGGAGAGGACCGCTGAATA | 791 | 115 |
| GGAGAGGACCGCTGAATAT | 792 | 116 |
| AGAGGACCGCTGAATATTA | 794 | 117 |
| AGGTCCAGAAGTTCGATGA | 983 | 118 |
| GTTCGATGAGAGGCTGGTA | 993 | 119 |
| TTCGATGAGAGGCTGGTAT | 994 | 120 |
| TCGATGAGAGGCTGGTATA | 995 | 121 |
| TTCAATCCGTCCTATGACA | 691 | 178 |
| CA12, |
Initial nucleotide position is with reference to SEQ ID NO:101 | SEQ ID NO： |
| TGTACTGCGGCAGGACTGA | 1039 | 122 |
| AGAGCGTGCTTTCAAGTGT | 1568 | 179 |
| GATGTCAAATCGTGGTTTA | 2326 | 180 |
| AAATCGTGGTTTAGATCAA | 2332 | 181 |
| ATGGAATGCTACTAAGATA | 2425 | 182 |
| CTACTAAGATACTCCATAT | 2433 | 183 |
| ACAACGATGGCAAGCCTTA | 2844 | 184 |
| CAACGATGGCAAGCCTTAT | 2845 | 185 |
| TTGCTAGGCAAAGTTACAA | 2880 | 186 |
| TAGGCAAAGTTACAAGTGA | 2884 | 187 |
| AGTTACAAGTGACCTAATG | 2891 | 188 |
| TGTGCACTCAAGACCTCTA | 2954 | 189 |
| GTGCACTCAAGACCTCTAA | 2955 | 190 |
| TGCACTCAAGACCTCTAAC | 2956 | 191 |
| GCACTCAAGACCTCTAACA | 2957 | 192 |
| AGACCTCTAACAGCCTCGA | 2964 | 193 |
| GACCTCTAACAGCCTCGAA | 2965 | 194 |
| TGCCATTAGCATGCCTCAT | 3006 | 195 |
| GCCATTAGCATGCCTCATG | 3007 | 196 |
| TAGCATGCCTCATGCATCA | 3012 | 197 |
| CATCATCAGATGACAAGGA | 3026 | 198 |
| CA12, variant 2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:134 | SEQ ID NO： |
| CTCCTTCAATCCGTCCTAT | 687 | 199 |
| AGAGCGTGCTTTCAAGTGT | 1535 | 200 |
| GATGTCAAATCGTGGTTTA | 2293 | 201 |
| AAATCGTGGTTTAGATCAA | 2299 | 202 |
| ATGGAATGCTACTAAGATA | 2392 | 203 |
| CTACTAAGATACTCCATAT | 2400 | 204 |
| ACAACGATGGCAAGCCTTA | 2811 | 205 |
| CAACGATGGCAAGCCTTAT | 2812 | 206 |
| TTGCTAGGCAAAGTTACAA | 2847 | 207 |
| TAGGCAAAGTTACAAGTGA | 2851 | 208 |
| AGTTACAAGTGACCTAATG | 2858 | 209 |
| TGTGCACTCAAGACCTCTA | 2921 | 210 |
| GTGCACTCAAGACCTCTAA | 2922 | 211 |
| TGCACTCAAGACCTCTAAC | 2923 | 212 |
| GCACTCAAGACCTCTAACA | 2924 | 213 |
| AGACCTCTAACAGCCTCGA | 2931 | 214 |
| GACCTCTAACAGCCTCGAA | 2932 | 215 |
| TGCCATTAGCATGCCTCAT | 2973 | 216 |
| GCCATTAGCATGCCTCATG | 2974 | 217 |
| TAGCATGCCTCATGCATCA | 2979 | 218 |
| CATCATCAGATGACAAGGA | 2993 | 219 |
Table 2 is listed the example of the siRNA target sequence in ADRB1 and the ADRB2DNA sequence (being respectively SEQ ID NO:3 and SEQ IDNO:4), by the mode of as above illustrating from wherein designing siRNA of the present invention.As mentioned above, encode respectively β 1-and beta 2-adrenergic receptor of ADRB1 and ADRB2.
The siRNA target sequence of table 2.ADRB1 and ADRB2
| The ADRB1 target sequence | Initial nucleotide position is with reference to SEQ ID NO:3 | SEQ ID NO： |
| TCCTTCTTCTGCGAGCTGT | 468 | 33 |
| TCGAGACCCTGTGTGTCAT | 523 | 34 |
| GCATCATGGCCTTCGTGTA | 799 | 35 |
| GAACGAGGAGATCTGTGTT | 1563 | 36 |
| ACGAGGAGATCTGTGTTTA | 1565 | 37 |
| GGAGATCTGTGTTTACTTA | 1569 | 38 |
| GATAGCAGGTGAACTCGAA | 1593 | 39 |
| CCCACAATCCTCGTCTGAA | 1613 | 40 |
| CCACAATCCTCGTCTGAAT | 1614 | 41 |
| TCTGAATCATCCGAGGCAA | 1626 | 42 |
| GCAATGTGCTGGTGATCGT | 310 | 220 |
| TGATCGTGGCCATCGCCAA | 322 | 221 |
| AAGTGCTGCGACTTCGTCA | 726 | 222 |
| CGTCCGTAGTCTCCTTCTA | 769 | 223 |
| CCGTAGTCTCCTTCTACGT | 772 | 224 |
| ATCATGGCCTTCGTGTACC | 801 | 225 |
| TCATGGCCTTCGTGTACCT | 802 | 226 |
| CCTCGGAATCCAAGGTGTA | 1501 | 227 |
| TGTGTTTACTTAAGACCGA | 1576 | 228 |
| GTGTTTACTTAAGACCGAT | 1577 | 229 |
| GTTTACTTAAGACCGATAG | 1579 | 230 |
| TTTACTTAAGACCGATAGC | 1580 | 231 |
| TTACTTAAGACCGATAGCA | 1581 | 232 |
| TAAGACCGATAGCAGGTGA | 1586 | 233 |
| ACCGATAGCAGGTGAACTC | 1590 | 234 |
| CGATAGCAGGTGAACTCGA | 1592 | 235 |
| ATAGCAGGTGAACTCGAAG | 1594 | 236 |
| CACAATCCTCGTCTGAATC | 1615 | 237 |
| ACAATCCTCGTCTGAATCA | 1616 | 238 |
| TCATCCGAGGCAAAGAGAA | 1632 | 239 |
| CATCCGAGGCAAAGAGAAA | 1633 | 240 |
| CCACGGACCGTTGCACAAA | 1654 | 241 |
| The ADRB2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:4 | SEQ ID NO： |
| GCATCGTCATGTCTCTCAT | 329 | 43 |
| GCTGGTCATCACAGCCATT | 375 | 44 |
| CCCTCAAGACGTTAGGCAT | 1031 | 45 |
| GCATCATCATGGGCACTTT | 1046 | 46 |
| CCTAAATTGGATAGGCTAT | 1149 | 47 |
| GCTATGTCAATTCTGGTTT | 1163 | 48 |
| GGAAGACTTTGTGGGCCAT | 1371 | 49 |
| GCCTAGCGATAACATTGAT | 1401 | 50 |
| GGGAGGAATTGTAGTACAA | 1426 | 51 |
| GCTGTGAACATGGACTCTT | 1880 | 52 |
| CACGACGTCACGCAGCAAA | 283 | 242 |
| GATCGCTACTTTGCCATTA | 607 | 243 |
| ATCGCTACTTTGCCATTAC | 608 | 244 |
| TCGCTACTTTGCCATTACT | 609 | 245 |
| GCCATTACTTCACCTTTCA | 619 | 246 |
| TTACTTCACCTTTCAAGTA | 623 | 247 |
| CCATTCAGATGCACTGGTA | 722 | 248 |
| TGATCATGGTCTTCGTCTA | 857 | 249 |
| AGACGTTAGGCATCATCAT | 1037 | 250 |
| TCGTTAACATTGTGCATGT | 1091 | 251 |
| AGGATAACCTCATCCGTAA | 1115 | 252 |
| TCATCCGTAAGGAAGTTTA | 1124 | 253 |
| AAGTTTACATCCTCCTAAA | 1136 | 254 |
| AGTTTACATCCTCCTAAAT | 1137 | 255 |
| TAAATTGGATAGGCTATGT | 1151 | 256 |
| CTATGTCAATTCTGGTTTC | 1164 | 257 |
| GGTACTGTGCCTAGCGATA | 1393 | 258 |
| GTACTGTGCCTAGCGATAA | 1394 | 259 |
| TACTGTGCCTAGCGATAAC | 1395 | 260 |
| GCGATAACATTGATTCACA | 1406 | 261 |
| CGATAACATTGATTCACAA | 1407 | 262 |
| GGAGGAATTGTAGTACAAA | 1427 | 263 |
| GAGGAATTGTAGTACAAAT | 1428 | 264 |
| AGGAATTGTAGTACAAATG | 1429 | 265 |
| CAAATGACTCACTGCTGTA | 1442 | 266 |
| GACCTGAGTCTGCTATATT | 1725 | 267 |
| ACCTGAGTCTGCTATATTT | 1726 | 268 |
| CCATGTATCTACCTCACTA | 1756 | 269 |
| CATGTATCTACCTCACTAT | 1757 | 270 |
| ATGTATCTACCTCACTATT | 1758 | 271 |
| CCTCACTATTCAAGTATTA | 1767 | 272 |
| TAATATATTGCTGCTGGTA | 1790 | 273 |
| AATATATTGCTGCTGGTAA | 1791 | 274 |
| ATATATTGCTGCTGGTAAT | 1792 | 275 |
| TATATTGCTGCTGGTAATT | 1793 | 276 |
| CTGGTAATTTGTATCTGAA | 1803 | 277 |
| GAGTATCTCGGACCTTTCA | 1861 | 278 |
| CGGACCTTTCAGCTGTGAA | 1869 | 279 |
| CGAGCAAAGGTCTAAAGTT | 1971 | 280 |
| GAGCAAAGGTCTAAAGTTT | 1972 | 281 |
| GGTCTAAAGTTTACAGTAA | 1979 | 282 |
Table 3 is listed the example of siRNA target sequence in the ACHE DNA sequence (being respectively SEQ IDNO:5 and SEQ ID NO:123) of splice variant E4-E5 and E4-E6, by the mode of as above illustrating from wherein designing siRNA of the present invention.As mentioned above, ACHE coding acetylcholinesterase.
The siRNA target sequence of table 3.ACHE
| ACHE E4-E5 target sequence | Initial nucleotide position is with reference to SEQ ID NO:5 | SEQ ID NO： |
| CCAGAGTGTCTGCTACCAA | 382 | 53 |
| GCTACCAATATGTGGACAC | 393 | 54 |
| CCAATATGTGGACACCCTA | 397 | 55 |
| GCTGGTGTCCATGAACTAC | 622 | 56 |
| TCATCAACGCGGGAGACTT | 1131 | 57 |
| GGTCTACGCCTACGTCTTT | 1459 | 58 |
| GCTACGAGATCGAGTTCAT | 1530 | 59 |
| GCTATAACGGTCAACCATT | 2251 | 60 |
| GGCTGCAAATAAACTGTTA | 2885 | 61 |
| GCTGCAAATAAACTGTTAC | 2886 | 62 |
| AGTGTCTGCTACCAATATG | 386 | 283 |
| AGACAACGAGTCTCTCATC | 1231 | 284 |
| GGCTGTGGTCCTGCATTAC | 1315 | 285 |
| CTTCCTCCTCAAACCGAGA | 2047 | 286 |
| TCCTCCTCAAACCGAGAGA | 2049 | 287 |
| CCTCAAACCGAGAGACTCA | 2053 | 288 |
| TCAAACCGAGAGACTCACA | 2055 | 289 |
| AAACCGAGAGACTCACACT | 2057 | 290 |
| CCACGCCTTTGTTGTTTGA | 2125 | 291 |
| CACGCCTTTGTTGTTTGAA | 2126 | 292 |
| ACGCCTTTGTTGTTTGAAT | 2127 | 293 |
| GGCTATAACGGTCAACCAT | 2250 | 294 |
| TATAACGGTCAACCATTTC | 2253 | 295 |
| CGGTCAACCATTTCTGTCT | 2258 | 296 |
| GTCAACCATTTCTGTCTCT | 2260 | 297 |
| CCGTCTTCCGGTCATTCTT | 2318 | 298 |
| CCTCTCGTCTTTCGCACAT | 2395 | 299 |
| TCTCGTCTTTCGCACATTC | 2397 | 300 |
| TTTCGCACATTCTCCTGAT | 2404 | 301 |
| TTCGCACATTCTCCTGATC | 2405 | 302 |
| AGAACCAGTTCGACCACTA | 2643 | 303 |
| AACCAGTTCGACCACTACA | 2645 | 304 |
| CTGCAAATAAACTGTTACA | 2887 | 305 |
| ACHE E4-E5 and E4-E6 target sequence | Initial nucleotide position is with reference to SEQ ID NO:5 | SEQ ID NO： |
| TAGACGCTACAACCTTCCA | 366 | 306 |
| CGCTACAACCTTCCAGAGT | 370 | 307 |
| AGAGTGTCTGCTACCAATA | 384 | 308 |
| GAGTGTCTGCTACCAATAT | 385 | 309 |
| CTGTCCTCGTCTGGATCTA | 525 | 310 |
| ATGGCCGCTTCTTGGTACA | 588 | 311 |
| CGACATCAGTGACGCTGTT | 768 | 312 |
| GCACGTGCTGCCTCAAGAA | 1045 | 313 |
| CACGTGCTGCCTCAAGAAA | 1046 | 314 |
| GAAAGCGTCTTCCGGTTCT | 1061 | 315 |
| TGTGGTAGATGGAGACTTC | 1090 | 316 |
| GACAACGAGTCTCTCATCA | 1232 | 317 |
| AGGCTGTGGTCCTGCATTA | 1314 | 318 |
| GCTGTGGTCCTGCATTACA | 1316 | 319 |
| GTCTACGCCTACGTCTTTG | 1460 | 320 |
| TCTACGCCTACGTCTTTGA | 1461 | 321 |
| CTACGCCTACGTCTTTGAA | 1462 | 322 |
| CGGCTACGAGATCGAGTTC | 1528 | 323 |
| CAGCGACTGATGCGATACT | 1607 | 324 |
| GGCTCAGCAGTACGTTAGT | 1705 | 325 |
| AGTACGTTAGTCTGGACCT | 1713 | 326 |
| ACHE E4-E6 target sequence | Initial nucleotide position is with reference to SEQ ID NO:123 | SEQ ID NO： |
| ACATGGTGCACTGGAAGAA | 1875 | 327 |
| AGAACCAGTTCGACCACTA | 1890 | 328 |
| GAACCAGTTCGACCACTAC | 1891 | 329 |
| GGCTATAACACAGACGAGC | 2011 | 330 |
| GCTATAACACAGACGAGCC | 2012 | 331 |
| GCTGCAAATAAACTGTTAC | 2133 | 332 |
| CTGCAAATAAACTGTTACA | 2134 | 333 |
Table 4 is listed Na
+/ K
+-ATP enzyme A and B subunit DNA sequence (ATP1A1 variant 1, SEQID NO:124; ATP1A1 variant 2, SEQ ID NO:125; ATP1A2, SEQ ID NO:6; ATP1A3, SEQ ID NO:126; ATP1A4 variant 1, SEQ ID NO:127; ATP1A4 variant 2, SEQ ID NO:128; ATP1B1 variant 1, SEQ ID NO:129; ATP1B1 variant 2, SEQ ID NO:130; ATP1B2, SEQ ID NO:131 and ATP1B3, SEQ IDNO:132) in the example of siRNA target sequence, by the mode of as above illustrating from wherein designing siRNA of the present invention.
The siRNA target sequence of table 4.ATP1A and ATP1B
| |
Initial nucleotide position is with reference to SEQ ID NO:124 | SEQ ID NO： |
| GCAATGAGACCGTGGAAGA | 2208 | 334 |
| TGCCAAGGCCTGCGTAGTA | 2275 | 335 |
| TAAAGGACATGACCTCCGA | 2307 | 336 |
| AGCAAGCTGCTGACATGAT | 2526 | 337 |
| ACATGATTCTTCTGGATGA | 2538 | 338 |
| GTCGTCTGATCTTTGATAA | 2592 | 339 |
| CTTATACCTTAACCAGTAA | 2628 | 340 |
| GGATCAACGATGTGGAAGA | 2979 | 341 |
| ACGATGTGGAAGACAGCTA | 2985 | 342 |
| CCGACTTGGTCATCTGTAA | 3093 | 343 |
| TAGGAAAGCACCGCAGCAT | 3474 | 344 |
| AGACGTCCTGGAATGAAGC | 3504 | 345 |
| GACGTCCTGGAATGAAGCA | 3505 | 346 |
| ACGTCCTGGAATGAAGCAT | 3506 | 347 |
| GAAGCATGTAGCTCTATGG | 3518 | 348 |
| The total target sequence of |
Initial nucleotide position is with reference to SEQ ID NO:124 | SEQ ID NO： |
| TTCAGAACAAGGTGATAAA | 343 | 349 |
| TGATGAACTTCATCGTAAA | 442 | 350 |
| GGTGCTATCAGCCGTTGTA | 700 | 351 |
| TCAGCCGTTGTAATCATAA | 707 | 352 |
| GATTCGAAATGGTGAGAAA | 811 | 353 |
| CAGAATCATATCTGCAAAT | 907 | 354 |
| CACGTGGTATTGTTGTCTA | 1059 | 355 |
| CTGCTTAGTGAAGAACTTA | 1363 | 356 |
| GTTTCAGGCTAACCAGGAA | 1594 | 357 |
| CACTCTTAAAGTGCATAGA | 1662 | 358 |
| AGTACCAGTTGTCTATTCA | 1758 | 359 |
| TACCAGTTGTCTATTCATA | 1760 | 360 |
| AGCTGAAAGACGCCTTTCA | 1896 | 361 |
| TCGATAATCTGTGCTTTGT | 2037 | 362 |
| ACAGGAGACCATCCAATCA | 2147 | 363 |
| ATP1A1 variant 2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:125 | SEQ ID NO： |
| TAGCCTTGATGAACTTCAT | 436 | 364 |
| TTGATGAACTTCATCGTAA | 441 | 365 |
| GATGAACTTCATCGTAAAT | 443 | 366 |
| CTACTCCTGAATGGATCAA | 552 | 367 |
| GGAGCGATTCTTTGTTTCT | 617 | 368 |
| GTGCTATCAGCCGTTGTAA | 701 | 369 |
| TGCTATCAGCCGTTGTAAT | 702 | 370 |
| GAGCATAAATGCGGAGGAA | 832 | 371 |
| GAAGGCAATGGACCTATGA | 2204 | 372 |
| CCGACTTGGTCATCTGTAA | 2291 | 373 |
| TATATGACGAAGTCAGAAA | 2495 | 374 |
| The ATP1A2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:6 | SEQ ID NO： |
| CCATCCAACGACAATCTAT | 471 | 63 |
| GCATCATATCAGAGGGTAA | 1990 | 64 |
| CCTCCTCATCTTCATCTAT | 3080 | 65 |
| GGAAGTGAGGTAGTGCCAA | 3797 | 66 |
| GGATGTCACTCATGTACTT | 4037 | 67 |
| GCTCCATGCTGTTCTGAAA | 4093 | 68 |
| GCTGGCCATTGGCTAGAAT | 4225 | 69 |
| GGTCAGAACCTTTGGACAA | 4323 | 70 |
| GCTAGAGGTGGCATGTTTA | 5213 | 71 |
| GCGAGTGCATGGGCTAATT | 5285 | 72 |
| TGGCAATGGATGACCACAA | 214 | 375 |
| TGAACCATCCAACGACAAT | 467 | 376 |
| ACCATCCAACGACAATCTA | 470 | 377 |
| CATCCAACGACAATCTATA | 472 | 378 |
| ATCCAACGACAATCTATAT | 473 | 379 |
| GCAGATCAACGCAGAGGAA | 632 | 380 |
| TGTTTCTTCTCCACCAACT | 825 | 381 |
| CCATAGCAATGGAGATTGA | 946 | 382 |
| AGATGCAAGATGCCTTTCA | 1693 | 383 |
| CTGAATCTGCCATCTGGAA | 1767 | 384 |
| TGAATCTGCCATCTGGAAA | 1768 | 385 |
| ATCGTCTTTGCTCGAACGT | 2157 | 386 |
| CTGCATTGAAGAAGGCTGA | 2263 | 387 |
| ATGAAGCGGCAGCCACGAA | 2589 | 388 |
| TGAAGCGGCAGCCACGAAA | 2590 | 389 |
| GGATGACCGGACCATGAAT | 2765 | 390 |
| GCTGCCTTTCTCTCTTACT | 2988 | 391 |
| TCTATGATGAGGTCCGAAA | 3094 | 392 |
| GTGGAGAAGGAGACATACT | 3144 | 393 |
| TGGAGAAGGAGACATACTA | 3145 | 394 |
| TAGACCTAACTGTGAACAA | 3344 | 395 |
| AGACCTAACTGTGAACAAT | 3345 | 396 |
| TCCACTATGTTGTCTATTT | 3418 | 397 |
| TGAGTGCAAGAGCCTGAGA | 3666 | 398 |
| TGACATGAGTCTCCAGATA | 3828 | 399 |
| GTCGTGGACTCCAGCTCTA | 3850 | 400 |
| TGTCACTCATGTACTTAAT | 4040 | 401 |
| GTCACTCATGTACTTAATA | 4041 | 402 |
| CACTTCACCTTCTGTAATA | 4061 | 403 |
| GTAGAGAGAGACCTAGATA | 4882 | 404 |
| CTAGATAGGTCATGCAAGT | 4894 | 405 |
| AGGTCATGCAAGTGAGAAA | 4900 | 406 |
| TATCAGAAGCAAGGAAGTA | 5040 | 407 |
| TCCGATTAATTGGAGATTA | 5114 | 408 |
| CCGATTAATTGGAGATTAC | 5115 | 409 |
| GATTACTAACTGTGGACAA | 5128 | 410 |
| ATTACTAACTGTGGACAAA | 5129 | 411 |
| TCAGGCACTTTAGAAATAT | 5253 | 412 |
| GGCTAATTATCATCAATCT | 5296 | 413 |
| AGTTTGAGGTACTACCTAT | 5375 | 414 |
| TACTACCTATGTACTTGAA | 5384 | 415 |
| ACTACCTATGTACTTGAAA | 5385 | 416 |
| The ATP1A3 target sequence | Initial nucleotide position is with reference to SEQ ID NO:126 | SEQ ID NO： |
| TGGCTATGACAGAGCACAA | 240 | 417 |
| GAGGTCTGCCGGAAATACA | 272 | 418 |
| CTCACGCCACCGCCTACCA | 362 | 419 |
| TCGACTGTGATGACGTGAA | 1836 | 420 |
| TGAACTTCACCACGGACAA | 1851 | 421 |
| CCAAGGCCTGCGTGATCCA | 2103 | 422 |
| GGACTTCACCTCCGAGCAA | 2137 | 423 |
| GACTTCACCTCCGAGCAAA | 2138 | 424 |
| ACTTCACCTCCGAGCAAAT | 2139 | 425 |
| TCGACGAGATCCTGCAGAA | 2157 | 426 |
| CGACGAGATCCTGCAGAAT | 2158 | 427 |
| ACGAGATCCTGCAGAATCA | 2160 | 428 |
| GATCTTCGACAACCTAAAG | 2425 | 429 |
| CCATCTCACTGGCGTACGA | 2580 | 430 |
| CTGCCGAAAGCGACATCAT | 2601 | 431 |
| CGGACAAATTGGTCAATGA | 2646 | 432 |
| CAAATTGGTCAATGAGAGA | 2650 | 433 |
| GGATGACCGCACCGTCAAT | 2794 | 434 |
| CACCGTCAATGACCTGGAA | 2803 | 435 |
| ATCTTCGTCTACGACGAAA | 3116 | 436 |
| CTACGACGAAATCCGCAAA | 3124 | 437 |
| ACGACGAAATCCGCAAACT | 3126 | 438 |
| ACGAAATCCGCAAACTCAT | 3129 | 439 |
| CCAAACCTCTCTCCTCTCT | 3377 | 440 |
| |
Initial nucleotide position is with reference to SEQ ID NO:127 | SEQ ID NO： |
| GGCACCTGGTTACGCTTCA | 113 | 441 |
| CATGGATGATCACAAATTA | 612 | 442 |
| AATCCTGACTCGAGATGGA | 702 | 443 |
| CCTACAGCATCCAGATATA | 833 | 444 |
| CCGGCTTATCTCTGCACAA | 1101 | 445 |
| AGCTCTGATACCTGGTTTA | 1732 | 446 |
| GCTCTGATACCTGGTTTAT | 1733 | 447 |
| AGGTGATGCTTCCGAGTCA | 1836 | 448 |
| GTACTCAATGAACGATGAA | 2070 | 449 |
| TACTCAATGAACGATGAAA | 2071 | 450 |
| GTGCTAGGCTTCTGCTTCT | 2143 | 451 |
| CATGGTAACAGGAGATCAT | 2328 | 452 |
| TGTGGTGCATGGTGCAGAA | 2475 | 453 |
| TGTTCATCATCCTCGGTAT | 2861 | 454 |
| GTTCATCATCCTCGGTATA | 2862 | 455 |
| GGCTTATGAGTCAGCTGAA | 2952 | 456 |
| GGACCTATGAGCAACGAAA | 3203 | 457 |
| CGGATCTCATCATCTCCAA | 3281 | 458 |
| TGGCTGCATTTCTGTCCTA | 3377 | 459 |
| GCTGCATTTCTGTCCTACA | 3379 | 460 |
| GTATTCTCATCTTCGTCTA | 3470 | 461 |
| TATTCTCATCTTCGTCTAT | 3471 | 462 |
| ACTAAACTCAGCAGATGAA | 3554 | 463 |
| GGCCAGAGATTATAAGTTT | 3614 | 464 |
| GCCAGAGATTATAAGTTTG | 3615 | 465 |
| CCAGAGATTATAAGTTTGA | 3616 | 466 |
| CAGAGATTATAAGTTTGAC | 3617 | 467 |
| ATAAGTTTGACACAACATC | 3625 | 468 |
| TAAGTTTGACACAACATCT | 3626 | 469 |
| TCTGAGACACTAGGATGAA | 3642 | 470 |
| AGACACTAGGATGAATTAT | 3646 | 471 |
| GACACTAGGATGAATTATC | 3647 | 472 |
| AGGATGAATTATCTTGGAT | 3653 | 473 |
| GATGAATTATCTTGGATGA | 3655 | 474 |
| CGTAGCCAGTCTAGACAGT | 3797 | 475 |
| GCCAGTCTAGACAGTAAAT | 3801 | 476 |
| CAGTCTAGACAGTAAATGT | 3803 | 477 |
| AGACAGTAAATGTCTGGAA | 3809 | 478 |
| GACAGTAAATGTCTGGAAA | 3810 | 479 |
| ATP1A4 variant 2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:128 | SEQ ID NO： |
| GCTGGATTCTTTACCTACT | 126 | 480 |
| GTGGACCTATGAGCAACGA | 251 | 481 |
| TGGACCTATGAGCAACGAA | 252 | 482 |
| GGACCTATGAGCAACGAAA | 253 | 483 |
| CGGATCTCATCATCTCCAA | 331 | 484 |
| TGGCTGCATTTCTGTCCTA | 427 | 485 |
| GCTGCATTTCTGTCCTACA | 429 | 486 |
| GTATTCTCATCTTCGTCTA | 520 | 487 |
| TATTCTCATCTTCGTCTAT | 521 | 488 |
| CTTCGTCTATGATGAAATC | 530 | 489 |
| ACTACTAAACTCAGCAGAT | 601 | 490 |
| CTACTAAACTCAGCAGATG | 602 | 491 |
| TACTAAACTCAGCAGATGA | 603 | 492 |
| ACTAAACTCAGCAGATGAA | 604 | 493 |
| GGCCAGAGATTATAAGTTT | 664 | 494 |
| GCCAGAGATTATAAGTTTG | 665 | 495 |
| CCAGAGATTATAAGTTTGA | 666 | 496 |
| CAGAGATTATAAGTTTGAC | 667 | 497 |
| ATAAGTTTGACACAACATC | 675 | 498 |
| TAAGTTTGACACAACATCT | 676 | 499 |
| TCTGAGACACTAGGATGAA | 692 | 500 |
| AGACACTAGGATGAATTAT | 696 | 501 |
| GACACTAGGATGAATTATC | 697 | 502 |
| TAGGATGAATTATCTTGGA | 702 | 503 |
| AGGATGAATTATCTTGGAT | 703 | 504 |
| GATGAATTATCTTGGATGA | 705 | 505 |
| TGAATTATCTTGGATGAGA | 707 | 506 |
| CGTAGCCAGTCTAGACAGT | 847 | 507 |
| GCCAGTCTAGACAGTAAAT | 851 | 508 |
| CAGTCTAGACAGTAAATGT | 853 | 509 |
| AGACAGTAAATGTCTGGAA | 859 | 510 |
| GACAGTAAATGTCTGGAAA | 860 | 511 |
| |
Initial nucleotide position is with reference to SEQ ID NO:129 | SEQ ID NO： |
| ACCTACTAGTCTTGAACAA | 1096 | 512 |
| TACTAGTCTTGAACAAACT | 1099 | 513 |
| GGACCTACACTTAATCTAT | 1130 | 514 |
| GACCTACACTTAATCTATA | 1131 | 515 |
| CTGCATTTAATAGGTTAGA | 1167 | 516 |
| CGTAACTGACTTGTAGTAA | 1299 | 517 |
| AGCAAGGTTTGCTGTCCAA | 1441 | 518 |
| TGCTGTCCAAGGTGTAAAT | 1450 | 519 |
| GCTGTCCAAGGTGTAAATA | 1451 | 520 |
| CTGTCCAAGGTGTAAATAT | 1452 | 521 |
| TTAACATACTCCATAGTCT | 1564 | 522 |
| GCCTTGTCCTCCGGTATGT | 1746 | 523 |
| TGTCCTCCGGTATGTTCTA | 1750 | 524 |
| GTCCTCCGGTATGTTCTAA | 1751 | 525 |
| TCCTCCGGTATGTTCTAAA | 1752 | 526 |
| CCATCACTTTGGCTAGTGA | 1795 | 527 |
| |
Initial nucleotide position is with reference to SEQ ID NO:129 | SEQ ID NO： |
| ACCGGTGGCAGTTGGTTTA | 203 | 528 |
| CCGGTGGCAGTTGGTTTAA | 204 | 529 |
| TTGGTTTAAGATCCTTCTA | 214 | 530 |
| AGATCCTTCTATTCTACGT | 222 | 531 |
| ATCCTTCTATTCTACGTAA | 224 | 532 |
| TCCTTCTATTCTACGTAAT | 225 | 533 |
| CCTTCTATTCTACGTAATA | 226 | 534 |
| GAAATTTCCTTTCGTCCTA | 380 | 535 |
| AACGAGGAGACTTTAATCA | 525 | 536 |
| GAAATTGCTCTGGATTAAA | 591 | 537 |
| ATGAAACTTATGGCTACAA | 612 | 538 |
| TGAAACTTATGGCTACAAA | 613 | 539 |
| AAACTTATGGCTACAAAGA | 615 | 540 |
| GGCAAACCGTGCATTATTA | 635 | 541 |
| GCAAACCGTGCATTATTAT | 636 | 542 |
| ACCGAGTTCTAGGCTTCAA | 663 | 543 |
| CCGAGTTCTAGGCTTCAAA | 664 | 544 |
| TTCTAGGCTTCAAACCTAA | 669 | 545 |
| ATGAGTCCTTGGAGACTTA | 699 | 546 |
| GCAAGCGAGATGAAGATAA | 765 | 547 |
| AGTTGGAAATGTGGAGTAT | 790 | 548 |
| CTGCAGTATTATCCGTACT | 839 | 549 |
| TGCAGTATTATCCGTACTA | 840 | 550 |
| GCAGTATTATCCGTACTAT | 841 | 551 |
| CCGTACAGTTCACCAATCT | 900 | 552 |
| TCACCAATCTTACCATGGA | 909 | 553 |
| AAATTCGCATAGAGTGTAA | 933 | 554 |
| TGTAAGGCGTACGGTGAGA | 947 | 555 |
| ATP1B1 variant 2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:130 | SEQ ID NO： |
| TGTGTTATGCTTGTATTGA | 1063 | 556 |
| GCCTTGTCCTCCGGTATGT | 1102 | 557 |
| TGTCCTCCGGTATGTTCTA | 1106 | 558 |
| GTCCTCCGGTATGTTCTAA | 1107 | 559 |
| TCCTCCGGTATGTTCTAAA | 1108 | 560 |
| CCTCCGGTATGTTCTAAAG | 1109 | 561 |
| TCCGGTATGTTCTAAAGCT | 1111 | 562 |
| CCATCACTTTGGCTAGTGA | 1151 | 563 |
| The ATP1B2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:131 | SEQ ID NO： |
| CCGAGGACGCACCAGTTTA | 653 | 564 |
| CGAGGACGCACCAGTTTAT | 654 | 565 |
| TGCAGACTGTCTCCGACCA | 771 | 566 |
| CAGACTGTCTCCGACCATA | 773 | 567 |
| CAAGACTGAGAACCTTGAT | 841 | 568 |
| AGAACCTTGATGTCATTGT | 849 | 569 |
| CCTTGATGTCATTGTCAAT | 853 | 570 |
| AAGTTCTTGGAGCCTTACA | 917 | 571 |
| AGTTCTTGGAGCCTTACAA | 918 | 572 |
| GAGCCTTACAACGACTCTA | 926 | 573 |
| AGCCTTACAACGACTCTAT | 927 | 574 |
| TTACAACGACTCTATCCAA | 931 | 575 |
| GCTATTACGAACAGCCAGA | 981 | 576 |
| TATTACGAACAGCCAGATA | 983 | 577 |
| ATTACGAACAGCCAGATAA | 984 | 578 |
| CAGATAATGGAGTCCTCAA | 996 | 579 |
| GATAATGGAGTCCTCAACT | 998 | 580 |
| AAACGTGCCTGCCAATTCA | 1022 | 581 |
| AACGTGCCTGCCAATTCAA | 1023 | 582 |
| AACCAGAGCATGAATGTTA | 1160 | 583 |
| CTCGGCAACTTCGTCATGT | 1214 | 584 |
| AATGTAGAATGTCGCATCA | 1355 | 585 |
| ATGTAGAATGTCGCATCAA | 1356 | 586 |
| CAACATCGCCACAGACGAT | 1381 | 587 |
| GACGATGAGCGAGACAAGT | 1394 | 588 |
| TGGCCTTCAAACTCCGCAT | 1425 | 589 |
| CCATCTCTCTCCTGTGGAT | 1474 | 590 |
| TTTGATAACAGAGCTATGA | 1550 | 591 |
| CCATTGCGGTTCCGTCACT | 1620 | 592 |
| AGGAGTTAGGAGCCTTTCT | 1707 | 593 |
| TGTGAGAGCTATCCACTCT | 1740 | 594 |
| CACTCTCCTGCCTGCATAT | 1753 | 595 |
| CGCCACACACACACACAAA | 1825 | 596 |
| TCTACACAGTCGCCATCTT | 1956 | 597 |
| TCGCCATCTTGGTGACTTT | 1965 | 598 |
| GGTTGACCTAGGCTGAATA | 2598 | 599 |
| GTTGACCTAGGCTGAATAT | 2599 | 600 |
| GGCTGAATATCCACTTTGT | 2608 | 601 |
| AGCAAGTTATCAACTAATC | 2828 | 602 |
| GCAAGTTATCAACTAATCA | 2829 | 603 |
| CCAAATCTAGCCTCTGAAT | 2888 | 604 |
| CTCCTGCTCTGAATATTCT | 3012 | 605 |
| TGTGTCAGATCTACTGTAA | 3251 | 606 |
| The ATP1B3 target sequence | Initial nucleotide position is with reference to SEQ ID NO:132 | SEQ ID NO： |
| TTGCTCTTCTACCTAGTTT | 292 | 607 |
| CAGTGACCGCATTGGAATA | 434 | 608 |
| GACCGCATTGGAATATACA | 438 | 609 |
| TTCAGTAGGTCTGATCCAA | 457 | 610 |
| CAGTAGGTCTGATCCAACT | 459 | 611 |
| GGTACATTGAAGACCTTAA | 488 | 612 |
| TACATTGAAGACCTTAAGA | 490 | 613 |
| AGACCTTAAGAAGTTTCTA | 498 | 614 |
| GACCTTAAGAAGTTTCTAA | 499 | 615 |
| GTTTATGTTGCATGTCAGT | 592 | 616 |
| TGGTATGAATGATCCTGAT | 639 | 617 |
| TGAAGGAGTGCCAAGGATA | 723 | 618 |
| TGTAGCAGTTTATCCTCAT | 774 | 619 |
| GTAGCAGTTTATCCTCATA | 775 | 620 |
| CTCATAATGGAATGATAGA | 788 | 621 |
| AGCCATTGGTTGCTGTTCA | 857 | 622 |
| GCCATTGGTTGCTGTTCAG | 858 | 623 |
| GTAACAGTTGAGTGCAAGA | 910 | 624 |
| TAACAGTTGAGTGCAAGAT | 911 | 625 |
| TGATGGATCAGCCAACCTA | 930 | 626 |
| GATGGATCAGCCAACCTAA | 931 | 627 |
| ATGGATCAGCCAACCTAAA | 932 | 628 |
| GCATAGTATGAGTAGGATA | 1009 | 629 |
| CATAGTATGAGTAGGATAT | 1010 | 630 |
| GGATATCTCCACAGAGTAA | 1023 | 631 |
| GATATCTCCACAGAGTAAA | 1024 | 632 |
| AGAAAGGTGTGTGGTACAT | 1111 | 633 |
| ATAACGTGCTTCCAGATCA | 1146 | 634 |
| TAACGTGCTTCCAGATCAT | 1147 | 635 |
| AGTGTACAGTCGCCAGATA | 1220 | 636 |
| GTGAACACCTGATTCCAAA | 1246 | 637 |
| AGCTTAATATGCCGTGCTA | 1321 | 638 |
| TAATATGCCGTGCTATGTA | 1325 | 639 |
| AATATGCCGTGCTATGTAA | 1326 | 640 |
| ATATGCCGTGCTATGTAAA | 1327 | 641 |
| GCCGTGCTATGTAAATATT | 1331 | 642 |
| TGCAAGAAATGTGGTATGT | 1437 | 643 |
| ATGCTGAATTAGCCTCGAT | 1548 | 644 |
| TTGATTAAGAGCACAAACT | 1571 | 645 |
| AGCAGACTGTGGACTGTAA | 1785 | 646 |
| GCAGACTGTGGACTGTAAT | 1786 | 647 |
| CAGACTGTGGACTGTAATA | 1787 | 648 |
Table 5 is listed the example of siRNA target sequence in the DNA sequence (being respectively SEQ ID NO:7 and SEQ ID NO:133) of SLC12A1 and SLC12A2, by the mode of as above illustrating from wherein designing siRNA of the present invention.As described above, SLC12A1 and SLC12A2 encode respectively Na-K-2Cl cotransporter NKCC2 and NKCC1.
The siRNA target sequence of table 5.SLC12A1
| The SLC12A1 target sequence | Initial nucleotide position is with reference to SEQ ID NO:7 | SEQ ID NO： |
| CCACCATAGTAACGACAAT | 675 | 73 |
| GGAATGGAATGGGAGGCAA | 974 | 74 |
| GGGATGAACTGCAATGGTT | 1373 | 75 |
| CCATGCCTCTTATGCCAAA | 1780 | 76 |
| CCTGCTCTCCTGGACATAA | 2102 | 77 |
| GCATCTGCTGTGAAGTCTT | 2151 | 78 |
| GCCTCAGGCTTAGGAAGAA | 2315 | 79 |
| GGAAGCGACTATCAAAGAT | 2542 | 80 |
| GCTGGCAAGTTGAACATTA | 2609 | 81 |
| GCAAGAAAGGGATCCATAT | 3197 | 82 |
| TAATACCAATCGCTTTCAA | 67 | 649 |
| ACCAATCGCTTTCAAGTTA | 71 | 650 |
| CAATCGCTTTCAAGTTAGT | 73 | 651 |
| ATAGAGTACTATCGTAACA | 353 | 652 |
| CCAGCCTGCTTGAGATTCA | 405 | 653 |
| CTGTAGTAGATCTACTTAA | 864 | 654 |
| ACCAATGACATCCGGATTA | 911 | 655 |
| CCAATGACATCCGGATTAT | 912 | 656 |
| CAATGACATCCGGATTATA | 913 | 657 |
| GGCTATGACTTCTCAAGAT | 1409 | 658 |
| GCCTCATATGCACTTATTA | 1748 | 659 |
| AGACCTGCGTATGGAATTT | 1811 | 660 |
| ACGTCTATGTGACTTGTAA | 1935 | 661 |
| GTCTATGTGACTTGTAAGA | 1937 | 662 |
| TTCCTACGTGAGTGCTTTA | 1993 | 663 |
| GACAATGCTCTGGAATTAA | 2012 | 664 |
| CTCTGGTGATTGGATATAA | 2346 | 665 |
| TGACAGAGATTGAGAACTA | 2388 | 666 |
| TGAGATTGGCGTGGTTATA | 2437 | 667 |
| GCATCCGAGGCTTGTTTAA | 2586 | 668 |
| ACCATATCGTCTCCATGAA | 3007 | 669 |
| CCATATCGTCTCCATGAAA | 3008 | 670 |
| TGAAAGCTGCAAAGATTTA | 3022 | 671 |
| TCGACTGAATGAACTCTTA | 3130 | 672 |
| CCATATCGGATTTGTTGTA | 3210 | 673 |
| GGTTGGAAATCCTCACAAA | 3237 | 674 |
| CTTACTAGTTAGAGGAAAT | 3271 | 675 |
| The SLC12A2 target sequence | Initial nucleotide position is with reference to SEQ ID NO:133 | SEQ ID NO： |
| ACCACCAGCACTACTATTA | 748 | 676 |
| CCACCAGCACTACTATTAT | 749 | 677 |
| CAGCACTACTATTATGATA | 753 | 678 |
| CTATCAGTCCTTGTAATAA | 1119 | 679 |
| ATTGTCTACTTCAGCAATA | 1169 | 680 |
| TATTGGTGATTTCGTCATA | 1499 | 681 |
| TTCGTCATAGGAACATTTA | 1509 | 682 |
| TAATGACACTATCGTAACA | 1820 | 683 |
| GATGTTTGCTAAAGGTTAT | 2081 | 684 |
| CTTCGTGGCTACATCTTAA | 2118 | 685 |
| TGCACTTGGATTCATCTTA | 2147 | 686 |
| GATGATCTGTGGCCATGTA | 2615 | 687 |
| CTCGAAGACAAGCCATGAA | 2644 | 688 |
| TGAAAGAGATGTCCATCGA | 2659 | 689 |
| AGAGATGTCCATCGATCAA | 2663 | 690 |
| CCATCGATCAAGCCAAATA | 2671 | 691 |
| CATCGATCAAGCCAAATAT | 2672 | 692 |
| GGTCGTATGAAGCCAAACA | 2793 | 693 |
| CACTTGTCCTTGGATTTAA | 2812 | 694 |
| TAGTGGTTATTCGCCTAAA | 2914 | 695 |
| ATCTCATCTTCAAGGACAA | 2948 | 696 |
| CGATTTAGATACTTCCAAA | 3044 | 697 |
| TCATTGGTGGAAAGATAAA | 3334 | 698 |
| TTAGCAAGTTCCGGATAGA | 3391 | 699 |
| GAAATCATTGAGCCATACA | 3480 | 700 |
| AGCAAGATATTGCAGATAA | 3520 | 701 |
| GATGAACCATGGCGAATAA | 3549 | 702 |
| CATTCAAGCACAGCTAATA | 3639 | 703 |
| TTCAGTGCCTAGTGTAGTA | 3840 | 704 |
| AGGAAAGTTGCTCCATTGA | 3941 | 705 |
| AAAGTTGCTCCATTGATAA | 3944 | 706 |
| CAATCTTAATGGTGATTCT | 4001 | 707 |
| TTGACATCATAGTCTAGTA | 4995 | 708 |
| GACATCATAGTCTAGTAAA | 4997 | 709 |
| GTGTGTGTGTGTGTATATA | 5141 | 710 |
| GTGTGTGTGTGTATATATA | 5143 | 711 |
| TAGGCAAACTTTGGTTTAA | 5249 | 712 |
| GGAGAATACTTCGCCTAAA | 5375 | 713 |
| TGAGTATGACCTAGGTATA | 5834 | 714 |
| AGAGATCTGATAACTTGAA | 5852 | 715 |
| GGTAAAGACAGTAGAAATA | 5981 | 716 |
| TTTAAGCTCTGGTGGATGA | 6678 | 717 |
As described in top example, the target sequence information that one of ordinary skill in the art in provides in can use table 1-5 designs RNA interfering, shorter or the longer length of sequence that provides than among the table 1-5 is provided this RNA interfering, by with reference at SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ IDNO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ IDNO:130, SEQ ID NO:131, SEQ ID NO:132, sequence location among SEQ ID NO:133 or the SEQ IDNO:134, and to respectively with SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, the increase of the complementary or approximate complementary nucleotide of SEQ ID NO:133 or SEQ ID NO:134 or disappearance and realize.
Target RNA cleavage reaction by siRNA and the guiding of other forms of RNA interfering is highly sequence-specific.Usually, described here is siRNA as the siRNA embodiment that suppresses mRNA, its be included on the sequence part with target mRNA consistent have adopted nucleotide chain and with the complete complementary antisense nucleotide chain of the part of target mRNA.Yet, do not need between antisense siRNA chain and the target mRNA or antisense siRNA chain and 100% sequence complementation is arranged between the adopted siRNA chain for implementing the present invention.Therefore, for example, the sequence variation that the present invention's permission can be estimated owing to gene mutation, bacterial strain polymorphism or evolutionary divergence.
In one embodiment of the invention, it is complementary that the antisense strand of siRNA and target mRNA have being close at least fully continuously of at least 19 nucleotide." be close to fully " used herein mean the antisense strand of siRNA and target mRNA to small part be " complementary substantially ", and the sense strand of siRNA and target mRNA to small part be " basically identical ".Known as all those of ordinary skill of this area, " homogeneity " is meant by the order of nucleotide between the matching sequence and feature and comes serial correlation degree between the definite kernel nucleotide sequence.In one embodiment, the antisense strand of siRNA and target mRNA sequence have 80% and 80%-100% between complementation, for example, 85%, 90% or 95% complementation, this is considered to be close to complementary completely and can uses in the present invention." completely " continuous complementation is meant the standard Watson-Crick base pairing of adjacent base pair." be close to completely " complementation continuously at least and be included in " completely " complementation of using here.Be designed for and measure homogeneity or maximum match degree that complementary computer approach comes the definite kernel nucleotide sequence, for example BLASTN (Altschul, people such as S.F. (1990) J.MoI.Biol.215:403-410).
The percentage ratio of continuous nucleotide in first nucleic acid molecules described in term " homogeneity percent ", and continuous nucleotide shared percentage ratio in second nucleic acid molecules of series of identical length also described in this term.The percentage ratio of continuous nucleotide in first nucleic acid molecules described in term " complementary percent ", and this nucleic acid molecules can carry out base pairing by a series of continuous nucleotides in Watson-Crick principle and second nucleic acid molecules.
Relation between the chain (sense strand) among target mRNA (sense strand) and the siRNA is exactly a homogeneity.The sense strand of siRNA if exist, is also referred to as transfer chain.Relation between another chain (antisense strand) of target mRNA (sense strand) and siRNA is exactly complementary.The antisense strand of siRNA is also referred to as the guiding chain.
With 5 ' the inferior terminal bases of the nucleotide sequence write to the direction of 3 ＇ is the penult base, i.e. the adjacent base of 3 ' end base.With 5 ' to 3 ' time terminal 13 bases of the nucleotide sequence write of direction be adjacent last 13 base sequences of 3 ' end base, and do not comprise 3 ' end base.Similarly, with 5 ' to 3 ' time terminal 14,15,16,17 or 18 bases of the nucleotide sequence write of direction be respectively adjacent last 14,15,16,17 or 18 base sequences of 3 ' end base, and do not comprise 3 ' end base.
Phrase " has the zone of containing 13 continuous nucleotides at least of at least 90% sequence complementarity or at least 90% sequence homogeneity with 3 ' terminal penultimate 13 nucleotide of any one (sequence identifier) " and allows a nucleotide to replace.The replacement of two nucleotide (being 11/13=85% homogeneity/complementarity) is not included in such phrase.
In one embodiment of the invention, the continuous nucleotide zone is the zone of at least 14 continuous nucleotides, and penultimate 14 nucleotide of sequence 3 ' end of this zone and the identification of each sequence identifier have the complementary or at least 85% sequence homogeneity of at least 85% sequence.The replacement of two nucleotide (being 12/14=86% homogeneity/complementarity) is not included in such phrase.
In another embodiment of the invention, the zone of continuous nucleotide is the zone of at least 15,16,17 or 18 continuous nucleotides, and terminal penultimate 14 nucleotide of sequence 3 ＇ of this zone and sequence identifier have the complementary or at least 80% sequence homogeneity of at least 80% sequence.The replacement of three nucleotide is not included in such phrase.
Among the mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, the target sequence of SEQ ID NO:133 or SEQ ID NO:134 may be at 5 of mRNA ' or 3 ' end untranslated region and in the coding region of mRNA.
One of double-chain interference RNA or two chains may contain 3 ' jag of 1 to 6 nucleotide, and this 3 ' jag may be ribonucleotide or deoxyribonucleotide or its mixture.The nucleotide of jag is not base pairing.In one embodiment of the invention, RNA interfering comprises 3 ' jag of TT or UU.In another embodiment of the invention, RNA interfering comprises that at least one is flat terminal.This end contains 5 ' phosphate or 3 ' hydroxyl usually, and in other embodiments, antisense strand has 5 ' phosphate, and sense strand contains 5 ' hydroxyl.In the other embodiment, this end adds other molecules or functional group by covalency and is further modified.
The sense strand of double-stranded siRNA and the Double helix form that antisense strand may be aforesaid two strands also may be independent molecules, and wherein complementary region is base pairing and covalently bound to form independent chain by hairpin loop.Generally believe that the protein that is called dicer cuts the RNA interfering of hair clip with the RNA molecule that forms two independent base pairings in cell.
By adding, delete, replace or modifying one or more nucleotide, RNA interfering can be different from the RNA that nature forms.The non-nucleotide material can be incorporated into 5 of RNA interfering ' end or 3 ' end or it is inner.The such modification of common design increases the nuclease resistance of RNA interfering, the absorption that improves cell, the targeting that strengthens cell, assistance tracking RNA interfering, the further activation potential that improves stability or minimizing interferon path.For example, RNA interfering may comprise purine nucleotides at the jag end.For example, cholesterol also provides stability to siRNA by 3 ' end that the pyrrolidine joint is connected to siRNA molecule sense strand.
Further modify and comprise for example 3 ' terminal biotin molecule, known peptide, nano-particle, plan peptide class, fluorescent dye or dendrimer with cell-penetrating performance.
Can also play a role in embodiments of the invention at base portion, glycosyl part or the phosphoric acid part modified nucleotide of molecule.Modification comprises with replacing as alkyl, alkoxyl, amino, denitrogenation, halogen, hydroxyl, sulfydryl or its compositions.Can use more stable analog substituted nucleotide, as replacing ribonucleotide, or have glycosyl modified 2 ' amino, 2 ' O-methyl, 2 ' methoxy ethyl or the 2 ＇-O of for example using with deoxyribonucleotide, 4 '-the C methylene bridge replaces 2 '-hydroxyl.The nucleotide of modifying with N-that the purine of nucleotide or the example of pyrimidine analogue comprise that xanthine, hypoxanthine, azapurine, methyl mercapto adenine, 7-denitrogenation adenosine and O-modify.The phosphate of nucleotide can be modified by the one or more oxygen that replace in the phosphate with nitrogen or sulfur (D2EHDTPA).Be modified with and be beneficial to, for example, enhancement function improves stability or permeability or instructs location or targeting.
Antisense RNA interfering chain can have one or more zones and SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ IDNO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO.128, SEQ IDNO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, the part of SEQ IDNO:133 or SEQ ID NO:134 is not complementary.The incomplementarity zone may be complementary region or two complementary regions between 3 ' end, 5 ＇ end or these two simultaneously terminal.
Can perhaps use dicer or other to have the similar active suitable long double-stranded RNA of nuclease cutting and exogenous generation RNA interfering by chemosynthesis, in vitro transcription.Can be from commercial supplier such as Ambion Inc. (Austin; TX), Invitrogen (Carlsbad; CA) or Dharmacon (Lafayette; CO) locate to obtain the RNA interfering of chemosynthesis, this RNA interfering is to use conventional DNA/RNA synthesizer, makes from the ribonucleotide phosphoramidite of protection.Can be by for example utilizing the extraction of solvent or resin, the combination of precipitation, electrophoresis, chromatography or these methods comes the purification RNA interfering.Alternatively, can use the loss of RNA interfering that does not have purification or the few purification of process to avoid causing because of the sample processed.
RNA interfering also can be carried out endogenous expression from plasmid or virus expression carrier or from minimum expression cassette (for example the fragment that is produced by PCR, it comprises the one or more suitable template of one or more promoteres and RNA interfering).The example of commercial shRNA based on the expression vector of plasmid comprise the pSilencer series of products (Ambion, Austin, TX) and pCpG-siRNA (InvivoGen, SanDiego, CA).The viral vector of expressing RNA interfering may derive from multiple virus, comprises adenovirus, adeno-associated virus, slow virus (as HIV, FIV and EIAV) and herpesvirus.The example of the viral vector of commercial expression ShRNA comprise pSilencer adeno (Ambion, Austin, TX) and pLenti6/BLOCK-iT
TM-DEST (Invitrogen, Carlsbad, CA).The selection of viral vector, from the method for vector expression RNA interfering and the method for transmitting viral vector all within those of ordinary skills' limit of power.As the example of the test kit of producing the shRNA expression cassette that PCR produces comprise Silencer Express (Ambion, Austin, TX) and siXpress (Mirus, Madison, WT).
Can comprise from the known multiple eukaryotic promoter of those of ordinary skills and express RNA interfering pol III promoter (as U6 or H1 promoter) or the polII promoter (as cytomegalovirus promoter).Those skilled in the art admit that these promoteres also are applicable to the abduction delivering that allows RNA interfering.
Under physiological condition, hybridize: in certain embodiments of the invention, the antisense strand of RNA interfering with in vivo as the mRNA hybridization of RISC complex part.
" hybridization " refers to wherein single-chain nucleic acid and complementation or is close to complementary base sequence interaction is called the bond complexes of crossbred with formation process.Hybridization has susceptiveness and selectivity.External, the specificity of hybridization (being stringency) is by the concentration and the hybridization temperature control of for example salt in prehybridization and hybridization solution or Methanamide, and these operations are known in the art.Especially, stringency strengthens by reducing salinity, increase Methanamide concentration or rising hybridization temperature.
For example, at about 50% Methanamide, produce high stringency condition 37 ℃-42 ℃ the time.Produce the stringency condition that reduces at about 35%-25% Methanamide, 30 ℃-35 ℃ the time.The example of the stringency condition that is used to hybridize is provided at Sambrook, J., and 1989, Molecular Cloning:ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor is among the N.Y..Other example of stringency hybridization conditions comprises 400mM NaCl, 40mMPIPES, and pH6.4,1mM EDTA, 50 ℃ or 70 ℃ are washing again after following 12-16 hour; Perhaps in 70 ℃, 1 * SSC or 50 ℃, 1 * SSC and 50% Methanamide after the hybridization, in 70 ℃, 0.3 * SSC, wash; Perhaps in 70 ℃, 4 * SSC or 50 ℃, 4 * SSC and 50% Methanamide after the hybridization, in 67 ℃, 1 * SSC, wash.Hybridization temperature is than the fusing point (T of crossbred
m) approximately low 5-10 ℃, the computational methods below using are determined the fusing point T of the crossbred of 19-49 base pair length
m: T
m℃=81.5+16.6 (log
10[Na
+])+0.41 (%G+C)-(600/N), wherein N is a base number in the crossbred, and [Na
+] be the concentration of sodium ion in the hybridization buffer.
Above-mentioned external hybridization assays provides the combination of prediction between candidate siRNA and target whether to have specific method.Yet, in the RISC complex systems, use the antisense strand of the high stringency that can not prove external hybridization also can carry out specific cutting to target.
The strand RNA interfering: as mentioned above, RNA interfering finally works as strand.Found that strand (ss) RNA interfering can influence the mRNA silence, but lower than double-stranded RNA efficient.Therefore, embodiment of the present invention also provide uses the ss RNA interfering, its under physiological condition with SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ IDNO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ IDNO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ IDNO:132, the part hybridization of SEQ ID NO:133 or SEQ ID NO:134, and at least 19 nucleotide are arranged be close to continuous complementary zone fully at least, hybridization portion corresponds respectively to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ IDNO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ IDNO:128, SEQ IDNO:129, SEQ IDNO:130, SEQ IDNO:131, SEQ IDNO:132, SEQ ID NO:133 or SEQ ID NO:134.The same with above-mentioned ds RNA interfering, ss RNA interfering length is 19-49 nucleotide.The ss RNA interfering contains 5 ' end phosphoric acid or in position or intravital 5 ' end position by phosphorylation.Term " 5 ' phosphorylation " is used for describing, for example, polynucleotide or oligonucleotide with phosphate group, this phosphate group is connected to via ester bond on the C5 hydroxyl of sugar (for example ribose, deoxyribose or identical analog) at 5 of described polynucleotide or oligonucleotide ' end.
The same with the ds RNA interfering, obtain the ss RNA interfering by chemosynthesis or in vitro transcription or from carrier or expression cassette endogenous expression.Can add 5 ' end phosphate by kinases, or 5 ' end phosphoric acid can be generated by the nuclease cutting of RNA.Use the same with the ds RNA interfering.In one embodiment, use the ss RNA interfering that end with protection and nuclease resistance modify and realize silence.The ss RNA interfering can dry storage or is dissolved in the aqueous solution.In order to suppress annealing or for stable, this solution can comprise buffer or salt.
The hair clip RNA interfering: the hair clip RNA interfering is individual molecule (as a single oligonucleotide chain), comprises the sense strand and the antisense strand (as shRNA) of the RNA interfering in stem ring or the hairpin structure.For example, shRNA can express from dna vector, and wherein coding has the DNA oligonucleotide of adopted RNA interfering chain to be connected by the DNA oligonucleotide of short spacer with the antisense RNA interfering chain of coding reverse complemental.If for the needs of selected expression vector, can add the nucleotide of 3 ' terminal T ' s and formation restriction site.The rna transcription thing that obtains self is folded to form loop-stem structure.
Mode of administration: can be by ocular tissue injection as near the eyes, the injection of (intracanalicular) in the conjunctiva, fascial bursa (subtenon), anterior chamber in (intracameral), the vitreous body under (intravitreal), ophthalmic, the retina, under the conjunctiva, behind the eyeball or in the tear stains; Or directly apply to eyes by using conduit or other apparatus for placing such as retina pill, ophthalmic insertion, suppository or comprising implant porous, non-porous or spawn; By topical ophthalmic drop or ointment; By in the blind pipe or adjacent sclera (sclera corpus ciliare (transcleral)) or the slow releasing device within the eye implanted RNA interfering directly is delivered to eye.Intracameral injection can enter the anterior chamber by cornea, allows medicament arrive girder.Injection can enter vein collecting pipe drain Schlemm's canal or enter Schlemm's canal in the tear stains.Expection can be carried out whole body or intestinal is used outward, includes but not limited to intravenous, subcutaneous and oral administration.
Experimenter: need the high intraocular pressure of treatment or have the experimenter who develops into high intraocular pressure danger to suffer from high intraocular pressure or have people or the mammal that develops into high intraocular pressure danger, described high intraocular pressure does not need or unsuitable expression or active relevant with target described here, and described target is carbonic anhydrase II, IV or XII; β 1-or beta 2-adrenergic receptor; Acetylcholinesterase; Na
+/ K
+-ATP enzyme or Na-K-2Cl cotransporter.The eye structure relevant with this class disease may comprise for example eye, retina, choroid, crystalline lens, cornea, girder, iris, optic nerve, optic disc, sclera, the eye-chamber, vitreous chamber or corpus ciliare.The experimenter also may be eye cell, cell culture, organ or (ex vivo) organ or tissue in vitro.
Preparation and dosage: pharmaceutical preparation comprises that weight ratio can be up to 99% RNA interfering of the present invention or its salt, and it mixes with the acceptable eye vector medium of physiology such as water, buffer, normal saline, glycine, hyaluronic acid, mannitol or the like.
RNA interfering of the present invention is used with the form of solution, suspension or Emulsion.Be the examples of formulations that the present invention may implement below.
| Weight content % RNA interfering reaches 99; 0.1-99; 0.1-50; 0.5-10.0 hydroxypropyl emthylcellulose 0.5 sodium chloride 0.8 benzalkonium chloride 0.01EDTA 0.01NaOH/HCl qs pH7.4 pure water (not containing the RNA enzyme) qs 100mL |
| Weight content % RNA interfering reaches 99; 0.1-99; 0.1-50; 0.5-10.0 |
| Weight content % RNA interfering reaches 99; 0.1-99; 0.1-50; 0.5-10.0 sodium dihydrogen phosphate 0.05 sodium hydrogen phosphate 0.15 (anhydrous) |
| Sodium chloride 0.75EDTA disodium salt 0.05 polyoxyethylene castor oil, 0.1 benzalkonium chloride 0.01HCl and/or NaOH pH7.3-7.4 pure water (not containing the RNA enzyme) q.s. reach 100% |
In general, the EC that the RNA interfering of effective dose produces on target cell surface in the embodiment of the present invention is from 100pM to 100nM, or from 1nM to 50nM, or about 25nM from 5nM to about 10nM or extremely.Reach the required dosage of this local concentration and depend on many factors, comprise that medication, administration site, the cell number of plies, topical between administration site and target cell or tissue still are whole body administration or the like.Concentration ratio on the administration site may exceed much in the concentration of target cell or tissue surface.Compositions is locally applied to ocular surface, every day one to four time, or according to clinicist's routine judge by wider administration time table such as every day, weekly, two week, every month or longer time use.The pH of preparation approximately is pH4-9 or pH4.5-pH7.4.
By the persistent period of increase effect, thereby allow to reduce administration number of times and strengthen experimenter's compliance, use at the siRNA of ocular hypertension targets mRNA experimenter's treatment than being supposed to more favourable with micromolecule part medicament for the eyes drop.
Though correct dosage regimen depends on clinicist's decision, RNA interfering can instruct the method for dripping in every eye to use by the clinicist.The effective dose of preparation may depend on many factors, for example experimenter's age, race and sex other, ratio, RNA interfering usefulness and the RNA interfering stability of ocular hypertensive seriousness, target gene transcript/protein conversion.In one embodiment, RNA interfering is locally applied to eyes by therapeutic dose and arrives girder, retina or optic disc, thereby improve the disease relevant with high intraocular pressure.
Acceptable carrier: the ophthalmology acceptable carrier refers to that those cause at the most and seldom arrives the carrier that anophthalmia stimulates, and provides suitable anticorrosion if desired, and carries one or more RNA interfering of the present invention with uniform dose.The carrier accepted as the RNA interfering of using embodiment of the present invention comprises cationic-liposome-mediated transfection reagent
TKO (Minis Corporation, Madison, WI), LIPOFECTIN
, Lipofectamine, OLIGOFECTAMINE
TM(Invitrogen, Carlsbad, CA) or DHARMAFECT
TM(Dharmacon, Lafayette, CO); Polycation such as polymine; Cationic peptide such as Tat, poly arginine or wear film peptide (Antp peptide); Or liposome.The vesicle that liposome is formed at standard forms lipid and sterin, as cholesterol, and can comprise that target molecules for example has the monoclonal antibody of binding affinity to surface endothelial cell antigens.In addition, liposome can be the liposome that adds PEG.
Can or in the doser of bioerodable or abiotic corrosion, transmit RNA interfering at solution, suspension.RNA interfering can be used separately, also be can be used as the constituent of covalent conjugates, with cationic-liposome, cationic polypeptide or cationic polymer complexation or be wrapped in nano-particle targeting or non-targeting and use.
For dosing eyes, the acceptable antiseptic of RNA interfering and ophthalmology, cosolvent, surfactant, viscosifier, penetration enhancer, buffer, sodium chloride or water can be combined, to form water-soluble aseptic ophthalmic suspension or solution.Can prepare the ophthalmic solution preparation by RNA interfering being dissolved in the molten buffer of physiologically acceptable isotonic water.In addition, ophthalmic solution may comprise that ophthalmologic acceptable surfactant assists dissolution inhibitor.Viscosifier such as hydroxy methocel, hydroxyethyl-cellulose, methylcellulose, polyvinylpyrrolidone or analog can be added in the compositions of the present invention, to improve the delay of chemical compound.
In order to prepare aseptic spongaion preparation, RNA interfering is combined on suitable carrier (as mineral oil, liquid lanolin or white vaseline) with antiseptic.According to the method that is used to prepare other ophthalmic preparation known in the art, by RNA interfering being suspended in by for example
-940 (BFGoodrich, Charlotte NC) or in the hydrophilic substrate of the combined preparation of its homologue can prepare aseptic eye-gel preparation.For example
(Fort Worth TX) can be used as intraocular injection for Alcon Laboratories, Inc..Other compositionss of the present invention may comprise infiltration promote medicament such as cremephor and
80 (polyoxyethylene sorbitan monolaurate, Sigma Aldrich, St.Louis, MO), wherein the less infiltration of RNA interfering pleasing to the eye in.
Test kit: embodiment of the present invention provide test kit, and it comprises and is used for weakening the reagent of described mRNA in the expression of cell here.Test kit comprises siRNA or shRNA expression vector.For the shRNA expression vector of siRNA and non-virus, this test kit also may comprise transfection reagent or other suitable transmission carriers.For the shRNA expression vector of virus, this test kit also may comprise viral vector and/or be used to produce the essential component of viral vector (for example, package cell line and the carrier that comprises viral vector template and extra assistant carrier that is used to pack).This test kit also may comprise positive and negative control siRNA or shRNA expression vector (for example, the siRNA of no targeting contrast siRNA or the irrelevant mRNA of targeting).This test kit also may comprise the reagent that knocks out (for example, be used to detect the primer of quantitative PCR of target mRNA and probe and/or at the antibody of the respective egg white matter of western trace) of specifying target gene as assessment.Alternatively, this test kit may comprise siRNA sequence or shRNA sequence and operation instruction and be used for producing siRNA or making up the essential material of shRNA expression vector by in vitro transcription.
The drug regimen of kit form also is provided, and it comprises that (in the combination of packing) is suitable for accepting the carrier arrangement of hermetic container device therewith and comprises the RNA interfering compositions and ophthalmology can be accepted first kind of case of carrier.If desired, this test kit can also comprise the materia medica reagent constituents that one or more are conventional, for example contains the container of one or more drug acceptable carriers, extra container or the like, and this is conspicuous to those skilled in the art.This test kit also can comprise as plug-in unit or as operation instruction, the guide that is used to use of the printing that is used to indicate the group component of using of label and/or be used for the guide of blending ingredients.
RNA interfering is for example knocking out the ability of endogenous target gene expression in external assessment in the following manner in human body girder (TM) cell.With the people TM cell that transforms, for example specified cell line GTM-3 or HTM-3 (see Pang, I.H. wait the people, 1994.Curr.Eye Res.13:51-63), in bed board cultivation in standard growth culture medium (as the DMEM that augments with 10% fetal bovine serum) in 24 hours before the transfection.According to the description of manufacturer, (Dharmacon, Lafayette CO) carry out transfection in RNA interfering concentration in 0.1nM to 100nM scope to use Dharmafect 1.No targeting contrast RNA interfering and the fine fibroin A/C RNA interfering of nuclear (Dharmacon) are in contrast.After transfection 24 hours, for example use
(Applied Biosystems, Foster City CA), assess target mRNA level by quantitative PCR with the probe series that comprises target site for forward and reverse primer.Can be after transfection about 72 hours (depending on proteinic turnover rate actual time) for example by western trace assessment target proteins matter level.Isolation of RNA and/or proteinic standard technique are well-known to those skilled in the art from cultured cell.The probability of, miss the target effect non-specific for reducing should use can produce the possible least concentration RNA interfering of the expression of target gene level that knocks out of expectation.
The ability that RNA interfering of the present invention knocks out the CA2 protein expression level is able to further illustration in following embodiment 1.
Be used for RNA interfering at the reticent CA2 of HeLa cell specificity
This research detects the CA2-RNA interfering knocks out endogenous CA2 expression in the Hela cell of cultivating ability.
The transfection of HeLa cell is to use the standard body extracellular concentration (100nM and 1nM) of CA2siRNA or does not have targeting contrast siRNA and DharmaFECT
TM(Dharmacon, Lafayette CO) finish 1 transfection reagent.All siRNA are dissolved in 1 * siRNA buffer, and this buffer is to contain 20mM KCl, 6mM HEPES (pH7.5), 0.2mM MgCl
2Aqueous solution.After transfection 72 hours, assess CA2 protein expression and actin protein expression (going up the sample contrast) by the western engram analysis.CA2siRNA is a double-chain interference RNA, and it has specificity to following target sequence: siCA2# 1 targeting SEQ ID NO:721; SiCA2# 3 targeting SEQ IDNO:15; SiCA2# 4 targeting SEQ ID NO:720; SiCA2# 5 targeting SEQ ID NO:141.As western trace data show among Fig. 1, with respect to non-targeting contrast siRNA, each among four CA2siRNA all significantly reduces CA2 at 100nM and 1nM and expresses.The siCA2# 5 of the SiCA2# 4 of targeting SEQ IDNO:720 and targeting SEQ ID NO:141 seems effective especially.
Here the list of references of quoting is incorporated herein by reference especially, and it provides exemplary operation or other details as replenishing those contents of illustrating at this.
Under guidance of the present disclosure, those of ordinary skills will approve, can significantly modify embodiment disclosed herein and do not deviate from the spirit or scope of the present invention.Under guidance of the present disclosure, disclosed here all embodiments can both accomplishedly need not over-drastic experiment with carrying out.In the disclosure and equivalent embodiments thereof, announce four corner of the present invention.Description should be by the too narrow claimed four corner of the present invention that is interpreted as.
Except as otherwise noted, term " a " and " an " that here uses means " one ", " at least one " or " one or more ".
Sequence table
＜110〉Alcon Universal Ltd.
＜120〉ocular hypertension targets of RNAi mediation suppresses
<130>45263P008WO2
＜140〉do not specify as yet
<141>2006-02-01
<150>US60/648,926
<151>2005-02-01
<150>US60/753,364
<151>2005-12-22
<160>724
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<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>34
<210>35
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉target sequence
<400>35
<210>36
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>36
<210>37
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>37
<210>38
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>38
<210>39
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>39
<210>40
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>40
<210>41
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>41
<210>42
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>42
<210>43
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>43
<210>44
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>44
<210>45
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>45
<210>46
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>46
<210>47
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>47
<210>48
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>48
<210>49
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>49
<210>50
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>50
<210>51
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>51
<210>52
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>52
<210>53
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>53
<210>54
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>54
<210>55
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>55
<210>56
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>56
<210>57
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>57
<210>58
<211>19
<212>DNA
＜213〉artificial
<220>
＜223〉target sequence
<400>58
<210>59
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>59
<210>60
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>60
<210>61
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>61
<210>62
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>62
<210>63
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>63
<210>64
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>64
<210>65
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>65
<210>66
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>66
<210>67
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>67
<210>68
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>68
<210>69
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>69
<210>70
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>70
<210>71
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>71
<210>72
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>72
<210>73
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>73
<210>74
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>74
<210>75
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>75
<210>76
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>76
<210>77
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>77
<210>78
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>78
<210>79
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>79
<210>80
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>80
<210>81
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>81
<210>82
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>82
<210>83
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>83
<210>84
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>84
<210>85
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>85
<210>86
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>86
<210>87
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>87
<210>88
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>88
<210>89
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>89
<210>90
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>90
<210>91
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>91
<210>92
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>92
<210>93
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>93
<210>94
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>94
<210>95
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>95
<210>96
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>96
<210>97
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>97
<210>98
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>98
<210>99
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>99
<210>100
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>100
<210>101
<211>3992
<212>DNA
＜213〉homo sapiens
<400>101
<210>102
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>102
<210>103
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>103
<210>104
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>104
<210>105
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>105
<210>106
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>106
<210>107
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>107
<210>108
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>108
<210>109
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>109
<210>110
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>110
<210>111
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>111
<210>112
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>112
<210>113
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>113
<210>114
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>114
<210>115
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>115
<210>116
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>116
<210>117
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>117
<210>118
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>118
<210>119
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>119
<210>120
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>120
<210>121
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>121
<210>122
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>122
<210>123
<211>2156
<212>DNA
＜213〉homo sapiens
<400>123
<210>124
<211>3713
<212>DNA
＜213〉homo sapiens
<400>124
<210>125
<211>2911
<212>DNA
＜213〉homo sapiens
<400>125
<210>126
<211>3587
<212>DNA
＜213〉homo sapiens
<400>126
<210>127
<211>3873
<212>DNA
＜213〉homo sapiens
<400>127
<210>128
<211>951
<212>DNA
＜213〉homo sapiens
<400>128
<210>129
<211>2212
<212>DNA
＜213〉homo sapiens
<400>129
<210>130
<211>1568
<212>DNA
＜213〉homo sapiens
<400>130
<210>131
<211>3350
<212>DNA
＜213〉homo sapiens
<400>131
<210>132
<211>1853
<212>DNA
＜213〉homo sapiens
<400>132
<210>133
<211>6891
<212>DNA
＜213〉homo sapiens
<400>133
<210>134
<211>3959
<212>DNA
＜213〉homo sapiens
<400>134
<210>135
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>135
<210>136
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>136
<210>137
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>137
<210>138
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>138
<210>139
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>139
<210>140
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>140
<210>141
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>141
<210>142
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>142
<210>143
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>143
<210>144
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>144
<210>145
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>145
<210>146
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>146
<210>147
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>147
<210>148
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>148
<210>149
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>149
<210>150
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>150
<210>151
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>151
<210>152
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>152
<210>153
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>153
<210>154
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>154
<210>155
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>155
<210>156
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>156
<210>157
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>157
<210>158
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>158
<210>159
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>159
<210>160
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>160
<210>161
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>161
<210>162
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>162
<210>163
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>163
<210>164
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>164
<210>165
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>165
<210>166
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>166
<210>167
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>167
<210>168
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>168
<210>169
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>169
<210>170
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>170
<210>171
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>171
<210>172
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>172
<210>173
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>173
<210>174
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>174
<210>175
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>175
<210>176
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>176
<210>177
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>177
<210>178
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>178
<210>179
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>179
<210>180
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>180
<210>181
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>181
<210>182
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>182
<210>183
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>183
<210>184
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>184
<210>185
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>185
<210>186
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>186
<210>187
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>187
<210>188
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>188
<210>189
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>189
<210>190
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>190
<210>191
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>191
<210>192
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>192
<210>193
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>193
<210>194
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>194
<210>195
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>195
<210>196
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>196
<210>197
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>197
<210>198
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>198
<210>199
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>199
<210>200
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>200
<210>201
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>201
<210>202
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>202
<210>203
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>203
<210>204
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>204
<210>205
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>205
<210>206
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>206
<210>207
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>207
<210>208
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>208
<210>209
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>209
<210>210
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>210
<210>211
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>211
<210>212
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>212
<210>213
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>213
<210>214
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>214
<210>215
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>215
<210>216
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>216
<210>217
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>217
<210>218
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>218
<210>219
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>219
<210>220
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>220
<210>221
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>221
<210>222
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>222
<210>223
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>223
<210>224
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>224
<210>225
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>225
<210>226
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>226
<210>227
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>227
<210>228
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>228
<210>229
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>229
<210>230
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>230
<210>231
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>231
<210>232
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>232
<210>233
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>233
<210>234
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>234
<210>235
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>235
<210>236
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>236
<210>237
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>237
<210>238
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>238
<210>239
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>239
<210>240
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>240
<210>241
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>241
<210>242
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>242
<210>243
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>243
<210>244
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>244
<210>245
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>245
<210>246
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>246
<210>247
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>247
<210>248
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>248
<210>249
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>249
<210>250
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>250
<210>251
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>251
<210>252
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>252
<210>253
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>253
<210>254
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>254
<210>255
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>255
<210>256
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>256
<210>257
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>257
<210>258
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>258
<210>259
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>259
<210>260
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>260
<210>261
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>261
<210>262
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>262
<210>263
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>263
<210>264
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>264
<210>265
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>265
<210>266
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>266
<210>267
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>267
<210>268
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>268
<210>269
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>269
<210>270
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>270
<210>271
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>271
<210>272
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>272
<210>273
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>273
<210>274
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>274
<210>275
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>275
<210>276
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>276
<210>277
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>277
<210>278
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>278
<210>279
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>279
<210>280
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>280
<210>281
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>281
<210>282
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>282
<210>283
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>283
<210>284
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>284
<210>285
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>285
<210>286
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>286
<210>287
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>287
<210>288
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>288
<210>289
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>289
<210>290
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>290
<210>291
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>291
<210>292
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>292
<210>293
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>293
<210>294
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>294
<210>295
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>295
<210>296
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>296
<210>297
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>297
<210>298
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>298
<210>299
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>299
<210>300
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>300
<210>301
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>301
<210>302
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>302
<210>303
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>303
<210>304
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>304
<210>305
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>305
<210>306
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>306
<210>307
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>307
<210>308
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>308
<210>309
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>309
<210>310
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>310
<210>311
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>311
<210>312
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>312
<210>313
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>313
<210>314
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>314
<210>315
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>315
<210>316
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>316
<210>317
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>317
<210>318
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>318
<210>319
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>319
<210>320
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>320
<210>321
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>321
<210>322
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>322
<210>323
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>323
<210>324
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>324
<210>325
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>325
<210>326
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>326
<210>327
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>327
<210>328
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>328
<210>329
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>329
<210>330
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>330
<210>331
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>331
<210>332
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>332
<210>333
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>333
<210>334
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>334
<210>335
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>335
<210>336
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>336
<210>337
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>337
<210>338
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>338
<210>339
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>339
<210>340
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>340
<210>341
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>341
<210>342
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>342
<210>343
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>343
<210>344
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>344
<210>345
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>345
<210>346
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>346
<210>347
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>347
<210>348
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>348
<210>349
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>349
<210>350
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>350
<210>351
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>351
<210>352
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>352
<210>353
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>353
<210>354
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>354
<210>355
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>355
<210>356
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>356
<210>357
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>357
<210>358
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>358
<210>359
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>359
<210>360
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>360
<210>361
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>361
<210>362
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>362
<210>363
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>363
<210>364
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>364
<210>365
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>365
<210>366
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>366
<210>367
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>367
<210>368
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>368
<210>369
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>369
<210>370
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>370
<210>371
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>371
<210>372
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>372
<210>373
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>373
<210>374
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>374
<210>375
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>375
<210>376
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>376
<210>377
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>377
<210>378
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>378
<210>379
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>379
<210>380
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>380
<210>381
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>381
<210>382
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>382
<210>383
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>383
<210>384
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>384
<210>385
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>385
<210>386
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>386
<210>387
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>387
<210>388
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>388
<210>389
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>389
<210>390
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>390
<210>391
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>391
<210>392
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>392
<210>393
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>393
<210>394
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>394
<210>395
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>395
<210>396
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>396
<210>397
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>397
<210>398
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>398
<210>399
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>399
<210>400
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>400
<210>401
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>401
<210>402
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>402
<210>403
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>403
<210>404
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>404
<210>405
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>405
<210>406
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>406
<210>407
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>407
<210>408
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>408
<210>409
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>409
<210>410
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>410
<210>411
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>411
<210>412
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>412
<210>413
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>413
<210>414
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>414
<210>415
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>415
<210>416
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>416
<210>417
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>417
<210>418
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>418
<210>419
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>419
<210>420
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>420
<210>421
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>421
<210>422
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>422
<210>423
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>423
<210>424
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>424
<210>425
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>425
<210>426
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>426
<210>427
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>427
<210>428
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>428
<210>429
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>429
<210>430
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>430
<210>431
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>431
<210>432
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>432
<210>433
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>433
<210>434
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>434
<210>435
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>435
<210>436
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>436
<210>437
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>437
<210>438
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>438
<210>439
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>439
<210>440
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>440
<210>441
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>441
<210>442
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>442
<210>443
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>443
<210>444
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>444
<210>445
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>445
<210>446
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>446
<210>447
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>447
<210>448
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>448
<210>449
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>449
<210>450
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>450
<210>451
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>451
<210>452
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>452
<210>453
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>453
<210>454
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>454
<210>455
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>455
<210>456
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>456
<210>457
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>457
<210>458
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>458
<210>459
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>459
<210>460
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>460
<210>461
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>461
<210>462
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>462
<210>463
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>463
<210>464
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>464
<210>465
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>465
<210>466
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>466
<210>467
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>467
<210>468
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>468
<210>469
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>469
<210>470
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>470
<210>471
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>471
<210>472
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>472
<210>473
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>473
<210>474
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>474
<210>475
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>475
<210>476
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>476
<210>477
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>477
<210>478
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>478
<210>479
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>479
<210>480
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>480
<210>481
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>481
<210>482
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>482
<210>483
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>483
<210>484
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>484
<210>485
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>485
<210>486
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>486
<210>487
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>487
<210>488
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>488
<210>489
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>489
<210>490
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>490
<210>491
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>491
<210>492
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>492
<210>493
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>493
<210>494
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>494
<210>495
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>495
<210>496
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>496
<210>497
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>497
<210>498
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>498
<210>499
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>499
<210>500
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>500
<210>501
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>501
<210>502
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>502
<210>503
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>503
<210>504
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>504
<210>505
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>505
<210>506
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>506
<210>507
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>507
<210>508
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>508
<210>509
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>509
<210>510
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>510
<210>511
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>511
<210>512
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>512
<210>513
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>513
<210>514
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>514
<210>515
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>515
<210>516
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>516
<210>517
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>517
<210>518
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>518
<210>519
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>519
<210>520
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>520
<210>521
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>521
<210>522
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>522
<210>523
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>523
<210>524
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>524
<210>525
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>525
<210>526
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>526
<210>527
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>527
<210>528
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>528
<210>529
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>529
<210>530
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>530
<210>531
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>531
<210>532
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>532
<210>533
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>533
<210>534
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>534
<210>535
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>535
<210>536
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>536
<210>537
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>537
<210>538
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>538
<210>539
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>539
<210>540
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>540
<210>541
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>541
<210>542
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>542
<210>543
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>543
<210>544
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>544
<210>545
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>545
<210>546
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>546
<210>547
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>547
<210>548
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>548
<210>549
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>549
<210>550
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>550
<210>551
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>551
<210>552
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>552
<210>553
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>553
<210>554
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>554
<210>555
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>555
<210>556
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>556
<210>557
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>557
<210>558
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>558
<210>559
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>559
<210>560
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>560
<210>561
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>561
<210>562
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>562
<210>563
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>563
<210>564
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>564
<210>565
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>565
<210>566
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>566
<210>567
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>567
<210>568
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>568
<210>569
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>569
<210>570
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>570
<210>571
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>571
<210>572
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>572
<210>573
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>573
<210>574
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>574
<210>575
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>575
<210>576
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>576
<210>577
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>577
<210>578
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>578
<210>579
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>579
<210>580
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>580
<210>581
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>581
<210>582
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>582
<210>583
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>583
<210>584
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>584
<210>585
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>585
<210>586
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>586
<210>587
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>587
<210>588
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>588
<210>589
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>589
<210>590
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>590
<210>591
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>591
<210>592
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>592
<210>593
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>593
<210>594
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>594
<210>595
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>595
<210>596
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>596
<210>597
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>597
<210>598
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>598
<210>599
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>599
<210>600
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>600
<210>601
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>601
<210>602
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>602
<210>603
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>603
<210>604
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>604
<210>605
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>605
<210>606
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>606
<210>607
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>607
<210>608
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>608
<210>609
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>609
<210>610
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>610
<210>611
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>611
<210>612
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>612
<210>613
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>613
<210>614
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>614
<210>615
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>615
<210>616
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>616
<210>617
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>617
<210>618
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>618
<210>619
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>619
<210>620
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>620
<210>621
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>621
<210>622
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>622
<210>623
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>623
<210>624
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>624
<210>625
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>625
<210>626
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>626
<210>627
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>627
<210>628
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>628
<210>629
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>629
<210>630
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>630
<210>631
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>631
<210>632
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>632
<210>633
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>633
<210>634
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>634
<210>635
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>635
<210>636
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>636
<210>637
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>637
<210>638
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>638
<210>639
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>639
<210>640
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>640
<210>641
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>641
<210>642
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>642
<210>643
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>643
<210>644
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>644
<210>645
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>645
<210>646
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>646
<210>647
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>647
<210>648
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>648
<210>649
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>649
<210>650
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>650
<210>651
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>651
<210>652
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>652
<210>653
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>653
<210>654
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>654
<210>655
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>655
<210>656
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>656
<210>657
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>657
<210>658
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>658
<210>659
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>659
<210>660
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>660
<210>661
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>661
<210>662
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>662
<210>663
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>663
<210>664
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>664
<210>665
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>665
<210>666
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>666
<210>667
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>667
<210>668
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>668
<210>669
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>669
<210>670
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>670
<210>671
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>671
<210>672
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>672
<210>673
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>673
<210>674
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>674
<210>675
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>675
<210>676
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>676
<210>677
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>677
<210>678
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>678
<210>679
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>679
<210>680
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>680
<210>681
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>681
<210>682
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>682
<210>683
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>683
<210>684
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>684
<210>685
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>685
<210>686
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>686
<210>687
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>687
<210>688
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>688
<210>689
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>689
<210>690
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>690
<210>691
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>691
<210>692
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>692
<210>693
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>693
<210>694
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>694
<210>695
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>695
<210>696
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>696
<210>697
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>697
<210>698
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>698
<210>699
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>699
<210>700
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>700
<210>701
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>701
<210>702
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>702
<210>703
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>703
<210>704
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>704
<210>705
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>705
<210>706
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>706
<210>707
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>707
<210>708
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>708
<210>709
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>709
<210>710
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>710
<210>711
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>711
<210>712
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>712
<210>713
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>713
<210>714
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>714
<210>715
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>715
<210>716
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>716
<210>717
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉target sequence
<400>717
<210>718
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＜223〉target sequence
<400>721
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＜223〉sense strand
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Claims (41)
1. weaken the method that ocular hypertension targets mRNA expresses in the experimenter, this method comprises: using the length that comprises effective dose to the experimenter is the RNA interfering of 19-49 nucleotide and the compositions of drug acceptable carrier, and this RNA interfering comprises:
The zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:8, SEQID NO:14-SEQ ID NO:32, SEQ ID NO:83-SEQ IDNO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ IDNO:135-SEQ ID NO:219, SEQ ID NO:720 and SEQ IDNO:721.
2. the process of claim 1 wherein ocular hypertension targets mRNA coding carbonic anhydrase II, and described RNA interfering comprises:
The zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:8, SEQ ID NO:14-SEQID NO:22, SEQ IDNO:83-SEQ ID NO:100, SEQ ID NO:135-SEQ ID NO:155, SEQ ID NO:720 and SEQ ID NO:721.
3. the process of claim 1 wherein ocular hypertension targets mRNA coding carbonic anhydrase IV, and described RNA interfering comprises:
The zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:23-SEQ ID NO:32 and SEQ ID NO:156-SEQ ID NO:177.
4. the process of claim 1 wherein ocular hypertension targets mRNA coding carbonic anhydrase XII, and described RNA interfering comprises:
The zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:102-SEQ ED NO:122 and SEQ ID NO:178-SEQ ID NO:219.
5. the method for claim 1, wherein the zone of continuous nucleotide is the zone of at least 14 continuous nucleotides, and terminal 14 nucleotide of 3 of the sequence of this zone and sequence identifier ' end time have complementary or at least 85% the sequence homogeneity of at least 85% sequence.
6. the method for claim 1, wherein the zone of continuous nucleotide is the zone of at least 15,16,17 or 18 continuous nucleotides, and this zone has complementary or at least 80% the sequence homogeneity of at least 80% sequence with 3 of the sequence of sequence identifier ' end time terminal 15,16,17 or 18 nucleotide respectively.
7. the process of claim 1 wherein that RNA interfering is shRNA.
8. the process of claim 1 wherein that RNA interfering is miRNA.
9. the process of claim 1 wherein that RNA interfering is siRNA.
10. compositions is used for the treatment of purposes in the ocular hypertensive medicine of experimenter in preparation, and the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide, and this RNA interfering comprises:
The zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ ID NO:8, SEQ ID NO:14-SEQID NO:32, SEQ ID NO:83-SEQ ID NO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ ID NO:135-SEQ ID NO:219, SEQ IDNO:720 and SEQ ID NO:721.
11. the purposes of claim 10, wherein RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ IDNO:8, SEQ ID NO:14-SEQ ID NO:22, SEQ ID NO:83-SEQ IDNO:100, SEQ ID NO:135-SEQ ID NO:155, SEQ ID NO:720 and SEQ IDNO:721.
12. the purposes of claim 10, wherein this RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ IDNO:23-SEQ ID NO:32 and SEQ ID NO:156-SEQ ID NO:177.
13. the purposes of claim 10, wherein RNA interfering comprises the zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and following arbitrary sequence ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence, and described sequence is SEQ IDNO:102-SEQ ID NO:122 and SEQ ID NO:178-SEQ ID NO:219.
14. the purposes of claim 10, wherein RNA interfering is shRNA.
15. the purposes of claim 10, prepare wherein that compositions is used in the part, vitreous body, the sclera corpus ciliare, near the eyes, in the conjunctiva, fascial bursa, anterior chamber, using under the retina, under the conjunctiva, behind the eyeball or in the tear stains.
16. the purposes of claim 10, the wherein compositions that is used to use via the expression in vivo preparation from the expression vector that can express RNA interfering.
17. the purposes of claim 10, wherein RNA interfering is miRNA.
18. the purposes of claim 10, wherein RNA interfering is siRNA.
19. weaken the method that second kind of variant expression of ocular hypertension targets mRNA expressed and do not weakened to first kind of variant of experimenter's ocular hypertension targets mRNA, it comprises:
The experimenter is used compositions, and the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide, and this RNA interfering comprises
The zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and first kind of variant ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence,
Wherein the expression of first kind of variant mRNA is weakened, and does not weaken the expression of second kind of variant mRNA, and wherein first kind of variant target mRNA is that SEQ ID NO:101 and second kind of variant target mRNA are SEQ ID NO:134.
20. weaken the method that second kind of variant expression of ocular hypertension targets mRNA expressed and do not weakened to first kind of variant of experimenter's ocular hypertension targets mRNA, it comprises:
The experimenter is used compositions, and the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide, and this RNA interfering comprises
The zone of at least 13 continuous nucleotides, penultimate 13 nucleotide of 3 of this continuous nucleotide and first kind of variant ' end have complementary or at least 90% the sequence homogeneity of at least 90% sequence,
Wherein the expression of first kind of variant mRNA is weakened, and does not weaken the expression of second kind of variant mRNA, and wherein first kind of variant target mRNA is that SEQ ID NO:134 and second kind of variant target mRNA are SEQ ID NO:101.
21. weaken the method that experimenter's ocular hypertension targets mRNA expresses, it comprises:
The experimenter is used compositions, and the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide, and this RNA interfering comprises:
The intimate at least continuous complementary zone fully that adopted nucleotide chain, antisense nucleotide chain and at least 19 nucleotide are arranged;
Wherein antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:101 or SEQ ID NO:134, and have at least 19 nucleotide, be close to continuous complementary zone fully at least with hybridization portion respectively corresponding to the mRNA of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:101 or SEQ ID NO:134
Wherein the expression of ocular hypertension targets mRNA is weakened.
22. the method for claim 21, ocular hypertension targets mRNA coding carbonic anhydrase II wherein, and antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:1, and contain at least 19 nucleotide, be close to continuous complementary zone fully at least with hybridization portion corresponding to the mRNA of SEQ ID NO:1.
23. the method for claim 21, ocular hypertension targets mRNA coding carbonic anhydrase IV wherein, and antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:2, and contain at least 19 nucleotide, be close to continuous complementary zone fully at least with hybridization portion corresponding to the mRNA of SEQ ID NO:2.
24. the method for claim 21, ocular hypertension targets mRNA coding carbonic anhydrase XII wherein, and antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:101 or SEQ ID NO:134, and contain at least 19 nucleotide, be close to continuous complementary zone fully at least with hybridization portion corresponding to the mRNA of SEQ ID NO:101 or SEQ ID NO:134.
25. the method for claim 21, wherein antisense strand is designed to the mRNA of targeting corresponding to SEQ IDNO:1, described mRNA comprises nucleotide 232,527,721,728,809,810,855,856,921,1139,506,547,548,740,911,1009,1140,1149,1150,1151,1188,1194,1195,1223,1239,1456,1457,1458,100,158,166,247,286,318,322,328,371,412,482,504,505,541,734,772,777,814,972,998,1232,317 or 401.
26. the method for claim 21, wherein antisense strand is designed to the mRNA of targeting corresponding to SEQ IDNO:2, described mRNA comprises nucleotide 213,252,258,266,399,457,463,490,595,1064,109,112,125,126,150,261,265,280,398,453,459,462,467,492,534,785,801,825,827,876,1003 or 1012.
27. the method for claim 21, wherein antisense strand is designed to the mRNA of targeting corresponding to SEQ ID NO:101, described mRNA comprises nucleotide 191,239,274,275,341,389,412,413,423,687,689,695,710,791,792,794,983,993,994,995,691,1039,1568,2326,2332,2425,2433,2844,2845,2880,2884,2891,2954,2955,2956,2957,2964,2965,3006,3007,3012 or 3026.
28. the method for claim 21, wherein antisense strand is designed to the mRNA of targeting corresponding to SEQ ID NO:134, described mRNA comprises nucleotide 687,1535,2293,2299,2392,2400,2811,2812,2847,2851,2858,2921,2922,2923,2924,2931,2932,2973,2974,2979 or 2993.
29. the method for claim 21, it also comprises to the experimenter uses second kind of RNA interfering that length is 19-49 nucleotide, and described second kind of RNA interfering comprises
The intimate at least complete complementary zone that adopted nucleotide chain, antisense nucleotide chain and at least 19 nucleotide are arranged;
Wherein the antisense strand of second kind of RNA interfering under physiological condition with second portion hybridization corresponding to the mRNA of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:101 or SEQ ID NO:134, and antisense strand contain at least 19 nucleotide, be close to continuous complementary zone fully at least with second hybridization portion of the mRNA that corresponds respectively to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:101 or SEQID NO:134.
30. the method for claim 21, wherein linking by the ringed nucleus nucleotide sequence has adopted nucleotide chain and antisense nucleotide chain.
31. compositions is used for the treatment of purposes in the ocular hypertensive medicine of experimenter in preparation, the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide, and this RNA interfering comprises:
The intimate at least complete complementary zone that adopted nucleotide chain, antisense nucleotide chain and at least 19 nucleotide are arranged;
Wherein antisense strand under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:101 or SEQ ID NO:134, and have at least 19 nucleotide, be close to continuous complementary zone fully at least with the hybridization portion of the mRNA that corresponds respectively to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:101 or SEQ ID NO:134.
32. the purposes of claim 31, wherein linking by the ringed nucleus nucleotide sequence has adopted nucleotide chain and antisense nucleotide chain.
33. the purposes of claim 31, prepare wherein that compositions is used in the part, vitreous body, the sclera corpus ciliare, near the eyes, in the conjunctiva, fascial bursa, anterior chamber, using under the retina, under the conjunctiva, behind the eyeball or in the tear stains.
34. the purposes of claim 31, the wherein compositions that is used to use via the expression in vivo preparation from the expression vector that can express RNA interfering.
35. weaken the method for the expression of experimenter's ocular hypertension targets mRNA, it comprises:
Use compositions to the experimenter, the length that said composition comprises effective dose is the RNA interfering and the drug acceptable carrier of 19-49 nucleotide,
Wherein the strand RNA interfering under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:1, described part comprises nucleotide 232,527,721,728,809,810,855,856,921,1139,506,547,548,740,911,1009,1140,1149,1150,1151,1188,1194,1195,1223,1239,1456,1457,1458,100,158,166,247,286,318,322,328,371,412,482,504,505,541,734,772,777,814,972,998,1232,317 or 401, and this RNA interfering contains with hybridization portion corresponding to the mRNA of SEQ ID NO:1 and is close to continuous complementary zone fully at least;
Or
Wherein the strand RNA interfering under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:2, described part comprises nucleotide 213,252,258,266,399,457,463,490,595,1064,109,112,125,126,150,261,265,280,398,453,459,462,467,492,534,785,801,825,827,876,1003 or 1012, and this RNA interfering contain at least 19 nucleotide, be close to continuous complementary zone fully at least with hybridization portion corresponding to the mRNA of SEQ ID NO:2;
Or
Wherein the strand RNA interfering under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:101, described part comprises nucleotide 191,239,274,275,341,389,412,413,423,687,689,695,710,791,792,794,983,993,994,995,691,1039,1568,2326,2332,2425,2433,2844,2845,2880,2884,2891,2954,2955,2956,2957,2964,2965,3006,3007,3012 or 3026, and this RNA interfering contains at least 19 nucleotide, at least be close to continuous complementary zone fully with hybridization portion corresponding to the mRNA of SEQ ID NO:101;
Or
Wherein the strand RNA interfering under physiological condition with part hybridization corresponding to the mRNA of SEQ ID NO:134, described part comprises nucleotide 687,1535,2293,2299,2392,2400,2811,2812,2847,2851,2858,2921,2922,2923,2924,2931,2932,2973,2974,2979 or 2993, and this RNA interfering contain at least 19 nucleotide, be close to continuous complementary zone fully at least with hybridization portion corresponding to the mRNA of SEQ ID NO:134;
Wherein the expression of ocular hypertension targets mRNA is weakened thus.
36. the method for claim 35, wherein RNA interfering is miRNA.
37. the method for claim 35, wherein RNA interfering is siRNA.
38. comprise the compositions of RNA interfering and drug acceptable carrier, described RNA interfering length is 19-49 nucleotide and contains arbitrary nucleotide sequence or its complementary series among SEQ ID NO:8, SEQ ID NO:14-SEQ IDNO:32, SEQ ID NO:83-SEQ ID NO:100, SEQ ID NO:102-SEQ IDNO:122, SEQ ID NO:135-SEQ ID NO:219, SEQ ID NO:720 and the SEQ IDNO:721.
39. the compositions of claim 38, wherein RNA interfering is shRNA.
40. the compositions of claim 38, wherein RNA interfering is siRNA.
41. the compositions of claim 38, wherein RNA interfering is miRNA.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US64892605P | 2005-02-01 | 2005-02-01 | |
| US60/648,926 | 2005-02-01 | ||
| US60/753,364 | 2005-12-22 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2006800073006A Division CN101137753A (en) | 2005-02-01 | 2006-02-01 | Rnai-mediated inhibition of ocular hypertension targets |
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| CN101444525A true CN101444525A (en) | 2009-06-03 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2008102131399A Pending CN101444525A (en) | 2005-02-01 | 2006-02-01 | RNAi-mediated inhibition of ocular targets |
| CNA2006800073006A Pending CN101137753A (en) | 2005-02-01 | 2006-02-01 | Rnai-mediated inhibition of ocular hypertension targets |
| CNA2006800102460A Pending CN101155918A (en) | 2005-02-01 | 2006-02-01 | Rnai-mediated inhibition of ocular hypertension targets |
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| CNA2006800073006A Pending CN101137753A (en) | 2005-02-01 | 2006-02-01 | Rnai-mediated inhibition of ocular hypertension targets |
| CNA2006800102460A Pending CN101155918A (en) | 2005-02-01 | 2006-02-01 | Rnai-mediated inhibition of ocular hypertension targets |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102604946A (en) * | 2011-01-21 | 2012-07-25 | 北京蛋白质组研究中心 | Small interfering ribonucleic acid (siRNA) for inhibiting tumor metastasis and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102757946B (en) * | 2011-04-28 | 2014-07-09 | 中国科学院生态环境研究中心 | Gobiocypris rarus alpha type Na+/K+-ATPase, and encoding gene and application thereof |
| JP6988481B2 (en) * | 2016-01-26 | 2022-01-05 | 日産化学株式会社 | Single-stranded oligonucleotide |
| CN109557309B (en) * | 2018-12-04 | 2021-09-10 | 九江学院附属医院 | Application of carbonic anhydrase-2 as detection marker in diagnosis of kidney stones |
-
2006
- 2006-02-01 CN CNA2008102131399A patent/CN101444525A/en active Pending
- 2006-02-01 CN CNA2006800073006A patent/CN101137753A/en active Pending
- 2006-02-01 CN CNA2006800102460A patent/CN101155918A/en active Pending
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604946A (en) * | 2011-01-21 | 2012-07-25 | 北京蛋白质组研究中心 | Small interfering ribonucleic acid (siRNA) for inhibiting tumor metastasis and application thereof |
| CN102604946B (en) * | 2011-01-21 | 2014-02-19 | 北京蛋白质组研究中心 | Small interfering ribonucleic acid (siRNA) for inhibiting tumor metastasis and application thereof |
Also Published As
| Publication number | Publication date |
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| ZA200706083B (en) | 2008-09-25 |
| CN101155918A (en) | 2008-04-02 |
| ZA200706084B (en) | 2009-04-29 |
| CN101137753A (en) | 2008-03-05 |
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