CN104910253A - Small peptide coupling peptide nucleic acid monomer composition used as nucleic acid cutting reagent and application thereof - Google Patents
Small peptide coupling peptide nucleic acid monomer composition used as nucleic acid cutting reagent and application thereof Download PDFInfo
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Abstract
The invention discloses a small peptide coupling peptide nucleic acid monomer composition used as a nucleic acid cutting reagent and application thereof, the small peptide coupling peptide nucleic acid monomer composition is characterized by being prepared by complexing of a small peptide coupling peptide nucleic acid monomer and a metal ion ligand, and the small peptide coupling peptide nucleic acid monomer is 2-(N-histadyl lysyl amine ethyl-2-(thymine-1)-acetyl amino) acetic acid (HLT for abbreviation) and (2-(N-amino ethyl-2-(thymine-1)-acetamido) acetamido) lysyl histidine (THL for abbreviation), the metal ion is <99 m>TcO4 <->. The agarose gel electrophoresis method is used for detecting the cutting effect of a nucleic acid cutting reagent, a good research foundation for researches on synthetic DNA fixed-point recognition and cutting are further provided, at the same time an effective test support for researches on excellent antisense reagents and diagnostic reagents for gene targeting effects is provided, and the small peptide coupling peptide nucleic acid monomer composition has great significance.
Description
Technical field
The present invention relates to little peptide coupling peptide nucleic acid monomer composition as nucleic acid cutting reagent and application thereof, belong to biological chemistry, molecular biology and molecular nuclear medicine technical field.
Background technology
Radiotherapy from so far, experienced by one from macroscopic view to microcosmic, from organizing level to cell levels, then to the evolution of the level of molecule and gene.Be the research field of radiotherapy forefront using gene as target position, compared with other cancer treatment method, its great advantage is exactly that purpose is strong, can be down to minimum to the side effect of normal gene element.At present, many drug mains will be studied using protein as target position, and gene is made another key areas that target position then becomes new drug development, small pieces DNA has wherein been proved to be up-and-coming research direction as medicine by.Reach this object, the medicine carrying radionuclide should have oncogene target position specificity on a molecular scale, can be attached to specifically on Oncogene Sequences target site, forms stable mixture, thus reaches the object for the treatment of or diagnosing cancer.Antisense/anti-gene radiation treatment is an emerging oncotherapy technology, and antisense nucleic acid prevents oncogene and radionuclide target to damage oncogene to organically combine by it.
PNA (Peptide Nucleic Acid, peptide nucleic acid(PNA)) as a kind of biological ployose, that a kind of radiation kernel that transmits carries out molecule cutting and damage to cancer cells target position to it, or carry out the effective tool of Molecular Graphs picture detection, the clinical application of cancer diagnosis and radio genetic therapy has the bright outlook (Nielsen P E, Egholm M, Berg R H, et al.Sequence-selective recognition of DNA by strand displacement with a thymine-substitutedpolyamide.Science, 1991, 254:1497-1450.He Y J, lgor G P, Alex K, et al.Sequence-specificDNA strand cleavage by
111in-labeled peptide nucleic acids.Eur J Nucl Med Mol Imaging, 2004,31 (6): 837-845.).PNA can replace ribose and the phosphoric acid of DNA with the glycine skeleton repeated, the same with n DNA (or RNA), PNA also can synthesize the oligomeric peptide nucleic acid of various Different Alkali basic sequence.Compare with native peptides with protease-sensitive natural acid with to nuclease, PNA has very high chemistry and biology stability, it is not easily by nuclease and protease hydrolyzed, be easy to be transferred in nucleus simultaneously, and can be combined with the form stable of double-strand or triple strand dna by the base pairing of the single-minded flight of steps leading to a palace hall with gene target position.Therefore, the PNA of active nucleus mark can instruct the radiation therapy of gene target position on a molecular scale, there is important application (Panyutin I G in diagnosis simultaneously, Winters T A, Feinendegen L E, et al.Development of DNA-based radiopharmaceuticals carryingAuger-electron emitters for anti-gene radiotherapy.Q J Nucl Med.2000, 44:256-267.Panyutin IV, Luu A N, Panyutin I G, et al.Strand breaks in whole plasmid DNA produced by the decay of125I in a triplex-forming oligonucleotide.Radiat Res.2001, 156:158-166.).
A subject matter of PNA mark is difficult to form stable coordination be good for; by after acylation reaction can on PNA coupling bifunctional chelating agent DTPA (Lewis M R; Jia F; Gallazzi F; et al.Radiometal-labeled peptide-PNAconjugates for targeting bcl-2expression:preparation; characterization; and in vitro mRNAbinding.Bioconjugate Chem.2002; 13:1176-1180.); this makes mark become easy, and marker is more stable.So far, the document in the world about radioisotope labeling pna molecule is very rare, and the researchist of association area has also just started to pay close attention to this direction.
Chinese Scientists is applied to PNA in the middle of Radionuclide antisense therapy, carried out a series of experimental study work (Zheng Junnian, Sun Xiaoqing, Chen Jiacun, etc.
125i marks Ki67 Antisense Peptide nucleic acid to the restraining effect of xenografts in nude mice kidney transplanted tumor. Chinese tumor biotherapy magazine, and 2005,12 (1): 18. Zheng Jun, Sun Xiaoqing, Chen Jiacun, etc.
125i marks the experimental study that Ki67 polypeptide-nucleic acid affects kidney cancer cell propagation and apoptosis. Chinese Journal of Nuclear Medicine, 2004,24 (3): 139-141.).Research shows,
125after I-PNAs is combined with oncogene DNA,
125the Auger electronics that I sends directly can destroy oncogene,
125i on average decays and once can produce a DNA break, reaches the effect of gene radiotherapy tumour.Research finds
125i-PNA can strengthen PNAs and prevent kidney cancer cell Ki67 genetic expression, Inhibit proliferaton, promotes apoptotic effect, is expected to treat for kidney.
External scientist attempt with [
99mtc (CO)
3(H
2o)
3]
+mark PNA monomer, research shows to form high yield and the composition of good stability (Xavier C, Pak J K, Santos I, et al.Evaluation of chelators for labeling a PNAmonomer with the fac-[
99mtc (CO)
3]
+moiety.Journal of organometallic chemistry, 2007,692:1332-1339.), likely become the antisense Radionuclide imaging probe of the target mRNA of PNA complementary with it.
Design and synthesis is connected with the α-PNA conjugate monomer of little peptide, adopts
99mtc mark makes title complex, and research marker ligand compound, to the cutting action of DNA, can be the researchs such as the fracture and damage reparation of researching DNA specific site, gene target diagnosis and provides effective approach, have important Research Significance.At present, yet there are no little peptide coupling peptide nucleic acid monomer composition as the application patent documentation of nucleic acid cutting reagent and non-patent literature report.
Summary of the invention
The object of the invention is the little peptide coupling peptide nucleic acid monomer composition and the application thereof that are provided as nucleic acid cutting reagent for the deficiencies in the prior art, be characterized in little peptide coupling peptide nucleic acid monomer composition being that little peptide coupling peptide nucleic acid monomer and metallic ion coordination form, little peptide coupling peptide nucleic acid monomer is 2-(N-histidyl-lysyl amine ethyl-2-(thymus pyrimidine-1)-kharophen) acetic acid (being abbreviated as HLT) of structural formula I and (2-(N-amine ethyl-2-(thymus pyrimidine-1)-kharophen) kharophen) lysyl Histidine (being abbreviated as THL) of formula II, english system called after 2-(N-((6-amino-2-(2-amino-3-(1H-imidazol-2-yl) propanamido) hexanamido)-ethyl)-2-(5-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H)-yl) acetamido) acetic acid and 3-(1H-imidazol-2-yl)-2-(6-amino-2-(2-(N-2-aminoethyl-2-(5-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H)-yl) acetamido) acetamido) hexanamido) propanonic acid, chemical formula is C
23h
35n
9o
7, structure is as follows:
Described metal ion is
99mtcO
4 -.
According to the first embodiment of the present invention, provide a kind of little peptide coupling peptide nucleic acid monomer composition as nucleic acid cutting reagent, said composition comprises little peptide coupling peptide nucleic acid monomer as ligand and metal ion
99mtcO
4 -or by as the little peptide coupling peptide nucleic acid monomer of ligand and metal ion
99mtcO
4 -composition, wherein
Little peptide coupling peptide nucleic acid monomer is selected from 2-(N-histidyl-lysyl amine ethyl-2-(thymus pyrimidine-1)-kharophen) acetic acid [being abbreviated as HLT] or 2-(N-amine ethyl-2-(thymus pyrimidine-1)-kharophen) kharophen) one or both in lysyl Histidine [being abbreviated as THL].
In general, little peptide coupling peptide nucleic acid monomer and metal ion
99mtcO
4 -mol ratio be 0.1 ~ 50:1, preferably 0.3 ~ 40:1, more preferably 0.4 ~ 30:1, is more preferably 0.5 ~ 20:1, more preferably 0.6 ~ 10:1, more preferably 0.7 ~ 5:1, more preferably 0.8 ~ 3:1, more preferably 0.9 ~ 2:1, more preferably 1 ~ 1.5:1.
According to the second embodiment of the present invention, provide a kind of method preparing above-described composition, the method comprises containing metal ion
99mtcO
4 -solution mix with the little peptide coupling peptide nucleic acid monomer as ligand.
Generally, containing metal ion
99mtcO
4 -the radioactive concentration of solution or radioactive activity be 0.5 × 10
9~ 5 × 10
9bq/mL, preferably 1.0 × 10
9~ 3 × 10
9bq/mL.
Generally, little peptide coupling peptide nucleic acid monomer uses with the solution form of 3.0 ~ 150 μMs, preferably 14.3 ~ 57.2 μM concentration.
According to the third embodiment the present invention, provide the composition described in the application as the purposes of nucleic acid cutting reagent.
The present invention can also adopt agarose gel electrophoresis method to detect nucleic acid cutting effect.Therefore, according to the 4th embodiment of the present invention, provide a kind of method adopting the composition cutting nucleic acid described in the application, the method comprises the following steps:
(1) agarose is added in Erlenmeyer flask, add 1 × TBE electrophoretic buffer again, the agarose concentration selected for cutting plasmid pUC 019DNA (genome sequence L09137) is 1.5wt%), the Erlenmeyer flask that agarose and 1 × TBE electrophoretic buffer are housed is placed in microwave oven and heats (such as 20 ~ 50s, as 30s makes agarose particle dissolve completely, transparent uniform solution, cooling (is such as cooled to 40 ~ 55 DEG C, as 50 DEG C), pour in the glue groove assembling band comb, the thickness of solution is 2 ~ 7cm (such as 4cm), and the bubble removed in glue-line, ambient temperatare is put (such as 30min), then glue groove is put into electrophoresis chamber,
(2) will add 1 × TBE electrophoretic buffer in above-mentioned electrophoresis chamber, add-on, to flood glue face top 1mm, then carefully takes out comb, makes sample well naturally be full of electrophoretic buffer;
(3) in centrifuge tube, add the electrophoresis Sample with the configuration of PBS damping fluid, then put into thermostat container, at 35 ~ 39 DEG C, hatch 20 ~ 50min, amount adds staining agent and developer 1 ~ 2 μ L per sample;
(4) sample micropipet is joined on gel sample hole, and cover electrophoresis chamber, at room temperature, electrophoretic voltage adopts 10V/cm ~ 40V/cm (such as 20V/cm or 30V/cm), DNA anode is moved, observe the electrophoresis situation (stop electrophoresis when sample arrives anode, be lasted for 15min) of tetrabromophenol sulfonphthalein display in developer;
(5) take out gel, observe in gel imaging instrument and electrophoresis result picture under taking;
(6) electrophoresis result is converted into linear DNA with super coiled DNA and represents.
In general, the process for preparation of described 5 × TBE electrophoretic buffer is: 54.0g Tutofusin tris (Tris), 27.5g H
3bO
3, 4.5g EDTA is dissolved in 1L deionized water, obtains the electrophoretic buffer of 5 × TBE, during use, takes out appropriate, dilutes 5 times, namely obtain 1 × TBE electrophoretic buffer.
The preparation of described PBS damping fluid is by 1.2164g NaH
2pO
4.2H
2o and 4.3685g Na
2hPO
4.12H
2o is dissolved in 100mL deionized water, and concentration is 0.1015mol/L, pH=6.4;
Generally, the preparation of described staining agent pipettes Goldview 1 μ L with micropipet, adds 1 × TBE electrophoretic buffer 9 μ L, be placed in refrigerator and keep in Dark Place.
Generally, the preparation of described developer pipettes Loading buffer 1 μ L with micropipet, adds 1 × TBE electrophoretic buffer 9 μ L, be placed in refrigerator and keep in Dark Place.
Nucleic acid is important life genetic material, is one of the molecular biology task of primarily solving to the fracture and damage reparation of its specific site.Along with the rising year by year of difficult and complicated cases (especially cancer) sickness rate, the medicine finding high specific selectivity just seems very important.The medicine of high specific selectivity not only can improve diagnosis and treatment efficiency, also can reduce the toxic side effect to patient simultaneously.
Structural characterization and performance test:
Adopt GelDoc-IT
tMthe effect of 300 type gel-electrophoretic apparatuses to different system cutting plasmid pUC 019DNA (genome sequence L09137) is analyzed, shown in Fig. 1 ~ 10.
Experimental result shows, such little peptide coupling peptide nucleic acid monomer composition close under physiological pH condition, at lower concentration, effectively can cut (pUC) 019DNA in the short period of time.Hatch in 37 DEG C of atmosphere, respectively at 2h, 5h, 12h tri-point in time sampling, observation cutting effect finds only have
99mtcO
4 -when, DNA is hatched 5h and is put and can find out cutting band, demonstrates obvious cutting effect just now after 12h;
99mtc (O) HLT and
99mtc (O) THL and DNA is hatched 2h and is namely demonstrated fairly obvious cutting effect, and 5h cuts substantially completely.The cutting effect of each factor of Integrated comparative hatch 1h in 37 DEG C of atmosphere after, finds, removes
99mtc (O) HLT and
99moutside the band that Tc (O) THL is corresponding and band, other are as SnCl
2the band that+GH is corresponding,
99mtcO
4 -corresponding band does not almost have cutting effect to show, and the band that the band that HLT is corresponding, THL are corresponding also only shows atomic weak cutting phenomenon, illustrates that marker ligand compound to the cutting effect of pUC019 should be
99mthe synergy of Tc and PNA.
The present invention has the following advantages:
The present invention is that the research of the preparation such as fracture and damage reparation, gene target diagnosis of further researching DNA specific site provides basis and effective approach preferably, has important Research Significance.
Accompanying drawing explanation
Fig. 1 is
99mtcO
4 -the agarose gel electrophoresis figure of cutting pUC 019DNA 2h
Fig. 2 is
99mtcO
4 -the agarose gel electrophoresis figure of cutting pUC 019DNA 5h
Fig. 3 is
99mtcO
4 -the agarose gel electrophoresis figure of cutting pUC 019DNA 12h
Fig. 4 is
99mthe agarose gel electrophoresis figure of Tc (O) HLT title complex cutting pUC 019DNA 1h
Fig. 5 is
99mthe agarose gel electrophoresis figure of Tc (O) HLT title complex cutting pUC 019DNA 2h
Fig. 6 is
99mthe agarose gel electrophoresis figure of Tc (O) HLT title complex cutting pUC 019DNA 5h
Fig. 7 is
99mthe agarose gel electrophoresis figure of Tc (O) THL title complex cutting pUC 019DNA 1h
Fig. 8 is
99mthe agarose gel electrophoresis figure of Tc (O) THL title complex cutting pUC 019DNA 2h
Fig. 9 is
99mthe agarose gel electrophoresis figure of Tc (O) THL title complex cutting pUC 019DNA 5h
Figure 10 is the agarose gel electrophoresis figure that each combined factors compares cutting pUC 019DNA 1h
Embodiment
Below by embodiment, the present invention is specifically described; be necessary that again this is pointed out that the present embodiment is only for further illustrating of carrying out the present invention; can not be interpreted as limiting the scope of the invention, person skilled in art can make some nonessential improvement and adjustment according to the content of foregoing invention.
Embodiment 1: little peptide coupling peptide nucleic acid monomer title complex carries out cutting experiment to plasmid DNA, cutting experiment adopts agarose gel electrophoresis method for detecting.
1, instrument
2, reagent
3, the preparation of test solution
The preparation of 5 × TBE electrophoretic buffer: by 54.0g Tris, 27.5g H
3bO
3, 4.5g EDTA is dissolved in 1L deionized water, obtains the electrophoretic buffer of 5 × TBE.During use, take out appropriate, dilute 5 times, namely obtain 1 × TBE electrophoretic buffer.
The preparation of PBS damping fluid: by 1.2164g NaH
2pO
4.2H
2o and 4.3685g Na
2hPO
4.12H
2o is dissolved in 100mL deionized water, and concentration is 0.1015mol/L, pH=6.4;
Staining agent: pipette Goldview 1 μ L with micropipet, adds 1 × TBE electrophoretic buffer 9 μ L, is placed in refrigerator and keeps in Dark Place;
Developer: pipette Loading buffer 1 μ L with micropipet, adds 1 × TBE electrophoretic buffer 9 μ L, is placed in refrigerator and keeps in Dark Place.
4, the preparation of little peptide coupling peptide nucleic acid monomer
HLT: 2-chlorine trityl chloride resin 10g is put into reaction tubes, adds dimethyl formamide 150mL, vibration 30min.Solvent is fallen by husky core suction filtration; add the 2-after amido protecting (N-(2-amine ethyl)-2-(thymus pyrimidine-1)-kharophen) acetic acid 1.30g; add diisopropylethylamine 3.25g again; add dimethyl formamide to dissolve; solvent is removed after vibration 30min; add the dimethyl formamide solution 200mL of 20% piperidines; after 5min, filtering filtrate adds the dimethyl formamide solution 200mL of 20% piperidines again; take out removing piperidine solution after 15min, solid is successively with dimethyl formamide, methyl alcohol, dimethyl formamide washing.Add the Methionin 3.50g after amido protecting; O-benzotriazole-tetramethyl-urea phosphofluoric acid ester 3.00g; dissolve with a small amount of dimethyl formamide; after adding N-methylmorpholine 2.60g. reaction 30min after adding reaction tubes at once, suction filtration removes liquid; add the Histidine 2.85g after amido protecting; O-benzotriazole-tetramethyl-urea phosphofluoric acid ester 3.00g; dissolve with a small amount of dimethyl formamide; add reaction tubes again; add at once after N-methylmorpholine 2.60g. reacts 30min and use dimethyl formamide, methyl alcohol, washed with dichloromethane successively, drain 10min.Add the dichloromethane solution 100mL rinse of 1% trifluoroacetic acid, dry up with nitrogen after filtration as far as possible, wash rear normal temperature with ether and volatilize.Thick product pure water is added a small amount of acetonitrile to dissolve, obtaining purity after HPLC purifying is 99.36% white solid little peptide coupling peptide nucleic acid monomer product HLT.HPLC-MS composes: 550.97 [M-H]
+; A (aqueous solution of 0.05% trifluoroacetic acid), B (acetonitrile solution of 0.05% trifluoroacetic acid); Phenomenex, Jupiter 5 μ L, C18,300A 150 × 4.6mm; 230nm; Voyager-DE RP.Conform to theoretical MS value 550.65.
TLH: 2-chlorine trityl chloride resin 10g is put into reaction tubes, adds dimethyl formamide 150mL, vibration 30min.Solvent is fallen by husky core suction filtration; add the Histidine 2.85g after amido protecting; add diisopropylethylamine 3.20g again; dissolve with dimethyl formamide; filtering solvent after vibration 30min, adds the dimethyl formamide solution 200mL of 20% piperidines, removes the dimethyl formamide solution 200mL that solution adds 20% piperidines again in solid after 5min; filtering piperidine solution after 15min, successively with dimethyl formamide, methyl alcohol, dimethyl formamide washing.Add the Methionin 3.50g after amido protecting, O-benzotriazole-tetramethyl-urea phosphofluoric acid ester 2.80g, dissolves with a small amount of dimethyl formamide, adds reaction tubes, adds N-methylmorpholine 2.50g. at once and reacts 30min; Add the 2-after amido protecting (N-(2-amine ethyl)-2-(thymus pyrimidine-1)-kharophen) acetic acid 1.28g after suction filtration, dissolve with a small amount of DMF, add reaction tubes, add N-methylmorpholine 2.50g. at once and react 30min; Use dimethyl formamide, methyl alcohol, washed with dichloromethane successively, drain 10min.Add the dichloromethane solution 100mL rinse of 1% trifluoroacetic acid, dry up with nitrogen after filtration as far as possible, wash rear normal temperature with ether and volatilize.Thick product pure water is added a small amount of acetonitrile to dissolve, obtaining purity after HPLC purifying is 95.89% white solid little peptide coupling peptide nucleic acid monomer product TLH.HPLC-MS composes: 550.85 [M-H]
+; A (aqueous solution of 0.05% trifluoroacetic acid), B (acetonitrile solution of 0.05% trifluoroacetic acid); Phenomenex, Jupiter 5 μ L, C18,300A 150 × 4.6mm; 230nm; Voyager-DE RP.Conform to theoretical MS value 550.65.
5, operation steps
Agarose gel electrophoresis experimental procedure is as follows:
(1) agarose is added in Erlenmeyer flask, add 1 × TBE electrophoretic buffer again, be 1.5wt% for the agarose concentration selected of cutting pUC19, the Erlenmeyer flask that agarose and 1 × TBE electrophoretic buffer are housed be placed in microwave oven and heat 30s agarose particle is dissolved completely, transparent uniform solution, be cooled to 50 DEG C, pour in the glue groove assembling band comb, thickness is about 4cm, and removes the bubble in glue-line, ambient temperatare puts 30min, then glue groove is put into electrophoresis chamber;
(2) add 1 × TBE electrophoretic buffer in electrophoresis chamber, add-on, to flood glue face top 1mm, then carefully takes out comb, makes sample well naturally be full of electrophoretic buffer;
(3) in centrifuge tube, add the electrophoresis Sample with the configuration of PBS damping fluid, then centrifuge tube is put into thermostat container, hatch 30min at 37 DEG C after, amount adds appropriate staining agent, developer etc. per sample;
(4) sample micropipet is joined on gel sample hole, and cover electrophoresis chamber, at room temperature, electrophoretic voltage adopts 20V/cm, DNA anode is moved, observe the electrophoresis situation of tetrabromophenol sulfonphthalein display in developer, stop electrophoresis (lasting about 15min) when sample arrives anode;
(5) take out gel, observe in gel imaging instrument and electrophoresis result picture under taking;
(6) electrophoresis result is converted into linear DNA (Form II) with ring-type super coiled DNA (Form I) and represents.
As shown in Fig. 1 ~ Figure 10, carry out cutting test by adopting different cutting agents.
In Fig. 1, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtcO
4 -in PBS, cut the experimental result picture of pUC 019DNA 2h, activity concentration is followed successively by: 1.11 × 10
9bq/mL, 2.22 × 10
9bq/mL, 3.33 × 10
9bq/mL, 4.44 × 10
9bq/mL.As we know from the figure,
99mtcO
4 -phenomenon during cutting pUC 019DNA 2h is very faint, and effect is also not obvious.
In Fig. 2, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtcO
4 -in PBS, cut the experimental result picture of pUC 019DNA 5h, activity concentration is followed successively by: 1.11 × 10
9bq/mL, 2.22 × 10
9bq/mL, 3.33 × 10
9bq/mL, 4.44 × 10
9bq/mL.As we know from the figure, concentration is 4.44 × 10
9bq/mL's
99mtcO
4 -can find out during cutting pUC 019DNA 5h and comparatively significantly cut band.
In Fig. 3, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtcO
4 -in PBS, cut the experimental result picture of pUC 019DNA 12h, activity concentration is followed successively by: 1.11 × 10
9bq/mL, 2.22 × 10
9bq/mL, 3.33 × 10
9bq/mL, 4.44 × 10
9bq/mL.As we know from the figure,
99mtcO
4 -cutting pUC 019DNA 12h time effect clearly, especially when concentration is 3.33 × 10
9bq/mL, 4.44 × 10
9during Bq/mL.
In Fig. 4, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtc (O) HLT (
99mtc (O): HLT=1:1 mol ratio) in PBS, cut the experimental result picture of pUC 019DNA 1h, concentration is followed successively by: 14.3 μMs, 28.6 μMs, 42.9 μMs, 57.2 μMs.As we know from the figure,
99mphenomenon during Tc (O) HLT cutting pUC 019DNA 1h is obvious.
In Fig. 5, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtc (O) HLT (
99mtc (O): HLT=1:1 mol ratio) in PBS, cut the experimental result picture of pUC 019DNA 2h, concentration is followed successively by: 14.3 μMs, 28.6 μMs, 42.9 μMs, 57.2 μMs.As we know from the figure,
99mtc (O) HLT cut pUC 019DNA 2h time phenomenon clearly, even if concentration is 14.3 μMs.
In Fig. 6, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtc (O) HLT (
99mtc (O): HLT=1:1 mol ratio) in PBS, cut the experimental result picture of pUC 019DNA 5h, concentration is followed successively by: 14.3 μMs, 28.6 μMs, 42.9 μMs, 57.2 μMs.As we know from the figure,
99mtc (O) HLT substantially reaches when cutting pUC 019DNA 5h and cuts completely, even if concentration is 14.3 μMs.
In Fig. 7, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtc (O) THL (
99mtc (O): THL=1:1 mol ratio) in PBS, cut the experimental result picture of pUC 019DNA 1h, concentration is followed successively by: 14.3 μMs, 28.6 μMs, 42.9 μMs, 57.2 μMs.As we know from the figure,
99mphenomenon during Tc (O) THL cutting pUC 019DNA 1h is obvious.
In Fig. 8, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtc (O) THL (
99mtc (O): THL=1:1 mol ratio) in PBS, cut the experimental result picture of pUC 019DNA 2h, concentration is followed successively by: 14.3 μMs, 28.6 μMs, 42.9 μMs, 57.2 μMs.As we know from the figure,
99mtc (O) THL cut pUC 019DNA 2h time phenomenon clearly, even if concentration is 14.3 μMs.
In Fig. 9, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5 are
99mtc (O) THL (
99mtc (O): THL=1:1 mol ratio) in PBS, cut the experimental result picture of pUC 019DNA 5h, concentration is followed successively by: 14.3 μMs, 28.6 μMs, 42.9 μMs, 57.2 μMs.As we know from the figure,
99mtc (O) THL substantially reaches when cutting pUC 019DNA 5h and cuts completely, even if concentration is 14.3 μMs.
In Figure 10, lane1 is the independent experimental result picture of pUC 019DNA in PBS from left to right, and lane2, lane3, lane4, lane5, lane6 are followed successively by tin chloride (SnCl
2) and glucose (GH),
99mtcO
4 -, HLT,
99mtc (O) HLT, THL,
99mtc (O) THL etc. cuts the experimental result picture of pUC 019DNA 1h in PBS.As we know from the figure, remove
99mlane5 corresponding to Tc (O) HLT and
99moutside the corresponding lane7 of Tc (O) THL, other are as SnCl
2the lane2 that+GH is corresponding,
99mtcO
4 -corresponding lane3 does not almost have cutting effect to show, and the lane6 that lane4, THL that HLT is corresponding are corresponding also only shows atomic weak cutting phenomenon.Known in conjunction with upper interpretation, title complex to the cutting effect of pUC019 should be
99mthe synergy of Tc and PNA.
Claims (8)
1., as the little peptide coupling peptide nucleic acid monomer composition of nucleic acid cutting reagent, it is characterized in that said composition comprises little peptide coupling peptide nucleic acid monomer as ligand and metal ion
99mtcO
4 -or by as the little peptide coupling peptide nucleic acid monomer of ligand and metal ion
99mtcO
4 -composition, wherein
Little peptide coupling peptide nucleic acid monomer is selected from 2-(N-histidyl-lysyl amine ethyl-2-(thymus pyrimidine-1)-kharophen) acetic acid of structural formula I or 2-(N-amine ethyl-2-(thymus pyrimidine-1)-kharophen) kharophen of formula II) one or both in lysyl Histidine
2., according to claim 1 as the little peptide coupling peptide nucleic acid monomer composition of nucleic acid cutting reagent, it is characterized in that little peptide coupling peptide nucleic acid monomer and metal ion
99mtcO
4 -mol ratio be 0.1 ~ 50:1.
3., according to claim 2 as the little peptide coupling peptide nucleic acid monomer composition of nucleic acid cutting reagent, it is characterized in that little peptide coupling peptide nucleic acid monomer and metal ion
99mtcO
4 -mol ratio be 0.3 ~ 40:1.
4., according to claim 3 as the little peptide coupling peptide nucleic acid monomer composition of nucleic acid cutting reagent, it is characterized in that little peptide coupling peptide nucleic acid monomer and metal ion
99mtcO
4 -mol ratio be 0.4 ~ 30:1.
5. prepare the method for the described little peptide coupling peptide nucleic acid monomer composition as nucleic acid cutting reagent of one of claim 1-5, it is characterized in that the method comprises containing metal ion
99mtcO
4 -solution mix with the little peptide coupling peptide nucleic acid monomer as ligand.
6., according to claim 5 as the method for the little peptide coupling peptide nucleic acid monomer composition of nucleic acid cutting reagent, it is characterized in that containing metal ion
99mtcO
4 -the radioactive concentration of solution or radioactive activity be 0.5 × 10
9~ 5 × 10
9bq/mL, preferably 1.0 × 10
9~ 3 × 10
9bq/mL.
7. according to claim 5 or 6 as the method for the little peptide coupling peptide nucleic acid monomer composition of nucleic acid cutting reagent, it is characterized in that little peptide coupling peptide nucleic acid monomer uses with the solution form of 3.0 ~ 150 μMs, preferably 14.3 ~ 57.2 μM concentration.
8. according to the purposes of the described little peptide coupling peptide nucleic acid monomer composition as nucleic acid cutting reagent of one of claim 1-4 as nucleic acid cutting reagent.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580252A (en) * | 2003-08-06 | 2005-02-16 | 上海师范大学 | Artificial endo enzyme containing peptide nucleic acid recognition element, and its preparing method and use |
| CN101014713A (en) * | 2004-07-07 | 2007-08-08 | 挪威镭医院研究基金会 | Method for introducing a pna molecule conjugated to a positively charged peptide into the cytosol and/or the nucleus by photochemical internalisation (PCI) |
| WO2009066513A1 (en) * | 2007-11-19 | 2009-05-28 | The University Of Tokyo | Method of site-selectively cleaving target nucleic acid |
| CN102286092A (en) * | 2011-09-14 | 2011-12-21 | 深圳翰宇药业股份有限公司 | Solid-phase synthesis method of liraglutide |
-
2015
- 2015-05-15 CN CN201510250729.9A patent/CN104910253A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1580252A (en) * | 2003-08-06 | 2005-02-16 | 上海师范大学 | Artificial endo enzyme containing peptide nucleic acid recognition element, and its preparing method and use |
| CN101014713A (en) * | 2004-07-07 | 2007-08-08 | 挪威镭医院研究基金会 | Method for introducing a pna molecule conjugated to a positively charged peptide into the cytosol and/or the nucleus by photochemical internalisation (PCI) |
| WO2009066513A1 (en) * | 2007-11-19 | 2009-05-28 | The University Of Tokyo | Method of site-selectively cleaving target nucleic acid |
| CN102286092A (en) * | 2011-09-14 | 2011-12-21 | 深圳翰宇药业股份有限公司 | Solid-phase synthesis method of liraglutide |
Non-Patent Citations (2)
| Title |
|---|
| WANG MING-QI ET AL: "Metal-free cleavage efficiency towards DNA by a novel PNA analog-bridged macrocyclic polyamine", 《BIOORGANIC ＆ MEDICINAL CHEMISTRY LETTERS》 * |
| 宋宏涛等: "99mTc(O)(PNA-dp)辐射切割质粒DNA 的性能研究", 《辐射研究与辐射工艺学报》 * |
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