TWI821192B - 非天然核苷酸之導入及其方法 - Google Patents
非天然核苷酸之導入及其方法 Download PDFInfo
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- TWI821192B TWI821192B TW107123898A TW107123898A TWI821192B TW I821192 B TWI821192 B TW I821192B TW 107123898 A TW107123898 A TW 107123898A TW 107123898 A TW107123898 A TW 107123898A TW I821192 B TWI821192 B TW I821192B
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Abstract
本文揭示用於利用突變tRNA合成包含非天然胺基酸之蛋白質之方法、組合物及套組。
Description
寡核苷酸且其應用已徹底改變生物技術。然而,包括DNA及RNA之寡核苷酸各自僅包括四個天然核苷酸腺苷(A)、鳥苷(G)、胞嘧啶(C)、胸腺嘧啶(T)用於DNA,及四個天然核苷酸腺苷(A)、鳥苷(G)、胞嘧啶(C)及尿苷(U)用於RNA,且其顯著限制寡核苷酸之潛在功能及應用。
定序能力,特別是,例如藉由PCR或等溫擴增系統,使用聚合酶合成/擴增寡核苷酸(DNA或RNA)(例如使用T7 RNA聚合酶轉錄)已徹底改變生物技術。除奈米技術中之所有潛在應用以外,此已實現多種多樣的新技術,諸如RNA及DNA適體及酶之經由藉由指數富集之配位體系統性進化(Systematic Evolution of Ligands by Exponential Enrichment,SELEX
)的活體外進化。參見,例如,Oliphant AR, Brandl CJ及Struhl K (1989), Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 proteins,Mol. Cell Biol.
, 9:2944-2949;Tuerk C及Gold L (1990), Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase,Science
, 249:505-510;Ellington AD及Szostak JW (1990),In vitro
selection of RNA molecules that bind specific ligands,Nature
, 346:818-822。
在一些態樣中,此等應用受天然基因字母表(DNA中之四個天然核苷酸A、C、G及T,及RNA中之四個天然核苷酸A、C、G及U)中所存在之有限化學/物理多樣性限制。本文揭示生成含有擴展遺傳字母表之核酸之其他方法。
在某些實施例中,本文揭示製備含有非天然胺基酸之蛋白質之方法,該方法包含:製備突變tRNA,其中該突變tRNA包含選自表1或2之突變反密碼子序列;製備突變mRNA,其中該突變mRNA包含選自表1或2之突變密碼子序列;及利用該突變tRNA及該突變mRNA合成含有非天然胺基酸之蛋白質。在一些情況下,在無細胞轉譯系統中合成蛋白質。在一些情況下,在細胞(半合成生物體或SSO)中合成蛋白質。在一些情況下,半合成生物體包含微生物。在一些情況下,半合成生物體包含細菌。在一些情況下,半合成生物體包含大腸桿菌(Escherichia coli
)。在一些情況下,突變tRNA之突變反密碼子與選自表1至3之突變密碼子配對。在一些情況下,非天然胺基酸包含至少一個非天然核苷酸。在一些情況下,非天然核苷酸包含非天然核鹼基。在一些情況下,非天然核苷酸之非天然鹼基選自由以下組成之群:2-胺基腺嘌呤-9-基、2-胺基腺嘌呤、2-F-腺嘌呤、2-硫尿嘧啶、2-硫基-胸腺嘧啶、2-硫胞嘧啶、腺嘌呤及鳥嘌呤之2-丙基及烷基衍生物、2-胺基-腺嘌呤、2-胺基-丙基-腺嘌呤、2-胺基吡啶、2-吡啶酮、2'-去氧尿苷、2-胺基-2'-去氧腺苷、3-去氮鳥嘌呤、3-去氮腺嘌呤、4-硫基-尿嘧啶、4-硫基-胸腺嘧啶、尿嘧啶-5-基、次黃嘌呤-9-基(I)、5-甲基-胞嘧啶、5-羥基甲基胞嘧啶、黃嘌呤、次黃嘌呤、5-溴及5-三氟甲基尿嘧啶及胞嘧啶;5-鹵基尿嘧啶、5-鹵基胞嘧啶、5-丙炔基-尿嘧啶、5-丙炔基胞嘧啶、5-尿嘧啶、5-取代、5-鹵基、5-取代嘧啶、5-羥基胞嘧啶、5-溴胞嘧啶、5-溴尿嘧啶、5-氯胞嘧啶、氯化胞嘧啶、環胞嘧啶、胞嘧啶***糖苷、5-氟胞嘧啶、氟嘧啶、氟尿嘧啶、5,6-二氫胞嘧啶、5-碘胞嘧啶、羥脲、碘尿嘧啶、5-硝基胞嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶及5-碘尿嘧啶、腺嘌呤及鳥嘌呤之6-烷基衍生物、6-氮雜嘧啶、6-偶氮-尿嘧啶、6-偶氮胞嘧啶、氮雜胞嘧啶、6-偶氮-胸腺嘧啶、6-硫基-鳥嘌呤、7-甲基鳥嘌呤、7-甲基腺嘌呤、7-去氮鳥嘌呤、7-去氮鳥苷、7-去氮-腺嘌呤、7-去氮-8-氮雜鳥嘌呤、8-氮雜鳥嘌呤、8-氮雜腺嘌呤、8-鹵基、8-胺基、8-巰基、8-硫基烷基及8-羥基取代腺嘌呤及鳥嘌呤;N4-乙基胞嘧啶、N-2取代嘌呤、N-6取代嘌呤、O-6取代嘌呤、彼等增加雙螺旋形成之穩定性者、通用核酸、疏水性核酸、混雜核酸、尺寸擴展核酸、氟化核酸、三環嘧啶、啡噁嗪胞苷([5,4-b][l,4]苯并噁嗪-2(3H)-酮)、啡噻嗪胞苷(1H-嘧啶并[5,4-b][l,4]苯并噻嗪-2(3H)-酮)、G-夾鉗、啡噁嗪胞苷(9-(2-胺基乙氧基)-H-嘧啶并[5,4-b][l,4]苯并噁嗪-2(3H)-酮)、咔唑胞苷(2H-嘧啶并[4,5-b]吲哚-2-酮)、吡啶并吲哚胞苷(H-吡啶并[3',2':4,5]吡咯并[2,3-d]嘧啶-2-酮)、5-氟尿嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-碘尿嘧啶、次黃嘌呤、黃嘌呤、4-乙醯基胞嘧啶、5-(羧基羥基甲基)尿嘧啶、5-羧甲基胺基甲基-2-硫尿苷、5-羧甲基胺基甲基尿嘧啶、二氫尿嘧啶、β-D-半乳糖苷基Q核苷、肌苷、N6-異戊烯基腺嘌呤、1-甲基鳥嘌呤、1-甲基肌苷、2,2-二甲基鳥嘌呤、2-甲基腺嘌呤、2-甲基鳥嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-腺嘌呤、7-甲基鳥嘌呤、5-甲基胺基甲基尿嘧啶、5-甲氧基胺甲基-2-硫尿嘧啶、β-D-甘露糖苷基Q核苷、5'-甲氧基羧基甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-N6-異戊烯基腺嘌呤、尿嘧啶-5氧基乙酸、Y丁氧苷(wybutoxosine)、假尿嘧啶、Q核苷、2-硫胞嘧啶、5-甲基-2-硫尿嘧啶、2-硫尿嘧啶、4-硫尿嘧啶、5-甲基尿嘧啶、尿嘧啶-5-乙醛酸甲酯、尿嘧啶-5-乙醛酸、5-甲基-2-硫尿嘧啶、3-(3-胺基-3-N-2-羧丙基)尿嘧啶、(acp3)w及2,6-二胺基嘌呤以及其中嘌呤或嘧啶鹼基經雜環替換之彼等。在一些情況下,非天然核苷酸選自由以下組成之群(為了清楚說明之目的僅顯示核鹼基部分,省略核糖及磷酸酯主鏈):。
在一些情況下,非天然核苷酸選自由以下組成之群(為了清楚說明之目的僅顯示核鹼基部分,省略核糖及磷酸主鏈):。
在一些情況下,非天然核苷酸另外包含非天然糖部分。在一些情況下,非天然核苷酸非天然糖部分選自由以下2'位置處之修飾所組成之群中:OH;經取代之低碳數烷基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3
、OCN、Cl、Br、CN、CF3
、OCF3
、SOCH3
、SO2
CH3
、ONO2
、NO2
、N3
、NH2
F;O-烷基、S-烷基、N-烷基;O-烯基、S-烯基、N-烯基;O-炔基、S-炔基、N-炔基;O-烷基-O-烷基、2'-F、2'-OCH3
、2'-O(CH2
)2
OCH3
,其中烷基、烯基及炔基可為經取代或未經取代之C1
-C10
、烷基、C2
-C10
烯基、C2
-C10
炔基、-O[(CH2
)O]m
CH3
、-O(CH2
)n
OCH3
、-O(CH2
)n
NH2
、-O(CH2
)n
CH3
、-O(CH2
)n
-ONH2
及-O(CH2
)n
ON[(CH2
)n
CH3
)]2
,其中n及m為1至約10;及/或在5'位置處之修飾:5'-乙烯基、5'-甲基(R或S);在4'位置處之修飾:4'-S、雜環烷基、雜環烷芳基、胺基烷胺基、聚烷基胺基、經取代矽烷基;RNA裂解基團;報導子基團;嵌入劑;改善寡核苷酸之藥物動力學特性之基團或改善寡核苷酸之藥力學特性基團,以及其任何組合。在一些情況下,突變反密碼子或突變密碼子另外包含非天然主鏈。在一些情況下,突變反密碼子及突變密碼子另外包含非天然主鏈。在一些情況下,非天然核苷酸由DNA聚合酶、RNA聚合酶或反轉錄酶識別。在一些情況下,在轉錄期間非天然核苷酸藉由RNA聚合酶導入至mRNA中,以生成含有突變密碼子之突變mRNA。在一些情況下,在轉錄期間非天然核苷酸藉由RNA聚合酶導入至tRNA中,以生成含有突變反密碼子之突變tRNA。在一些情況下,在轉錄期間非天然核苷酸藉由RNA聚合酶導入至mRNA中,以生成突變mRNA。在一些情況下,在轉錄期間非天然核苷酸藉由RNA聚合酶導入至tRNA中,以生成突變tRNA。在一些情況下,突變tRNA帶有非天然胺基酸殘基。在一些情況下,在轉譯期間利用突變tRNA及突變mRNA生成含有非天然胺基酸之蛋白質。
交叉參考
本申請案主張於2017年7月11日申請之美國臨時申請案第62/531,325號之權益,其以全文引用之方式併入本文中。某些術語
除非另外定義,否則本文所用之所有技術及科學術語均具有與所主張主題之所屬領域之熟習此項技術者通常所理解的相同的含義。應理解,前述一般描述及以下詳細描述僅為例示性及解釋性且不限制所主張之任何主題。在本申請案中,除非另外明確陳述,否則單數之使用包括複數。必須注意,除非上下文明確另外指定,否則如本說明書及隨附申請專利範圍中所用之單數形式「一(a/an)」及「該(the)」包括複數個指示物。在本申請案中,除非另外說明,否則使用「或」意指「及/或」。此外,使用術語「包括(including)」以及其他形式,諸如「包括(include)」、「包括(includes)」及「包括(included)」,不具限制性。
如本文所使用,範圍及量可表示為「約」特定數值或範圍。約亦包括準確量。因此「約5 µL」意指「約5 µL」亦及「5 µL」。一般而言,術語「約」包括將預期在實驗誤差內的量。
本文中所用之各部分標題僅出於組織目的而不應理解為限制所描述之主題。
概述
生命資訊由四字母基因字母表編碼,其因兩個鹼基對(d)G-(d)C及(d)A-dT/U之選擇性形成而成為可能。在兩個合成核苷酸之間形成之第三非天然鹼基對(UBP)擴展此系統,從而增加資訊儲存之潛能,且具有深遠理論及實踐影響。在已報導之多種多樣的合成核苷酸類似物中,有幾種在其他方面天然的DNA雙螺旋內彼此穩定配對,但不由聚合酶識別,且表明在雙螺旋DNA中控制穩定配對之力與控制聚合酶介導之複製之力不同。因此,已採取不同方法發展可複製UBP,例如經設計以經由不被天然核苷酸採用之補充的氫鍵結(H-鍵結)模式相互作用的UBP。儘管天然鹼基對經由H-鍵結形成,沒有原因假設H-鍵結為唯一足以支撐遺傳資訊之儲存(或檢索)之力的先驗。舉例而言,已表明,大腸桿菌(E. coli) DNA聚合酶I之克列諾(Klenow)片段(Kf)以dA配對非天然核苷酸dF,其二氟甲苯核鹼基為不能有效H-鍵結之胸腺嘧啶之形狀模擬。此支持DNA複製之「幾何選擇」機制且表明除H-鍵結以外之力亦促進複製。
活體外複製、轉錄及轉譯成蛋白質之UBP之發展為支撐天然資訊之儲存及檢索之力提供了見解,且亦實現在化學及合成生物學中之廣泛應用。然而,發展UBP之多種工作之最終目標係其作為半合成生物體(SSO)(一種穩定儲存及檢索大量資訊之生物體(非天然的或合成的,意指人工製得的))之基礎之活體內用途。而且,此類SSO具有革命性的實踐應用,包括用於人類健康。最值得注意的是,SSO徹底改變蛋白質治療劑之生長領域。然而,相比於傳統小分子治療劑,由於可由二十種天然胺基酸獲得之化學多樣性有限,蛋白質治療劑受其分子特性嚴重限制。
吾人最近報導了大腸桿菌SSO之創建,其藉助於來自三角褐指藻(Phaeodactylum tricornutum,PtNTT2)之核苷三磷酸運輸蛋白,自培養基輸入必需非天然三磷酸,且隨後使用他們複製含有UBP dNaM-dTPT3之質體。吾人已展示,含有UBP之DNA可藉由T7RNA聚合酶在SSO中轉錄,且當mRNA之密碼子中導入非天然核苷酸時,帶有ncAA且在其反密碼子中含有同源非天然核苷酸之不同tRNA可以有效地且選擇性解碼非天然密碼子。因為UBP可在不同密碼子之不同位置合併,此表明UBP可用於編碼具有多個不同ncAA之蛋白質。
本文在某些實施例中揭示用於利用突變tRNA合成包含非天然胺基酸之蛋白質的方法、組合物及套組。在一些情況下,在無細胞轉譯系統中合成蛋白質。在一些情況下,在細胞或半合成生物體(SSO)中合成蛋白質。在一些情況下,半合成生物體包含微生物。在一些情況下,半合成生物體包含細菌。在一些情況下,半合成生物體包含大腸桿菌。在一些情況下,突變tRNA含有突變反密碼子序列。在一些情況下,突變反密碼子序列係表1中所說明之反密碼子序列。在一些情況下,突變反密碼子序列係表2中所說明之反密碼子序列。在一些情況下,突變反密碼子序列係表3中所說明之反密碼子序列。表 1 表 2 表 3
在一些情況下,突變tRNA之突變反密碼子與突變密碼子配對。在一些實施例中,突變密碼子為表1中所說明之突變密碼子。在一些實施例中,突變密碼子為表2中所說明之突變密碼子。在一些實施例中,突變密碼子為表3中所說明之突變密碼子。
在一些實施例中,表1、表2及表3中所說明之Y及X表示非天然核苷酸之非天然鹼基。在一些實施例中,非天然核苷酸之非天然鹼基選自由以下組成之群:2-胺基腺嘌呤-9-基、2-胺基腺嘌呤、2-F-腺嘌呤、2-硫尿嘧啶、2-硫基-胸腺嘧啶、2-硫胞嘧啶、腺嘌呤及鳥嘌呤之2-丙基及烷基衍生物、2-胺基-腺嘌呤、2-胺基-丙基-腺嘌呤、2-胺基吡啶、2-吡啶酮、2'-去氧尿苷、2-胺基-2'-去氧腺苷、3-去氮鳥嘌呤、3-去氮腺嘌呤、4-硫基-尿嘧啶、4-硫基-胸腺嘧啶、尿嘧啶-5-基、次黃嘌呤-9-基(I)、5-甲基-胞嘧啶、5-羥基甲基胞嘧啶、黃嘌呤、次黃嘌呤、5-溴及5-三氟甲基尿嘧啶及胞嘧啶;5-鹵基尿嘧啶、5-鹵基胞嘧啶、5-丙炔基-尿嘧啶、5-丙炔基胞嘧啶、5-尿嘧啶、5-取代、5-鹵基、5-取代嘧啶、5-羥基胞嘧啶、5-溴胞嘧啶、5-溴尿嘧啶、5-氯胞嘧啶、氯化胞嘧啶、環胞嘧啶、胞嘧啶***糖苷、5-氟胞嘧啶、氟嘧啶、氟尿嘧啶、5,6-二氫胞嘧啶、5-碘胞嘧啶、羥脲、碘尿嘧啶、5-硝基胞嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶及5-碘尿嘧啶、腺嘌呤及鳥嘌呤之6-烷基衍生物、6-氮雜嘧啶、6-偶氮-尿嘧啶、6-偶氮胞嘧啶、氮雜胞嘧啶、6-偶氮-胸腺嘧啶、6-硫基-鳥嘌呤、7-甲基鳥嘌呤、7-甲基腺嘌呤、7-去氮鳥嘌呤、7-去氮鳥苷、7-去氮-腺嘌呤、7-去氮-8-氮雜鳥嘌呤、8-氮雜鳥嘌呤、8-氮雜腺嘌呤、8-鹵基、8-胺基、8-巰基、8-硫基烷基及8-羥基取代腺嘌呤及鳥嘌呤;N4-乙基胞嘧啶、N-2取代嘌呤、N-6取代嘌呤、O-6取代嘌呤、彼等增加雙螺旋形成之穩定性者、通用核酸、疏水性核酸、混雜核酸、尺寸擴展核酸、氟化核酸、三環嘧啶、啡噁嗪胞苷([5,4-b][l,4]苯并噁嗪-2(3H)-酮)、啡噻嗪胞苷(1H-嘧啶并[5,4-b][l,4]苯并噻嗪-2(3H)-酮)、G-夾鉗、啡噁嗪胞苷(9-(2-胺基乙氧基)-H-嘧啶并[5,4-b][l,4]苯并噁嗪-2(3H)-酮)、咔唑胞苷(2H-嘧啶并[4,5-b]吲哚-2-酮)、吡啶并吲哚胞苷(H-吡啶并[3',2':4,5]吡咯并[2,3-d]嘧啶-2-酮)、5-氟尿嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-碘尿嘧啶、次黃嘌呤、黃嘌呤、4-乙醯基胞嘧啶、5-(羧基羥基甲基)尿嘧啶、5-羧甲基胺基甲基-2-硫尿苷、5-羧甲基胺基甲基尿嘧啶、二氫尿嘧啶、β-D-半乳糖苷基Q核苷、肌苷、N6-異戊烯基腺嘌呤、1-甲基鳥嘌呤、1-甲基肌苷、2,2-二甲基鳥嘌呤、2-甲基腺嘌呤、2-甲基鳥嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-腺嘌呤、7-甲基鳥嘌呤、5-甲基胺基甲基尿嘧啶、5-甲氧基胺甲基-2-硫尿嘧啶、β-D-甘露糖苷基Q核苷、5'-甲氧基羧基甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-N6-異戊烯基腺嘌呤、尿嘧啶-5氧基乙酸、Y丁氧苷(wybutoxosine)、假尿嘧啶、Q核苷(queosine)、2-硫胞嘧啶、5-甲基-2-硫尿嘧啶、2-硫尿嘧啶、4-硫尿嘧啶、5-甲基尿嘧啶、尿嘧啶-5-乙醛酸甲酯、尿嘧啶-5-乙醛酸、5-甲基-2-硫尿嘧啶、3-(3-胺基-3-N-2-羧丙基)尿嘧啶、(acp3)w及2,6-二胺基嘌呤以及彼等其中嘌呤或嘧啶鹼基被雜環置換者。
在一些情況下,非天然核苷酸選自由以下組成之群(為了清楚說明之目的僅顯示核鹼基部分,省略核糖及磷酸主鏈):。
在一些情況下,非天然核苷酸選自由以下組成之群(為了清楚說明之目的僅顯示核鹼基部分,省略核糖及磷酸主鏈):。
在一些情況下,非天然核苷酸另外包含非天然糖部分。在一些情況下,非天然核苷酸非天然糖部分選自由以下2'位置處之修飾所組成之群中:OH;經取代之低碳數烷基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3
、OCN、Cl、Br、CN、CF3
、OCF3
、SOCH3
、SO2
CH3
、ONO2
、NO2
、N3
、NH2
F;O-烷基、S-烷基、N-烷基;O-烯基、S-烯基、N-烯基;O-炔基、S-炔基、N-炔基;O-烷基-O-烷基、2'-F、2'-OCH3
、2'-O(CH2
)2
OCH3
,其中烷基、烯基及炔基可為經取代或未經取代之C1
-C10
、烷基、C2
-C10
烯基、C2
-C10
炔基、-O[(CH2
)O]m
CH3
、-O(CH2
)n
OCH3
、-O(CH2
)n
NH2
、-O(CH2
)n
CH3
、-O(CH2
)n
-ONH2
及-O(CH2
)n
ON[(CH2
)n
CH3
)]2
,其中n及m為1至約10;及/或在5'位置處之修飾:5'-乙烯基、5'-甲基(R或S);在4'位置處之修飾:4'-S、雜環烷基、雜環烷芳基、胺基烷胺基、聚烷基胺基、經取代矽烷基;RNA裂解基團;報導子基團;嵌入劑;改善寡核苷酸之藥物動力學特性之基團或改善寡核苷酸之藥力學特性基團,以及其任何組合。
在一些情況下,突變反密碼子或突變密碼子另外包含非天然主鏈。在一些情況下,突變反密碼子另外包含非天然主鏈。在一些情況下,突變密碼子另外包含非天然主鏈。在一些情況下,非天然主鏈係選自由以下各者組成之群:硫代磷酸酯、對掌性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、胺基烷基磷酸三酯、C1
-C10
膦酸酯、3'-伸烷基膦酸酯、對掌性膦酸酯、亞膦酸酯、胺基磷酸酯、3'-胺基胺基磷酸酯、胺基烷基胺基磷酸酯、硫羰基胺基磷酸酯、硫羰基烷基膦酸酯、硫羰基烷基磷酸三酯及硼烷磷酸酯。
在一些情況下,非天然核苷酸由聚合酶識別。在一些情況下,聚合酶為DNA聚合酶、RNA聚合酶或反轉錄酶。在一些情況下,聚合酶包含Φ29、B103、GA-1、PZA、Φ15、BS32、M2Y、Nf、G1、Cp-1、PRD1、PZE、SF5、Cp-5、Cp-7、PR4、PR5、PR722、L17、ThermoSequenase®、9°Nm™、Therminator™ DNA聚合酶、Tne、Tma、TfI、Tth、TIi、Stoffel片段、Vent™及Deep Vent™ DNA聚合酶、KOD DNA聚合酶、Tgo、JDF-3、Pfu、Taq、T7 DNA聚合酶、T7 RNA聚合酶、PGB-D、UlTma DNA聚合酶、大腸桿菌DNA聚合酶I、大腸桿菌DNA聚合酶III、古細菌(archaeal) DP1I/DP2 DNA聚合酶II、9°N DNA聚合酶、Taq DNA聚合酶、Phusion® DNA聚合酶、Pfu DNA聚合酶、SP6 RNA聚合酶、RB69 DNA聚合酶、鳥成髓細胞瘤病毒(Avian Myeloblastosis Virus,AMV)反轉錄酶、莫洛尼鼠類白血病病毒(Moloney Murine Leukemia Virus,MMLV) 反轉錄酶、SuperScript® II反轉錄酶及SuperScript® III反轉錄酶。
在一些情況下,聚合酶為DNA聚合酶1-克列諾片段、Vent聚合酶、Phusion® DNA聚合酶、KOD DNA聚合酶、Taq聚合酶、T7 DNA聚合酶、T7 RNA聚合酶、Therminator™ DNA聚合酶、POLB聚合酶、SP6 RNA聚合酶、大腸桿菌DNA聚合酶I、大腸桿菌DNA聚合酶III、鳥成髓細胞瘤病毒(AMV)反轉錄酶、莫洛尼鼠類白血病病毒(MMLV)反轉錄酶、SuperScript® II反轉錄酶或SuperScript® III反轉錄酶。
在一些情況下,在轉錄期間非天然核苷酸藉由聚合酶導入至mRNA中,以產生含有突變密碼子之突變mRNA。在一些情況下,在轉錄期間非天然核苷酸藉由聚合酶導入至mRNA中,以生成突變mRNA。
在一些情況下,在轉錄期間非天然核苷酸藉由聚合酶導入至tRNA中,以生成含有突變反密碼子之突變tRNA。在一些情況下,在轉錄期間非天然核苷酸藉由聚合酶導入至tRNA中,以生成突變tRNA。
在一些情況下,突變tRNA表示非天然胺基酸殘基。在一些情況下,非天然胺基酸殘基為非天然胺基酸,諸如Liu C.C., Schultz, P.G.Annu . Rev . Biochem
. 2010, 79, 413中所描述之非天然胺基酸。
在一些情況下,在轉譯期間利用突變tRNA及突變mRNA生成含有非天然胺基酸之蛋白質。在一些情況下,在無細胞轉譯系統中生成含有非天然胺基酸之蛋白質。在一些情況下,在細胞或半合成生物體(SSO)中合成蛋白質。在一些情況下,半合成生物體包含微生物。在一些情況下,半合成生物體包含細菌。在一些情況下,半合成生物體包含大腸桿菌。核酸
核酸(例如,在本文中亦稱為靶核酸、靶核苷酸序列、所關注之核酸序列或所關注之核酸區域)可來自任何源或組合物,諸如DNA、cDNA、gDNA (基因組DNA)、RNA、siRNA (短抑制性RNA)、RNAi、tRNA或mRNA,且可呈任何形式(例如,線性、環狀、超螺旋、單股、雙股及其類似者)。核酸可包含核苷酸、核苷或多核苷酸。核酸可包含天然及非天然核酸。核酸亦可包含非天然核酸,諸如DNA或RNA類似物(例如,含有鹼基類似物、糖類似物及/或非自然主鏈及類似者)。應理解,術語“核酸”並不係指或表示特定長度之多核苷酸鏈,因此多核苷酸及寡核苷酸亦包括在定義中。例示性天然核苷酸包括但不限於:ATP、UTP、CTP、GTP、ADP、UDP、CDP、GDP、AMP、UMP、CMP、GMP、dATP、dTTP、dCTP、dGTP、dADP、dTDP、dCDP、dGDP、dAMP、dTMP、dCMP及dGMP。例示性天然去氧核糖核苷酸包括:dATP、dTTP、dCTP、dGTP、dADP、dTDP、dCDP、dGDP、dAMP、dTMP、dCMP及dGMP。例示性天然核糖核苷酸包括:ATP、UTP、CTP、GTP、ADP、UDP、CDP、GDP、AMP、UMP、CMP及GMP。對於RNA,尿嘧啶鹼基為尿苷。核酸有時為載體、質體、噬菌體、自主複製序列(autonomously replicating sequence,ARS)、著絲點、人工染色體、酵母人工染色體(例如YAC)或其他能夠複製或被複製的核酸。非天然核酸可為核酸類似物。 非天然核酸
核苷酸類似物或非天然核苷酸包含含有對鹼基、糖或磷酸酯部分之某種修飾的核苷酸。修飾可包含化學修飾。修飾可為,例如3'OH或5'OH基團、主鏈、糖組分或核苷酸鹼基之修飾。修飾可包括添加非天然存在之連接子分子及/或股間或股內交聯。在一個態樣中,經修飾核酸包含3'OH或5'OH基團、主鏈、糖組分或核苷酸鹼基中之一或多者之修飾,及/或非天然存在之連接子分子之添加。在一個態樣中,經修飾主鏈包含除磷酸二酯主鏈以外之主鏈。在一個態樣中,經修飾糖包含除去氧核糖(在經修飾DNA中)以外或除核糖(經修飾RNA)以外之糖。在一個態樣中,修飾鹼基包含除腺嘌呤、鳥嘌呤、胞嘧啶或胸腺嘧啶(在經修飾DNA中)以外之鹼基或除腺嘌呤、鳥嘌呤、胞嘧啶或尿嘧啶(在經修飾RNA中)以外之鹼基。
核酸可包含至少一個經修飾鹼基。對鹼基部分之修飾應包括A、C、G及T/U以及不同嘌呤或嘧啶鹼基之天然及合成修飾。在一些實施例中,修飾係對於腺嘌呤、鳥嘌呤胞嘧啶或胸腺嘧啶(在經修飾DNA中)之經修飾形式或腺嘌呤、鳥嘌呤胞嘧啶或尿嘧啶(經修飾RNA)之經修飾形式的修飾。
非天然核酸之經修飾鹼基包括但不限於:尿嘧啶-5-基、次黃嘌呤-9-基(I)、2-胺基腺嘌呤-9-基、5-甲基胞嘧啶(5-me-C)、5-羥基甲基胞嘧啶、黃嘌呤、次黃嘌呤、2-胺基腺嘌呤、腺嘌呤及鳥嘌呤之6-甲基及其他烷基衍生物、腺嘌呤及鳥嘌呤之2-丙基及其他烷基衍生物、2-硫尿嘧啶、2-硫胸腺嘧啶及2-硫胞嘧啶、5-鹵基尿嘧啶及胞嘧啶、5-丙炔基尿嘧啶及胞嘧啶、6-偶氮尿嘧啶、胞嘧啶及胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-鹵基、8-胺基、8-巰基、8-硫基烷基、8-羥基及其他8-取代腺嘌呤及鳥嘌呤、5-鹵基(尤其為5-溴、5-三氟甲基)及其他5-取代尿嘧啶及胞嘧啶、7-甲基鳥嘌呤及7-甲基腺嘌呤、8-氮雜鳥嘌呤及8-氮雜腺嘌呤、7-去氮鳥嘌呤及7-去氮腺嘌呤及3-去氮鳥嘌呤及3-去氮腺嘌呤。某些非天然核酸,諸如5-取代嘧啶、6-氮雜嘧啶及N-2取代嘌呤、N-6取代嘌呤、O-6取代嘌呤、2-胺基丙基腺嘌呤、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、5-甲基胞嘧啶、彼等增加雙螺旋形成之穩定性者、通用核酸、疏水性核酸、混雜核酸、尺寸擴展核酸、氟化核酸、5-取代嘧啶、6-氮雜嘧啶及N-2、N-6及0-6取代嘌呤,包括2-胺基丙基腺嘌呤、5-丙炔基尿嘧啶及5-丙炔基胞嘧啶。5-甲基胞嘧啶(5-me-C)、5-羥基甲基胞嘧啶、黃嘌呤、次黃嘌呤、2-胺基腺嘌呤、6-甲基、腺嘌呤及鳥嘌呤之其他烷基衍生物、腺嘌呤及鳥嘌呤之2-丙基及其他烷基衍生物、2-硫尿嘧啶、2-硫胸腺嘧啶及2-硫胞嘧啶、5-鹵基尿嘧啶、5-鹵基胞嘧啶、5-丙炔基(-C≡C-CI¼)尿嘧啶、5-丙炔基胞嘧啶、嘧啶核酸之其他炔基衍生物、6-偶氮尿嘧啶、6-偶氮胞嘧啶、6-偶氮胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-鹵基、8-胺基、8-巰基、8-硫基烷基、8-羥基及其他8-取代腺嘌呤及鳥嘌呤、5-鹵基(尤其5-溴、5-三氟甲基)、其他5-取代尿嘧啶及胞嘧啶、7-甲基鳥嘌呤、7-甲基腺嘌呤、2-F-腺嘌呤、2-胺基-腺嘌呤、8-氮雜鳥嘌呤、8-氮雜腺嘌呤、7-去氮鳥嘌呤、7-去氮腺嘌呤、3-去氮鳥嘌呤、3-去氮腺嘌呤、三環嘧啶、啡噁嗪胞苷、([5,4-b][l,4]苯并噁嗪-2(3H)-酮)、啡噻嗪胞苷(1H-嘧啶并[5,4-b][l,4]苯并噻嗪-2(3H)-酮)、G-夾鉗、啡噁嗪胞苷(例如9-(2-胺基乙氧基)-H-嘧啶并[5,4-b][l,4]苯并噁嗪-2(3H)-酮)、咔唑胞苷(2H-嘧啶并[4,5-b]吲哚-2-酮)、吡啶并吲哚胞苷(H-吡啶并[3',2':4,5]吡咯并[2,3-d]嘧啶-2-酮)、其中嘌呤或嘧啶鹼基經其他雜環替換之彼等、7-去氮-腺嘌呤、7-去氮鳥苷、2-胺基吡啶、2-吡啶酮、氮雜胞嘧啶、5-溴胞嘧啶、溴尿嘧啶、5-氯胞嘧啶、氯化胞嘧啶、環胞嘧啶、胞嘧啶***糖苷、5-氟胞嘧啶、氟嘧啶、氟尿嘧啶、5,6-二氫胞嘧啶、5-碘胞嘧啶、羥脲、碘尿嘧啶、5-硝基胞嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶及5-碘尿嘧啶、2-胺基-腺嘌呤、6-硫基-鳥嘌呤、2-硫基-胸腺嘧啶、4-硫基-胸腺嘧啶、5-丙炔基-尿嘧啶、4-硫基-尿嘧啶、N4-乙基胞嘧啶、7-去氮鳥嘌呤、7-去氮-8-氮雜鳥嘌呤、5-羥基胞嘧啶、2'-去氧尿苷、2-胺基-2'-去氧腺苷,及以下中所描述之彼等:美國專利第3,687,808號;第4,845,205號;第4,910,300號;第4,948,882號;第5,093,232號;第5,130,302號;第5,134,066號;第5,175,273號;第5,367,066號;第5,432,272號;第5,457,187號;第5,459,255號;第5,484,908號;第5,502,177號;第5,525,711號;第5,552,540號;第5,587,469號;第5,594,121號;第5,596,091號;第5,614,617號;第5,645,985號;第5,681,941號;第5,750,692號;第5,763,588號;第5,830,653號及第6,005,096號;WO 99/62923;Kandimalla等人(2001) Bioorg. Med. Chem. 9:807-813;The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., 編, John Wiley & Sons, 1990, 858- 859;Englisch等人,Angewandte Chemie, International版, 1991, 30, 613;及Sanghvi, Y.S., 第15章, Antisense Research and Applications, Crooke, S.T.及Lebleu, B., 編., CRC Press, 1993, 273-288。其他鹼基修飾可以參見,例如,美國專利第3,687,808號;Englisch等人,Angewandte Chemie, International 版, 1991, 30, 613;及Sanghvi, Y. S., 第15章, Antisense Research and Applications, 第289-302頁, Crooke, S. T.及Lebleu, B. 編, CRC Press, 1993。
在此項技術中可獲得包含各種雜環鹼基及各種糖部分(及糖類似物)之非天然核酸,且核酸可包括除天然存在之核酸之主要五種鹼基組分以外的一種或若干種雜環鹼基。舉例而言,雜環鹼基可包括:尿嘧啶-5-基、胞嘧啶-5-基、腺嘌呤-7-基、腺嘌呤-8-基、鳥嘌呤-7-基、鳥嘌呤-8-基、4-胺基吡咯并[2,3-d]嘧啶-5-基、2-胺基-4-側氧基吡咯并[2,3-d]嘧啶-5-基、2-胺基-4-側氧基吡咯并[2,3-d]嘧啶-3-基,其中嘌呤係經由9位置、嘧啶經由1位置、吡咯并嘧啶經由7位置且吡唑并嘧啶經由1位置附接至核酸之糖部分。
核苷酸類似物亦可在磷酸酯部分處經修飾。經修飾磷酸酯部分包括但不限於可經修飾以使得兩種核苷酸之間的鍵含有以下各者的磷酸酯部分:硫代磷酸酯;對掌性硫代磷酸酯;二硫代磷酸酯;磷酸三酯;胺基烷基磷酸三酯;甲基及其他烷基膦酸酯,包括3'-伸烷基膦酸酯及對掌性膦酸酯;亞膦酸酯;胺基磷酸酯,包括3'-胺基胺基磷酸酯及胺基烷基胺基磷酸酯;硫羰基胺基磷酸酯;硫羰基烷基膦酸酯;硫羰基烷基磷酸三酯及硼烷磷酸酯。應理解,在兩種核苷酸之間的此等磷酸酯或經修飾磷酸酯鍵可經由3'-5'鍵或2'-5'鍵,且該鍵可含有反轉極性,諸如3'-5'至5'-3'或2'-5'至5'-2'。亦包括各種鹽、混合鹽及游離酸形式。大量美國專利教示如何製備及使用含有經修飾磷酸酯之核苷酸,且該等美國專利包括但不限於:3,687,808、4,469,863、4,476,301、5,023,243、5,177,196、5,188,897、5,264,423、5,276,019、5,278,302、5,286,717、5,321,131、5,399,676、5,405,939、5,453,496、5,455,233、5,466,677、5,476,925、5,519,126、5,536,821、5,541,306、5,550,111、5,563,253、5,571,799、5,587,361及5,625,050,其中各者均以引用之方式併入本文中。
非天然核酸可包括2',3'-二去氧基-2',3'-二去氫-核苷(PCT/US2002/006460)、5'-取代DNA及RNA衍生物(PCT/US2011/033961;Saha等人,J. Org Chem., 1995, 60, 788-789;Wang等人,Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890;及Mikhailov等人,Nucleosides & Nucleotides, 1991, 10(1-3), 339-343;Leonid等人,1995, 14(3-5), 901-905;及Eppacher等人,Helvetica Chimica Acta, 2004, 87, 3004-3020;PCT/JP2000/004720;PCT/JP2003/002342;PCT/JP2004/013216;PCT/JP2005/020435;PCT/JP2006/315479;PCT/JP2006/324484;PCT/JP2009/056718;PCT/JP2010/067560)或製成為具有經修飾之鹼基之單磷酸酯的5'-取代單體(Wang等人,Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337)。
非天然核酸可包括在糖環之5'位置及2'位置處之修飾(PCT/US94/02993),諸如5'-CH2
取代2'-O-保護核苷(Wu等人,Helvetica Chimica Acta, 2000, 83, 1127-1143及Wu等人,Bioconjugate Chem. 1999, 10, 921-924)。非天然核酸可包括醯胺連接之核苷二聚體,其已經製備用於導入寡核苷酸中,其中在該二聚體(5'至3')中之3'連接核苷包含2'-OCH3
及5'-(S)-CH3
(Mesmaeker等人,Synlett, 1997, 1287-1290)。非天然核酸可包括2'-取代5'-CH2
(或O)修飾核苷(PCT/US92/01020)。非天然核酸可包括5'-亞甲基膦酸酯DNA及RNA單體及二聚體(Bohringer等人,Tet. Lett., 1993, 34, 2723-2726;Collingwood等人,Synlett, 1995, 7, 703-705;及Hutter等人,Helvetica Chimica Acta, 2002, 85, 2777-2806)。非天然核酸可包括具有2'-取代之5'-膦酸酯單體(US 2006/0074035)及其他經修飾5'-膦酸酯單體(WO 97/35869)。非天然核酸可包括5'-修飾亞甲基膦酸酯單體(EP614907及EP629633)。非天然核酸可包括在5'及/或6'位置處包含羥基之5'或6'-膦酸酯核糖核苷之類似物(Chen等人,Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786;Jung等人,Bioorg. Med. Chem., 2000, 8, 2501-2509;Gallier等人,Eur. J. Org. Chem., 2007, 925-933及Hampton等人,J. Med. Chem., 1976, 19(8), 1029-1033)。非天然核酸可包括具有5'-磷酸酯基團之5'-膦酸酯去氧核苷單體及二聚體(Nawrot等人,Oligonucleotides, 2006, 16(1), 68-82)。非天然核酸可包括核苷,其具有6'-膦酸酯基團,其中5'或/及6'位置未經取代或經硫基-第三丁基(SC(CH3
)3
)(及其類似物)取代;亞甲基胺基(CH2
NH2
) (及其類似物)或氰基(CN)(及其類似物)(Fairhurst等人,Synlett, 2001, 4, 467-472;Kappler等人,J. Med. Chem., 1986, 29, 1030-1038及J. Med. Chem., 1982, 25, 1179-1184;Vrudhula等人,J. Med. Chem., 1987, 30, 888-894;Hampton 等人,J. Med. Chem., 1976, 19, 1371-1377;Geze等人,J. Am. Chem. Soc, 1983, 105(26), 7638-7640;及Hampton等人,J. Am. Chem. Soc, 1973, 95(13), 4404-4414)。
非天然核酸亦可包括糖部分之修飾。本發明之核酸可視情況含有一或多種其中糖基團已經修飾的核苷。此類糖修飾核苷可賦予提高的核酸酶穩定性、提高的結合親和力或某些其他有利生物特性。在某些實施例中,核酸包含經化學修飾之核呋喃糖環部分。經化學修飾之核呋喃糖環之實例包括但不限於:添加取代基(包括5'及/或2'取代基);橋接兩個環原子以形成雙環核酸(BNA);由S、N(R)或C(R1
)(R2
)(R=H、C1
-C12
烷基或保護基)替換核糖基環氧原子;及其組合。經化學修飾之糖之實例可參見WO 2008/101157、US 2005/0130923及WO 2007/134181。
經修飾核酸可包含經修飾之糖或糖類似物。因此,除核糖及去氧核糖以外,糖部分可為戊醣、去氧戊醣、己醣、去氧已醣、葡萄糖、***糖、木糖、來蘇糖或糖「類似物」環戊基。糖可呈哌喃糖基或呋喃糖基形式。糖部分可為核糖、去氧核糖、***糖或2'-O-烷基核糖之呋喃糖苷,且該糖可以[α]或[β]變旋異構組態經附接至相應的雜環鹼基。糖修飾包括但不限於2'-烷氧基-RNA類似物、2'-胺基-RNA類似物、2'-氟-DNA及2'-烷氧基-或胺基-RNA/DNA嵌合體。舉例而言,糖修飾可包括2'-O-甲基-尿苷及2'-O-甲基-胞苷。糖修飾包括2'-O-烷基-取代去氧核糖核苷及2'-O-乙二醇類核糖核苷。此等糖或糖類似物及對應的「核苷」(其中此類糖或類似物附接至雜環鹼基(核酸鹼基))之製備係已知的。糖修飾亦可經其他修飾且與其他修飾組合。
對糖部分之修飾包括核糖及去氧核糖之天然修飾以及非天然修飾。糖修飾包括但不限於在2'位置處之以下修飾:OH;F;O-、S-或N-烷基;O-、S-或N-烯基;O-、S-或N-炔基;或O-烷基-O-烷基,其中烷基、烯基及炔基可為經取代或未經取代之C1
至C10
烷基或C2
至C10
烯基及炔基。2'糖修飾亦包括但不限於-O[(CH2
)n
O]m
CH3
、-O(CH2
)n
OCH3
、-O(CH2
)n
NH2
、-O(CH2
)n
CH3
、-O(CH2
)n
-ONH2
及-O(CH2
)n
ON[(CH2
)n CH3
)J2
,其中n及m為1至約10。
其他在2'位置處之修飾包括但不限於:C1
至C10
低碳數烷基、經取代低碳數烷基、烷芳基、芳烷基、O-烷芳基、O-芳烷基、SH、SCH3
、OCN、Cl、Br、CN、CF3
、OCF3
、SOCH3
、SO2
CH3
、ONO2
、NO2
、N3
、NH2
、雜環烷基、雜環烷芳基、胺基烷胺基、聚烷基胺基、經取代矽烷基、RNA裂解基團、報導子基團、嵌入劑、改善寡核苷酸之藥物動力學特性之基團或改善寡核苷酸之藥力學特性基團及具有類似特性之其他取代基。也可以在糖上之其他位置處作出類似修飾,尤其為3'末端核苷酸上或2'-5'連接寡核苷酸中之糖之3'位置及5'末端核苷酸之5'位置。經修飾糖應亦包括含有在橋環氧處之修飾(諸如CH2
及S)的經修飾糖。核苷酸糖類似物亦可具有糖模擬物,諸如環丁基部分,來替代呋喃戊醣基糖。有大量教示此類經修飾之糖結構之製備且詳述及描述一系列鹼基修飾的美國專利,諸如美國專利第4,981,957號;第5,118,800號;第5,319,080號;第5,359,044號;第5,393,878號;第5,446,137號;第5,466,786號;第5,514,785號;第5,519,134號;第5,567,811號;第5,576,427號;第5,591,722號;第5,597,909號;第5,610,300號;第5,627,053號;第5,639,873號;第5,646,265號;第5,658,873號;第5,670,633號;第4,845,205號;第5,130,302號;第5,134,066號;第5,175,273號;第5,367,066號;第5,432,272號;第5,457,187號;第5,459,255號;第5,484,908號;第5,502,177號;第5,525,711號;第5,552,540號;第5,587,469號;第5,594,121號;第5,596,091號;第5,614,617號;第5,681,941號;及第5,700,920號,其中各者均以全文引用的方式併入本文中。此等專利中之每一者均以引用之方式併入本文中。
具有經修飾糖部分之核酸之實例包括但不限於包含5'-乙烯基、5'-甲基(R或S)、4'-S、2'-F、2'-OCH3
及2'-O(CH2
)2
OCH3
取代基之核酸。在2'位置處之取代基亦可選自:烯丙基、胺基、疊氮基、硫基、O-烯丙基、O-C1
-C1o
烷基、OCF3
、O(CH2
)2
SCH3
、O(CH2
)2
-O-N(Rm
)(Rn
)及O-CH2
-C(=O)-N(Rm
)(Rn
),其中各Rm
及Rn
獨立地為H或經取代或未經取代之C1
-C10
烷基。
在某些實施例中,本發明之核酸包括一或多種雙環核酸。在某些此類實施例中,雙環核酸包含在4'核糖基環原子與2'核糖基環原子之間的橋。在某些實施例中,本文中所提供之核酸包括一或多種雙環核酸,其中橋包含4'至2'雙環核酸。此類4'至2'雙環核酸之實例包括但不限於下式中之一者:4'-(CH2
)-O-2' (LNA);4'-(CH2
)-S-2';4'-(CH2
)2
-O-2' (ENA);4'-CH(CH3
)-O-2'及4'-CH(CH2
OCH3
)-0-2',及其類似物(參見美國專利7,399,845,發佈於2008年7月15日);4'-C(CH3
)(CH3
)-0-2'及其類似物(參見WO2009/006478;WO2008/150729;US2004/0171570;美國專利7,427,672;Chattopadhyaya等人,J. Org. Chem.,2 09, 74, 118-134;及WO 2008/154401,發表於2008年12月8)。亦參見,例如,Singh等人,Chem. Commun., 1998, 4, 455-456;Koshkin等人,Tetrahedron, 1998, 54, 3607-3630;Wahlestedt等人,Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 5633-5638;Kumar等人,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh等人,J. Org. Chem., 1998, 63, 10035-10039;Srivastava等人,J. Am. Chem. Soc, 129(26) 8362-8379 (Jul. 4, 2007);Elayadi等人,Curr. Opinion Invens. Drugs, 2001, 2, 558-561;Braasch等人,Chem. Biol, 2001, 8, 1-7;Oram等人,Curr. Opinion Mol Ther., 2001, 3, 239-243;美國專利第7,053,207號、第6,268,490號、第6,770,748號、第6,794,499號、第7,034,133號、第6,525,191號、第6,670,461號及第7,399,845號;國際申請案WO 2004/106356、WO 1994/14226、WO 2005/021570及WO 2007/134181;美國專利編號US2004/0171570、US2007/0287831及US2008/0039618;美國專利第12/129,154、號、第60/989,574號、第61/026,995號、第61/026,998號、第61/056,564號、第61/086,231號、第61/097,787號及第61/099,844號;及PCT國際申請案編號PCT/US2008/064591、PCT US2008/066154及PCT US2008/068922、PCT/DK98/00393;及美國專利第4,849,513號、第5,015,733號、第5,118,800號及第5,118,802號。
在某些實施例中,核酸可包含經連接核酸。核酸可使用任何核酸間鍵連接在一起。兩個主要類別之核酸間連接基團係由存在或不存在磷原子來界定。代表性含磷核酸間鍵包括但不限於:磷酸二酯、磷酸三酯、甲基膦酸酯、胺基磷酸酯及硫代磷酸酯(P=S)。代表性不含磷核酸間連接基團包括但不限於:亞甲基甲基亞胺基(-CH2
-N(CH3
)-O-CH2
-)、硫代二酯(-O-C(O)-S-)、硫羰基胺基甲酸酯(-O-C(O)(NH)-S-);矽氧烷(-O-Si(H)2
-O-);及N,N*-二甲基肼(-CH2
-N(CH3
)-N(CH3
)-)。在某些實施例中,具有對掌性原子之核酸間鍵可製備為外消旋混合物、獨立的對映異構體(例如磷酸烷基酯及硫代磷酸酯)。非天然核酸可含有單個修飾。非天然核酸可含有在其中之一個部分中或在不同部分之間的多個修飾。
對核酸之主鏈磷酸酯修飾包括但不限於:甲基膦酸酯、硫代磷酸酯、胺基磷酸酯(橋接或非橋接)、磷酸三酯、二硫代磷酸酯、二硫代磷酸酯(phosphodithioate)及硼烷磷酸酯,且可以任何組合形式使用。亦可使用其他非磷酸酯鍵。
在一些實施例中,主鏈修飾(例如,甲基膦酸酯、硫代磷酸酯、磷醯胺酸及二硫代磷酸酯核苷酸間鍵)可對經修飾核酸賦予免疫調節活性及/或增強其活體內穩定性。
磷衍生物(或經修飾磷酸酯基團)可附接至糖或糖類似物部分且可為單磷酸、二磷酸酯、三磷酸酯、膦酸烷基酯、硫代磷酸酯、二硫代磷酸酯、胺基磷酸酯或類似者。含有經修飾之磷酸酯鍵或非磷酸酯鍵之例示性多核苷酸可參見於:Peyrottes等人 (1996) Nucleic Acids Res. 24: 1841-1848;Chaturvedi等人 (1996) Nucleic Acids Res. 24:2318-2323;及Schultz等人 (1996) Nucleic Acids Res. 24:2966-2973;Matteucci(1997)「Oligonucleotide Analogs: an Overview」,於Oligonucleotides as Therapeutic Agents, (DJ. Chadwick及G. Cardew編) John Wiley and Sons, New York, NY中;(Zon (1993)「Oligonucleoside Phosphorothioates」,於Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, 編) Humana Press, 第165-190頁中);(Miller等人 (1971) JACS 93:6657-6665);(Jager等人 (1988) Biochem. 27:7247-7246);(Nelson等人 (1997) JOC 62:7278-7287)(美國專利第5,453,496號);Micklefield, J. 2001, Current Medicinal Chemistry 8: 1157-1179。
主鏈修飾可包含用諸如陰離子、中性或陽離子基團之替代部分替換磷酸二酯鍵。此類修飾之實例包括:陰離子核苷間鍵;N3'至P5'胺基磷酸酯修飾;硼烷磷酸酯DNA;促寡核苷酸;中性核苷間鍵,諸如甲基膦酸酯;醯胺連接DNA;亞甲基(甲基亞胺基)鍵;甲縮醛及硫基甲縮醛鍵;含有磺醯基基團之主鏈;N-嗎啉基寡核苷酸;肽核酸(PNA);及帶正電的去氧核糖核酸胍(DNG)寡核苷酸(Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179)。經修飾核酸可包含嵌合或混合主鏈,其包含一或多個修飾,例如磷酸酯鍵之組合,諸如磷酸二酯鍵與硫代磷酸酯鍵之組合。
用於磷酸酯之替代物可為例如短鏈烷基或環烷基核苷間鍵、混合雜原子及烷基或環烷基核苷間鍵或一或多個短鏈雜原子或雜環核苷間鍵。此等主鏈包括具有N-嗎啉基鍵(一部分由核苷之糖部分形成的主鏈);矽氧烷主鏈;硫化物、亞碸及碸主鏈;甲醯基及硫甲醯基主鏈;亞甲基甲醯基及硫甲醯基主鏈;含有烯烴之主鏈;胺基磺酸酯主鏈;亞甲基亞胺基及亞甲基肼基主鏈;磺酸酯及磺醯胺主鏈;醯胺主鏈;及具有混合N、O、S及CH2
組成部分之其他主鏈。大量美國專利揭示如何製備及使用此等類型之磷酸酯替代物,該等美國專利包括但不限於美國專利第5,034,506號;第5,166,315號;第5,185,444號;第5,214,134號;第5,216,141號;第5,235,033號;第5,264,562號;第5,264,564號;第5,405,938號;第5,434,257號;第5,466,677號;第5,470,967號;第5,489,677號;第5,541,307號;第5,561,225號;第5,596,086號;第5,602,240號;第5,610,289號;第5,602,240號;第5,608,046號;第5,610,289號;第5,618,704號;第5,623,070號;第5,663,312號;第5,633,360號;第5,677,437號及第5,677,439號,其中各者均以引用之方式併入本文中。亦應理解,在核苷酸替代物中,核苷酸之糖及磷酸酯部分兩者均可經,例如,醯胺類鍵(胺基乙基甘胺酸) (PNA)替換。美國專利5,539,082;5,714,331;及5,719,262教示如何製備及使用PNA分子,其中各者均以引用之方式併入本文中。(以參見Nielsen等人,Science, 1991, 254, 1497-1500)。結合物可化學連接至核苷酸或核苷酸類似物。此類結合物包括但不限於脂質部分,諸如,膽固醇部分(Letsinger等人,Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556)、膽酸(Manoharan等人,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060)、硫醚,例如己基-S-三苯硫醇 (Manoharan等人,Ann. KY. Acad. Sci., 1992, 660, 306-309;Manoharan等人,Bioorg. Med. Chem. Let., 1993, 3, 2765-2770)、硫代膽固醇(Oberhauser等人,Nucl. Acids Res., 1992, 20, 533-538)、脂族鏈,例如十二烷二醇十一烷基殘基(Saison-Behmoaras等人,EM5OJ, 1991, 10, 1111-1118;Kabanov等人,FEBS Lett., 1990, 259, 327-330;Svinarchuk等人,Biochimie, 1993, 75, 49-54)、磷脂,例如雙十六基-rac-甘油或l,2-雙-O-十六基-rac-甘油-S-H-膦酸三乙基銨(Manoharan等人,Tetrahedron Lett., 1995, 36, 3651-3654;Shea等人,Nucl. Acids Res., 1990, 18, 3777-3783)、多元胺或聚乙二醇鏈(Manoharan等人,Nucleosides & Nucleotides, 1995, 14, 969-973)或金剛烷乙酸(Manoharan等人,Tetrahedron Lett., 1995, 36, 3651-3654)、十六烷基部分(Mishra等人,Biochem. Biophys. Acta, 1995, 1264, 229-237)或十八烷基胺或己基胺基-羰基-氧基膽固醇部分(Crooke等人,J. Pharmacol. Exp. Ther., 1996, 277, 923-937)。大量美國專利教示此類結合物之製備,該等美國專利包括但不限於美國專利第4,828,979號;第4,948,882號;第5,218,105號;第5,525,465號;第5,541,313號;第5,545,730號;第5,552,538號;第5,578,717號;第5,580,731號;第5,580,731號;第5,591,584號;第5,109,124號;第5,118,802號;第5,138,045號;第5,414,077號;第5,486,603號;第5,512,439號;第5,578,718號;第5,608,046號;第4,587,044號;第4,605,735號;第4,667,025號;第4,762,779號;第4,789,737號;第4,824,941號;第4,835,263號;第4,876,335號;第4,904,582號;第4,958,013號;第5,082,830號;第5,112,963號;第5,214,136號;第5,082,830號;第5,112,963號;第5,214,136號;第5,245,022號;第5,254,469號;第5,258,506號;第5,262,536號;第5,272,250號;第5,292,873號;第5,317,098號;第5,371,241號;第5,391,723號;第5,416,203號;第5,451,463號;第5,510,475號;第5,512,667號;第5,514,785號;第5,565,552號;第5,567,810號;第5,574,142號;第5,585,481號;第5,587,371號;第5,595,726號;第5,597,696號;第5,599,923號;第5,599,928號;及第5,688,941號,其中各者均以引用之方式併入本文中。聚合酶
一種特別有用的聚合酶功能係使用現存核酸作為模板催化核酸股之聚合。其他有用的功能在本文中別處描述。適用的聚合酶之實例包括DNA聚合酶及RNA聚合酶。
在例如需要導入非天然核酸之多種情形中,包括擴增、定序、標記、偵測、選殖及諸多其他,將特別需要改善聚合酶非天然核酸之特異性、持續合成能力或其他特徵之能力。本發明提供針對非天然核酸之具有經修飾特性的聚合酶、製備此類聚合酶之方法、使用此類聚合酶之方法及將在下文之完整審查之後變得顯而易見的許多其他特徵。
在一些情況下,本文所揭示之內容包括,例如在DNA擴增期間將非天然核酸導入生長模板複本中的聚合酶。在一些實施例中,聚合酶可經修飾以使得聚合酶之活性位點經修飾以減小非天然核酸向活性位點中之立體進入抑制。在一些實施例中,聚合酶可經修飾以藉由非天然核酸之一或多種非天然特徵提供補充。因此,本發明包括含異源或重組聚合酶之組合物及其使用方法。
可使用與蛋白質工程改造相關之方法修飾聚合酶。舉例而言,可依據晶體結構進行分子模型化來識別可進行突變以修飾標靶活性之聚合酶位置。被識別為用於置換之標靶之殘基可由使用能量最小化模式、同源性模式及/或保守胺基酸替代所選擇之殘基來置換,諸如在Bordo等人,J Mol Biol 217: 721-729 (1991)及Hayes等人 Proc Natl Acad Sci, USA 99: 15926- 15931 (2002)中所描述。
本文中闡述之方法或組合物可採用多種聚合酶中之任一種,包括例如自生物系統分離之蛋白質類酵素及其功能變體。除非另外指示,否則提及特定聚合酶(諸如下文所例示之聚合酶)應理解為包括其功能變體。在一些實施例中,聚合酶為野生型聚合酶。在一些實施例中,聚合酶為經修飾或突變之聚合酶。
亦可使用具有用於促進非天然核酸進入活性位點區域及用於協調活性位點區域中之非天然核苷酸的特徵的聚合酶。在一些實施例中,經修飾聚合酶具有經修飾之核苷酸結合位點。
在一些實施例中,經修飾聚合酶具有針對非天然核酸之特異性,其為野生型聚合酶對於非天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾或野生型聚合酶具有針對包含經修飾之糖之非天然核酸之特異性,其為野生型聚合酶對於不具有經修飾之糖之天然核酸及/或非天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾或野生型聚合酶具有針對包含經修飾鹼基之非天然核酸之特異性,其為野生型聚合酶對於不具有經修飾鹼基之天然核酸及/或非天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾或野生型聚合酶具有針對包含三磷酸酯之非天然核酸之特異性,其為野生型聚合酶對於包含三磷酸酯之核酸及/或不具有三磷酸酯之非天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。舉例而言,經修飾或野生型聚合酶可具有針對包含三磷酸酯之非天然核酸之特異性,其為野生型聚合酶對於具有二磷酸酯或單磷酸酯或無磷酸酯或其組合之非天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。
在一些實施例中,經修飾或野生型聚合酶針對非天然核酸具有寬鬆的特異性。在一些實施例中,經修飾或野生型聚合酶具有針對非天然核酸之特異性及針對天然核酸之特異性,其為野生型聚合酶對於天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾或野生型聚合酶具有針對包含經修飾之糖之非天然核酸之特異性及針對天然核酸之特異性,其為野生型聚合酶對於天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾或野生型聚合酶具有針對包含經修飾鹼基之非天然核酸之特異性及針對天然核酸之特異性,其為野生型聚合酶對於天然核酸之特異性的至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。
缺乏核酸外切酶活性可為野生型特徵或由變體聚合酶或工程改造聚合酶賦予之特徵。舉例而言,exo minus克列諾片段為缺乏3'至5'校正核酸外切酶活性之克列諾片段之突變版本。
本發明方法可用於擴展缺乏固有3'至5'核酸外切酶校正活性或3'至5'核酸外切酶校正活性已(例如經由突變)停用的任何DNA聚合酶的受質範圍。DNA聚合酶之實例包括polA、polB (參見,例如Parrel及Loeb, Nature Struc Biol 2001)、polC、polD、polY、polX及反轉錄酶(RT),但較佳為前進型高保真聚合酶(PCT/GB2004/004643)。在一些實施例中,經修飾或野生型聚合酶實質上缺乏3'至5'校正核酸外切酶活性。在一些實施例中,經修飾或野生型聚合酶實質上缺乏對於非天然核酸之3'至5'校正核酸外切酶活性。在一些實施例中,經修飾或野生型聚合酶具有3'至5'校正核酸外切酶活性。在一些實施例中,經修飾或野生型聚合酶具有針對天然核酸之3'至5'校正核酸外切酶活性且實質上缺乏針對非天然核酸之3'至5'校正核酸外切酶活性。
在一些實施例中,經修飾聚合酶具有3'至5'校正核酸外切酶活性,其為野生型聚合酶之校正核酸外切酶活性的至少約60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾聚合酶具有針對非天然核酸之3'至5'校正核酸外切酶活性,其為野生型聚合酶對於天然核酸之校正核酸外切酶活性的至少約60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾聚合酶具有針對非天然核酸之3'至5'校正核酸外切酶活性及針對天然核酸之3'至5'校正核酸外切酶活性,其為野生型聚合酶對於天然核酸之校正核酸外切酶活性的至少約60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些實施例中,經修飾聚合酶具有針對天然核酸之3'至5'校正核酸外切酶活性,其為野生型聚合酶對於天然核酸之校正核酸外切酶活性的至少約60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。
在一相關態樣中,本發明提供以下各者之方法:製備經修飾聚合酶,其包括在結構上模型化親本聚合酶(例如DNA聚合酶);識別一或多種複合穩定性或影響複合穩定性之核苷酸相互作用特徵、或在活性位點中之核苷酸進入或結合、或針對在活性位點處的核苷酸類似物之補充特徵;及使親本聚合酶突變以包括或移除此等特徵。舉例而言,聚合酶可經突變以改善非天然核苷酸至活性位點之立體進入或改善在非天然核苷酸與聚合酶之間的電荷-電荷或疏水相互作用。該等方法亦包括確定所得經修飾聚合酶是否顯示與親本聚合酶相比,核苷酸或非天然核苷酸向生長核酸複製物中之導入增加。
聚合酶可根據其自核酸之解離速率來表徵。在一些實施例中,對於一或多種天然及非天然核酸,聚合酶具有相對較低的解離速率。在一些實施例中,對於一或多種天然及非天然核酸,聚合酶具有相對較高的解離速率。解離速率為可經調節以在本文中所闡述的方法中調節反應速率的聚合酶活性。
聚合酶可根據其在與特定天然及/或非天然核酸或天然及/或非天然核酸之集合一起使用時的保真度來表徵。保真度通常係指在製備核酸模板之複本時聚合酶將正確核酸導入至生長核酸鏈中之精確度。DNA聚合酶保真度可以當存在天然及非天然核酸(例如以相同濃度存在)時為在聚合酶-股-模板核酸二元複合物中之相同位點處競爭股合成而導入之正確與不正確天然及非天然核酸之比率來度量。DNA聚合酶保真度可計算為(kcat
/Km
)(針對天然及非天然核酸)與(kcat
/Km
) (針對不正確天然及非天然核酸)之比率;其中kcat
及Km
為在穩態酶動力學中之Michaelis-Menten參數(Fersht, A. R. (1985) Enzyme Structure and Mechanism, 第2版, 第350頁, W. H. Freeman & Co., New York.,以引用之方式併入本文中)。在一些實施例中,在具有或不具有校正活性之情況下,聚合酶具有至少約100、1000、10,000、100,000或1×106
之保真度值。
來自自然來源的聚合酶或其變體可使用偵測具有特定結構的非天然核酸之導入之分析來篩檢。在一個實例中,聚合酶可針對導入非天然核酸或UBP (例如d5SICSTP、dNaMTP或d5SICSTP-dNaMTP UBP)之能力來篩檢。可使用與野生型聚合酶相比,顯示針對非天然核酸之經修飾的特性的聚合酶(例如異源聚合酶)。舉例而言,經修飾特性可為例如在非天然核酸(或天然存在的核苷酸)存在下之Km
、kcat
、Vmax
、聚合酶持續合成能力;在非天然核酸存在下之藉由聚合酶之平均模板讀取-長度;聚合酶對於非天然核酸之特異性;非天然核酸之結合速率、產物(焦磷酸酯、三磷酸酯等)釋放速率、分支化速率或其任何組合。在一個實施例中,經修飾特性為針對非天然核酸之減小的Km
及/或針對非天然核酸之增加的kcat
/Km
或Vmax
/Km
。類似地,與野生型聚合酶相比,該聚合酶視情況具有增加的非天然核酸結合速率、增加的產物釋放速率及/或降低的分支化速率。
同時,聚合酶可將天然核酸(例如A、C、G及T)導入至生長核酸複本中。舉例而言,聚合酶視情況顯示針對天然核酸之特異活性,其為對應的野生型聚合酶之至少約5%高(例如5%、10%、25%、50%、75%、100%或更高);及在天然核酸存在下模板之持續合成能力,其為在天然核酸存在下野生型聚合酶之至少5%高(例如5%、10%、25%、50%、75%、100%或更高)。視情況,聚合酶顯示kcat
/Km
或Vmax
/Km
(針對天然存在核苷酸),其為野生型聚合酶之至少約5%高(例如5%、10%、25%、50%、75%、100%或更高)。
本文中所用之可具有導入具有特定結構之非天然核酸之能力的聚合酶亦可使用定向進化方法產生。核酸合成分析可用於篩檢具有對於多種非天然核酸中之任一者之特異性的聚合酶變體。舉例而言,聚合酶變體可針對向核酸中導入非天然核酸或UBP (例如dTPT3、dNaM類似物或dTPT3-dNaM UBP)之能力來篩檢。在一些實施例中,此類分析為活體外分析,例如使用重組聚合酶變體。此類定向進此類化技術可用於篩檢任何適合的聚合酶之變體對於本文中所闡述的非天然核酸中之任一者之活性。
所描述之組合物之經修飾聚合酶可視情況為經修飾及/或重組Φ29型DNA聚合酶。視情況,聚合酶可為經修飾及/或重組Φ29、B103、GA-1、PZA、Φ15、BS32、M2Y、Nf、G1、Cp-1、PRD1、PZE 、SF5、Cp-5、Cp-7、PR4、PR5、PR722或L17聚合酶。
通常適用於本發明之核酸聚合酶包括DNA聚合酶、RNA聚合酶、反轉錄酶及其突變形式或變化形式。DNA聚合酶及其特性詳細描述在(尤其) DNA Replication第2版, Kornberg及Baker, W. H. Freeman, New York, N. Y. (1991)中。已知的適用於本發明之習知DNA聚合酶包括但不限於:強烈火球菌(Pyrococcus furiosus,Pfu) DNA聚合酶(Lundberg等人,1991, Gene, 108: 1, Stratagene);烏茲火球菌(Pyrococcus woesei,Pwo) DNA聚合酶(Hinnisdaels等人,1996, Biotechniques, 20:186-8, Boehringer Mannheim);嗜熱棲熱菌(Thermus thermophilus,Tth) DNA聚合酶(Myers及Gelfand 1991, Biochemistry 30:7661);嗜熱脂肪芽孢桿菌(Bacillus stearothermophilus) DNA聚合酶(Stenesh及McGowan, 1977, Biochim Biophys Acta 475:32);海濱嗜熱球菌(Thermococcus litoralis,TIi) DNA聚合酶(亦稱為Vent™ DNA聚合酶, Cariello等人,1991, Polynucleotides Res, 19: 4193, New England Biolabs);9°Nm™ DNA聚合酶(New England Biolabs)、Stoffel片段;Thermo Sequenase®
(Amersham Pharmacia Biotech UK);Therminator™ (New England Biolabs);海棲熱孢菌(Thermotoga maritima,Tma) DNA聚合酶(Diaz及Sabino, 1998 Braz J Med. Res, 31 :1239);水生棲熱菌(Thermus aquaticus,Taq) DNA聚合酶(Chien等人,1976, J. Bacteoriol, 127: 1550);DNA聚合酶;鹿兒島火球菌(Pyrococcus kodakaraensis) KOD DNA聚合酶(Takagi等人,1997, Appl. Environ. Microbiol. 63:4504);JDF-3 DNA聚合酶(來自熱球菌屬JDF-3,專利申請案WO 0132887);GB-D火球菌(PGB-D) DNA聚合酶(亦稱為Deep Vent™ DNA聚合酶,Juncosa-Ginesta等人,1994, Biotechniques, 16:820, New England Biolabs);UlTma DNA聚合酶(來自嗜熱海棲熱孢菌(thermophile Thermotoga maritima);Diaz及Sabino, 1998 Braz J. Med. Res, 31 :1239; PE Applied Biosystems);Tgo DNA聚合酶(來自柳珊瑚熱球菌(thermococcus gorgonarius),Roche Molecular Biochemicals);大腸桿菌DNA聚合酶I (Lecomte及Doubleday, 1983, Polynucleotides Res. 11 :7505);T7 DNA聚合酶 (Nordstrom等人,1981, J Biol. Chem. 256:3112);及古細菌DP1I/DP2 DNA聚合酶II (Cann等人,1998, Proc. Natl. Acad. Sci. USA 95:14250)。涵蓋嗜溫性聚合酶與嗜熱性聚合酶兩者。嗜熱DNA聚合酶包括但不限於:ThermoSequenase®
、9°Nm™、Therminator™、Taq、Tne、Tma、Pfu、TfI、Tth、TIi、Stoffel片段、Vent™及Deep Vent™ DNA聚合酶、KOD DNA聚合酶、Tgo、JDF-3及其突變體、變體及衍生物。亦涵蓋作為3'核酸外切酶缺失型突變體之聚合酶。適用於本發明之反轉錄酶包括但不限於來自以下之反轉錄酶:HIV、HTLV-I、HTLV-II、FeLV、FIV、SIV、AMV、MMTV、MoMuLV及其他反轉錄病毒(參見Levin, Cell 88:5-8 (1997);Verma, Biochim Biophys Acta. 473:1-38 (1977);Wu等人,CRC Crit Rev Biochem. 3:289- 347(1975))。聚合酶之其他實例包括但不限於:9°NDNA聚合酶、TaqDNA聚合酶、Phusion®DNA聚合酶、Pfu DNA聚合酶、RB69 DNA聚合酶、KOD DNA聚合酶及VentR® DNA聚合酶(Gardner等人 (2004) 「Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase」J. Biol. Chem., 279(12), 11834-11842;Gardner及Jack 「Determinants of nucleotide sugar recognition in an archaeon DNA polymerase」 Nucleic Acids Research, 27(12) 2545-2553)。自非嗜熱性生物體分離之聚合酶可為可熱滅活的。實例為來自噬菌體之DNA聚合酶。應理解,來自各種來源中之任一者的聚合酶可經修飾以增加或降低其對高溫條件之耐受性。在一些實施例中,聚合酶可為嗜熱性的。在一些實施例中,嗜熱性聚合酶可為可熱滅活的。嗜熱性聚合酶典型地適用於高溫條件或熱循環條件,諸如用於聚合酶鏈反應(PCR)技術之熱循環條件。
在一些實施例中,聚合酶包含:Φ29、B103、GA-1、PZA、Φ15、BS32、M2Y、Nf、G1、Cp-1、PRD1、PZE、SF5、Cp-5、Cp-7、PR4、PR5、PR722、L17、ThermoSequenase®、9°Nm™、Therminator™ DNA聚合酶、Tne、Tma、TfI、Tth、TIi、Stoffel片段、Vent™及Deep Vent™ DNA聚合酶、KOD DNA聚合酶、Tgo、JDF-3、Pfu、Taq、T7 DNA聚合酶、T7 RNA聚合酶、PGB-D、UlTma DNA聚合酶、大腸桿菌DNA聚合酶I、大腸桿菌DNA聚合酶III、古細菌DP1I/DP2 DNA聚合酶II、9°N DNA聚合酶、Taq DNA聚合酶、Phusion® DNA聚合酶、Pfu DNA聚合酶、SP6 RNA聚合酶、RB69 DNA聚合酶、鳥成髓細胞瘤病毒(AMV)反轉錄酶、莫洛尼鼠類白血病病毒(MMLV) 反轉錄酶、SuperScript® II反轉錄酶及SuperScript® III反轉錄酶。
在一些實施例中,聚合酶為DNA聚合酶1-克列諾片段、Vent聚合酶、Phusion® DNA聚合酶、KOD DNA聚合酶、Taq聚合酶、T7 DNA聚合酶、T7 RNA聚合酶、Therminator™ DNA聚合酶、POLB聚合酶、SP6 RNA聚合酶、大腸桿菌DNA聚合酶I、大腸桿菌DNA聚合酶III、鳥成髓細胞瘤病毒(AMV)反轉錄酶、莫洛尼鼠類白血病病毒(MMLV)反轉錄酶、SuperScript® II反轉錄酶或SuperScript® III反轉錄酶。
另外,此類聚合酶可用於DNA擴增及/或定序應用,包括(例如在擴增或定序之情況下)包括藉由聚合酶將非天然核酸殘基導入至DNA中的實時應用。在其他實施例中,導入之非天然核酸可與天然殘基相同,例如其中非天然核酸之標記或其他部分在導入期間藉由聚合酶之作用經移除,或非天然核酸可具有一或多種將其與天然核酸區分之特徵。
至少自地球上所有生命之最後的共同祖先起,遺傳資訊便已儲存在藉由形成兩鹼基對來傳播及檢索的四字母字母表中。合成生物學之核心目標係創建新的生命形式及功能,且通向此目標之最普遍的途徑係創建其DNA具有形成第三非天然鹼基對(UBP)的兩個額外字母的半合成生物體(SSO)。先前,吾人為生成此類SSO之工作在大腸桿菌菌株之創建中達到頂點,其藉助於來自三角褐指藻(Pt
NTT2)之核苷三磷酸運輸蛋白,自培養基輸入必需非天然三磷酸酯,且隨後使用其複製含有UBP dNaM-dTPT3之質體 (圖 1A
)。雖然SSO儲存更多資訊,但其不檢索資訊,此需要活體內將UBP轉錄至mRNA及tRNA、使用非天然胺基酸胺醯化tRNA,及最終,UBP在核糖體處高效參與解碼。在此,吾人報導含有dNaM及dTPT3之DNA向具有兩種不同非天然密碼子之mRNA及具有同源非天然反密碼子之tRNA的活體內轉錄;及其在核糖體處之高效解碼,該解碼係用以定向天然或非典型胺基酸(non-canonical amino acids,ncAA)向superfolder綠色螢光蛋白(superfolder green fluorescent protein,sfGFP)中之定點導入。結果表明,除氫鍵結以外之相互作用可以促進資訊儲存及檢索之每一步驟。所得SSO編碼及檢索更多資訊且將充當創建新的生命形式及功能之平台。
諸如sfGFP之綠色螢光蛋白及變體已充當用於使用琥珀抑制系統導入ncAA (包括在Y151位置處)之研究之模型系統,其已顯示出包容各種天然及ncAA (圖 4
)。為探究非天然密碼子之解碼,吾人首先聚焦於在sfGFP之位置151處之Ser之導入,因為大腸桿菌絲胺酸胺基醯基-tRNA合成酶(SerRS)不依賴於反密碼子識別用於tRNA胺醯化,因此排除低效填充之潛在併發情況。SSO菌株YZ3經編碼sfGFP及大腸桿菌tRNASer
基因(ser
T)之質體轉型,其中sfGFP密碼子151 (TAC)由非天然密碼子AXC替代(sfGFP(AXC)151
;X=NaM),且ser
T之反密碼子由非天然反密碼子GYT替代(tRNASer
(GYT);Y=TPT3)(圖 1B
)。在補充有dNaMTP及dTPT3TP之培養基生長中轉型體,隨後另外補充NaMTP及TPT3TP以及異丙基-β-D-硫代半乳糖苷(IPTG),以誘導T7 RNA聚合酶(T7 RNAP)及tRNASer
(GYT)之表現。在短暫的一段時間之tRNA誘導之後,添加無水四環素(aTc),以誘導sfGFP(AXC)151
之表現。
在誘導之後,相比於經編碼在151位置處具有天然Ser密碼子之sfGFP (sfGFP(AGT)151
;圖 1C
)之質體轉型的細胞,經編碼sfGFP(AXC)151
但缺乏tRNASer
(GYT)之對照組質體轉型之細胞,顯示顯著降低的螢光。而且,在誘導sfGFP(AXC)151
(圖 1D
)時細胞生長開始進入平台期,很可能係由於核糖體之停止及隔離。使用抗GFP抗體對此等細胞之溶解物進行西方墨點法,其顯示sfGFP表現顯著降低且存在在非天然密碼子之位置處截短之sfGFP (圖 1E
)。相比之下,經編碼sfGFP(AXC)151
及tRNASer
(GYT)之質體轉型之細胞展現與表現sfGFP(AGT)151
(圖 1C
)之對照組細胞幾乎等於的螢光,在誘導sfGFP(AXC)151
(圖 1D
)時細胞生長不進入平台期,且來自此等細胞之溶解物之西方墨點法僅顯示全長sfGFP蛋白質(圖 1E
)。另外,吾人評估經以相同形式表現之全部四種天然近同源tRNA (tRNASer
(GNT);N=G、C、A或T)解碼AXC密碼子之能力。在各情況下,幾乎觀測不到螢光且保留有生長缺陷(圖 5A
以及圖 5B
)。此等資料表明,Pt
NTT2能夠輸入兩種非天然核苷酸之去氧及核糖三磷酸酯,T7RNA聚合酶能夠活體內轉錄含有非天然核苷酸之mRNA及tRNA,且核糖體僅有效地藉由非天然反密碼子解碼非天然密碼子。
為評估解碼之保真度,吾人經由LC/MS-MS及經由峰強度之相對定量分析自表現sfGFP(AXC)151
及tRNASer
(GYT)之細胞純化的蛋白質,其顯示在位置151處之Ser導入為98.5±0.7% (95% CI,n
=4),其中Ile/Leu為主要雜質(圖 1F
,表 4
)。鑒於sfGFP(AXC)151
基因中之UBP之保留度為98±2% (95% CI,n
=4) (表 5
)且在複製期間X→T通常係主要突變(對於AXC之情況將產生Ile密碼子ATC),吾人將複製期間之UBP之損失歸因於在位置151處不含有Ser之大多數蛋白質,且得出結論,藉由非天然密碼子之轉譯之保真度較高。表 4 表 5
*對應於圖 7A
至圖 7D
中所分析之培養物。
為展示編碼具有UBP之ncAA,吾人構築了與上文所使用之質體類似但tRNASer
基因替換為馬氏甲烷八疊球菌(Methanosarcina mazei
) tRNAPyl
(GYT)基因的質體。tRNAPyl
可以選擇性地藉由巴氏甲烷八疊球菌(Methanosarcina barkeri
)吡咯離胺酸胺基醯基tRNA合成酶(PylRS)帶有ncAAN 6
-[(2-丙炔基氧基)羰基]-l-離胺酸(PrK)。除密碼子AXC以外,吾人亦分析了密碼子GXC及對應的tRNAPyl
(GYC)。攜帶編碼IPTG-誘導型PylRS之單獨質體的SSO經所需質體轉型且在添加或不添加PrK之情況下生長。在對照組實驗中,其具有在不存在PylRS、同源非天然tRNAPyl
或PrK之情況下表現sfGFP(AXC)151
或sfGFP(GXC)151
之細胞,吾人觀測到僅有較低細胞螢光(圖 2A
)、sfGFP截短(圖 6A
及圖 6B
)及細胞生長處於平台期(圖 6B
)。相比之下,對於具有其同源非天然tRNA之任一非天然mRNA,當存在PylRS且添加PrK時,吾人觀測到較高螢光(針對AXC及GXC,分別為sfGFP(TAC)151
之64%及69%)(圖 2A
及圖 2B
)、全長sfGFP之穩定產生(圖 6A
)及正常生長(圖 6B
)。
為驗證PrK之導入,自細胞溶解物使用C端鏈黴素(Strep
)標籤II親和純化sfGFP且進行銅催化點擊化學以附接羧基四甲基若丹明(TAMRA)染料(TAMRA-PEG4
-N3
),發現其在SDS-PAGE期間改變sfGFP之電泳流動性,因此允許吾人藉由西方墨點法評估PrK導入之保真度(圖 2C
)。當自表現sfGFP(AXC)151
及tRNAPyl
(GYT)或sfGFP(GXC)151
及tRNAPyl
(GYC)之細胞純化時,吾人觀測到強TAMRA信號且幾乎所有sfGFP均改變,且該等細胞在補充有PrK之培養基中培養(圖 2C
)。相比之下,當不存在NaMTP、TPT3TP或兩者時,觀測到幾乎無TAMRA信號或改變的sfGFP (圖 7A
及圖 7B
)。最後,在自具有任一非天然tRNA之表現sfGFP(TAC)151
之細胞純化的蛋白質中觀測到無TAMRA信號或改變的sfGFP (圖 2C
)。此資料表明,PrK經由藉由具有非天然反密碼子之tRNAs解碼非天然密碼子而特異性地導入至sfGFP中。
以最佳PrK濃度(圖 8A
至圖 8D
),吾人純化54±4及55±6 µg/mL之sfGFP (s.d.,n
=4,約40%之sfGFP(TAC)151
對照組(表6
),分別針對AXC及GXC密碼子。而且,基於質譜分析,具有PrK之sfGFP之純度針對AXC密碼子為96.2±0.3% (95% CI,n
=4)且針對GXC密碼子為97.5±0.7% (95% CI,n
=4)(圖 2D
)。儘管經純化之sfGFP蛋白質之產率略微低於具有琥珀抑制之情況(87±6 µg/mL,s.d.n
=4 (表 6
)),因為添加非天然核糖三磷酸酯情況下生長之中度降低(圖 7C
及圖 7D
),當正規化至細胞密度時兩種非天然密碼子之解碼產生高於琥珀抑制的螢光(圖 2A
及圖 2B
),意味著藉由非天然密碼子解碼比琥珀抑制更高效。
為探究具有UBP之其他ncAA之編碼,吾人檢查了具有AXC密碼子及進化詹氏甲烷球菌(Methanococcus jannaschii
) TyrRS/tRNATyr
對之對疊氮-***酸(p
AzF)的編碼(pAzFRS/tRNA p AzF
)。藉由誘導合成酶及向生長培養基中添加p
AzF,吾人觀測到穩定螢光,其等效於表現天然sfGFP(TAC)151
之細胞之螢光,及具有sfGFP(AXC)151
及tRNA p AzF
(GYT)之正常生長(圖 3A
,圖 9
)。純化全長sfGFP (86±6 µg/mL,s.d.,n
=4;sfGFP(TAC)151
對照組之68%,表 6
),且使用二苯并環辛基(DBCO)基團進行無銅點擊化學以附接TAMRA (TAMRA-PEG4
-DBCO)。吾人觀測到與自表現sfGFP(AXC)151
及tRNA p AzF
(GYT)之細胞分離且在p
AzF存在下培養之sfGFP之穩定TAMRA結合(圖 3B
)。儘管吾人未能精確評估p
AzF導入之保真度,但是由於疊氮部分之分解,約93%之sfGFP蛋白質改變,其相比於經由琥珀抑制產生之約95%改變的sfGFP而言有利(圖 3B
)。表 6
至少自地球上所有生命之最後的共同祖先起,蛋白質便已經由僅藉由四核苷酸基因字母表寫入之密碼子之解碼而產生。吾人現已展示之藉由擴展遺傳字母表寫入之兩個新密碼子之解碼,且使用新的密碼子位點特異性地將ncAA導入至蛋白質中。吾人發現,對於資訊儲存及檢索之每一步驟,氫鍵(對天然鹼基對顯然十分重要)可至少部分地替換為補充的封裝力及疏水性力。儘管其解碼機制新穎,非天然密碼子可以如其完全天然的對應物一樣有效地被解碼。雖然吾人僅檢查兩個非天然密碼子之解碼,UBP不大可能限制於此,且當與最近報導之Cas9編輯系統(其加強UBP保留度)組合時,將很可能使比向來可以使用之密碼子更多的密碼子可用。因此,所報導之SSO可能僅係能夠獲得天然生物體不可用之大範圍形式及功能的新形式的半合成生命的第一個。
儘管已經在本文中展示且描述本發明之較佳實施例,但對熟習此項技術者而言很明顯此等實施例僅例示性提供。在不脫離本發明之情況下,熟習此項技術者現應能夠想到諸多變化、改變及替代。應理解,本文中所描述之本發明實施例之各種替代方案可用於實踐本發明。預期以下申請專利範圍定義本發明之範疇,且由此涵蓋此等申請專利範圍及其等效物之範疇內的方法及結構。
本發明之各種態樣在隨附申請專利範圍中細緻地闡述。參考闡述利用本發明之原理的說明性實施例及其附圖的以下詳細描述將更好地理解本發明之特性及優勢:
圖 1A
說明dNaM
-dTPT3
UBP及天然dA-dT鹼基對之化學結構。
圖 1B
說明用於表現sfGFP(AX
C)151
及tRNA(GY
T)Ser
之基因卡匣。PT7
及TT7
分別表示T7 RNAP啟動子及終止子。在其中sfGFP在不存在serT
之情況下表現的對照組中,不存在sfGFP T7終止子之後之序列。
圖 1C
說明表現分別具有指定位置151密碼子及反密碼子之sfGFP及tRNASer
之細胞之螢光圖。負號表示在表現卡匣中不存在serT
。t=0對應於添加IPTG以誘導T7 RNAP及tRNASer
(若存在)之表現;在t=0.5 h添加aTc以誘導sfGFP之表現。AGT,天然Ser密碼子;TAG,琥珀終止密碼子;CTA琥珀抑制反密碼子。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 1D
說明表現分別具有指定位置151密碼子及反密碼子之sfGFP及tRNASer
之細胞之生長的圖。負號表示在表現卡匣中不存在serT
。t=0對應於添加IPTG以誘導T7 RNAP及tRNASer
(若存在)之表現;在t=0.5 h添加aTc以誘導sfGFP之表現。AGT,天然Ser密碼子;TAG,琥珀終止密碼子;CTA琥珀抑制反密碼子。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 1E
說明來自在圖 1C
及圖 1D
中所示之最後時間點收集之細胞之溶解物的西方墨點法(由OD600
正規化),使用α-GFP抗體(N端抗原決定基)探測。
圖 1F
說明在自表現sfGFP(AGT)151
或sfGFP(AX
C)151
及tRNASer
(GY
T)之細胞純化的sfGFP的151位置處偵測的胺基酸之相對豐度(由圖例中之其單個字母編碼指示)的圖,如藉由LC-MS/MS及基於前驅體離子強度之定量所測定(在<0.1% (平均,兩種密碼子)下偵測之胺基酸未示出;細節參見方法且所偵測之胺基酸之完整列表參見表4)。資料顯示為與個別資料點之平均,n
=4個純化sfGFP樣品,各自來自自個別群落繁殖且在圖 1C
及圖 1D
中所示之最後時間點收集之培養物。
圖 2A
說明在培養基中存在(+)或不存在(-)具有同源反密碼子之tRNAPyl
、PylRS或20 mM PrK (N 6
-[(2-丙炔基氧基)羰基]-L-離胺酸)之情況下表現具有指定位置151密碼子之sfGFP之細胞之螢光圖。在圖 2B
中所示之最後時間點測定螢光。星號表示在表現sfGFP(TAC)151
之細胞中不存在tRNAPyl
。TAC,天然Tyr密碼子;TAG,琥珀終止密碼子;n.d.,未測定。資料顯示為與個別資料點之平均,各自自個別群落繁殖。
圖 2B
說明圖 2A
中所示之資料之一子集之時間過程分析。加號及減號分別表示培養基中存在或不存在20 mM PrK。t=0對應於添加IPTG以誘導PylRS、T7 RNAP及tRNAPyl
之表現;在t=1 h添加aTc以誘導sfGFP之表現。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 2C
說明在具有或不具有TAMRA之點擊結合及/或向培養基添加20 mM PrK之情況下,自表現分別具有指定位置151密碼子及反密碼子之sfGFP及tRNAPyl
之細胞純化之sfGFP之西方墨點法。在表現sfGFP(TAC)151
之細胞中不存在tRNAPyl
。自在圖 2B
中所示之最後時間點收集之培養物純化sfGFP。西方墨點法使用α-GFP抗體探測且成像以偵測sfGFP及所結合之TAMRA。
圖 2D
說明在自表現分別具有指定位置151密碼子及同源反密碼子之sfGFP(TAC)151
或sfGFP及tRNAPyl
之細胞純化的sfGFP的151位置處偵測的胺基酸之相對豐度(由圖例中之其單個字母編碼指示)的圖,如藉由LC-MS/MS及基於前驅體離子強度之定量所測定(在<0.1% (平均,兩種密碼子)下偵測之胺基酸未示出;細節參見方法且所偵測之胺基酸之完整列表參見表4) 資料顯示為與個別資料點之平均,n
=4個純化sfGFP樣品,各自來自自個別群落繁殖之培養物。
圖 3A
說明在培養基中存在(+)或不存在(-)5 mMp
AzF之情況下,表現分別具有指定位置151密碼子及同源反密碼子之sfGFP(TAC)151
或sfGFP及tRNA p AzF
之細胞之螢光圖。t=0對應於添加IPTG以誘導p
AzFRS、T7 RNAP及tRNA p AzF
之表現;在t=0.5 h添加aTc以誘導sfGFP之表現。TAC,天然Tyr密碼子;TAG,琥珀終止密碼子。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。在不存在p
AzF之情況下藉由sfGFP(AX
C)151
觀測到之螢光歸因於tRNApAzF
(GY
T)帶有具有天然胺基酸(可能Tyr)。
圖 3B
說明在具有或不具有TAMRA之點擊結合及/或向培養基添加5 mMp
AzF之情況下,自表現分別具有指定位置151密碼子及反密碼子之sfGFP及tRNA p AzF
之細胞純化之sfGFP之西方墨點法。其中指示,負號表示在表現sfGFP(TAC)151
之細胞中不存在tRNA p AzF
。自在圖 3A
中示出之最後時間點收集之培養物純化sfGFP。西方墨點法使用α-GFP抗體探測且成像以偵測sfGFP及所結合之TAMRA。
圖 4
說明表現具有位置151處之各種密碼子之sfGFP之細胞的螢光。生長攜帶具有指定位置151密碼子之sfGFP質體之細胞至OD600
為約0.5且使用IPTG及aTc誘導。在誘導3 h之後進行螢光量測。資料顯示為與個別資料點之平均,n
=3個培養物,自單個群落***且同時生長。
圖 5A
藉由在存在或不存在具有指定反密碼子之tRNASer
之情況下之表現sfGFP(AX
C)151
的細胞的螢光圖說明具有天然近同源反密碼子之AXC密碼子之解碼。細胞如圖 1C
及圖 1D
中所描述地誘導且螢光量測對應於圖 1
C中所示之最後時間點。針對GY
T反密碼子且不存在tRNASer
(-tRNA)之情況下之數值對應於圖1C、圖1D中相同的數值。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 5B
藉由在存在或不存在具有指定反密碼子之tRNASer
之情況下之表現sfGFP(AX
C)151
的細胞的生長的圖說明具有天然近同源反密碼子之AXC密碼子之解碼。細胞如圖 1C
及圖 1D
中所描述地誘導且螢光量測對應於圖 1C
中所示之最後時間點。針對GYT反密碼子且不存在tRNASer
(-tRNA)之情況下之數值對應於圖 1C
及圖 1D
中相同的數值。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 6A
說明藉由tRNAPyl
解碼AXC及GXC密碼子之細胞之西方墨點法及生長。在培養基中存在(+)或不存在(-)具有同源反密碼子之tRNAPyl
、PylRS或20 mM PrK之情況下,來自表現具有指定位置151密碼子之sfGFP之細胞之溶解物之西方墨點法(由OD600
正規化)。使用α-GFP抗體(N端抗原決定基)探測墨點。誘導細胞且在如圖 2B
中所描述之等效時間點收集。
圖 6B
說明圖 6A
中所分析之培養物之生長。當存在胺醯化tRNAPyl
所必需之所有組分時,誘導sfGFP (t=1h)與最終時間點之間之OD600 之
倍數變化最大。OD600
之絕對值之變化係由於在開始T7 RNAP (及(若存在) tRNAPyl
)誘導時細胞密度之微小變化(t=0)。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 7A
說明藉由tRNAPyl
解碼AXC及GXC密碼子及細胞生長,其隨所添加之非天然核糖三磷酸酯而變。在培養基中存在(+)或不存在(-)各非天然核糖三磷酸酯之情況下且在存在或不存在20 mM PrK之情況下,來自表現具有指定位置151-密碼子/反密碼子之sfGFP及tRNAPyl
之細胞的純化sfGFP的螢光(下圖)。如圖 2B
中所描述地誘導細胞,且在誘導結束(約3.5h)時採集螢光量測結果,之後收集細胞且純化sfGFP蛋白質用於TAMRA之點擊結合及西方墨點法。
圖 7B
說明藉由tRNAPyl
解碼AXC及GXC密碼子之膠,其隨所添加之非天然核糖三磷酸酯而變。西方墨點法使用α-GFP抗體探測且成像以偵測sfGFP及所結合之TAMRA;所有色帶對應於自與所添加之PrK一起生長之細胞純化之sfGFP。資料顯示為與個別資料點之平均,n
=3個培養物,各自自個別群落繁殖;n.d.,未測定。
圖 7C
說明在存在(+)或不存在(-)兩種非天然去氧核糖三磷酸酯及各非天然核糖三磷酸酯之情況下,表現sfGFP(TAC)151
之細胞之螢光及生長之圖。t=0對應於添加IPTG以誘導T7 RNAP之表現;在t=1 h時添加aTc以誘導sfGFP之表現。資料顯示為平均值±s.d.,n
=3個培養物,各自自個別群落繁殖。在所使用之濃度下(參見方法),dNaM
TP及dTPT3
TP不抑制細胞生長,然而兩種非天然核糖三磷酸酯,尤其TPT3
TP,顯示一定程度之對生長之抑制。
圖 7D
說明對應於具有所添加之PrK (20 mM)之培養物(其螢光在圖 2B
中顯示)的細胞生長之圖。在無任何非天然三磷酸酯之情況下生長表現具有天然密碼子之sfGFP之細胞,然而使用兩種非天然去氧及核糖三磷酸酯生長表現具有非天然密碼子之sfGFP之細胞。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 8A
說明藉由tRNAPyl
解碼AXC及GXC密碼子之膠,其隨培養基中之PrK濃度而變。自表現具有指定位置151密碼子/反密碼子之sfGFP及tRNAPyl
之細胞純化的sfGFP的西方墨點法,在具有TAMRA之點擊結合及以指示濃度向培養基中添加PrK之情況下。誘導sfGFP且自如圖 2B
中所描述地收集之細胞純化。西方墨點法使用α-GFP抗體探測且成像以偵測sfGFP及所結合之TAMRA。
圖 8B
說明藉由tRNAPyl
解碼AXC及GXC密碼子之圖,其隨培養基中之PrK濃度而變。表現分別具有指定位置151密碼子及反密碼子之sfGFP及tRNAPyl
之細胞之螢光(在c
中所示之最後時間點量測),其隨培養基中之PrK濃度而變。對於0及20 mM PrK之螢光值分別與(-)及(+)PrK值相同,圖 2B
中所示。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
圖 8C
說明圖 8B
中之螢光及細胞生長之時間過程分析。為了清楚說明之目的,各密碼子/反密碼子對及PrK濃度僅顯示一個代表性培養物。不受理論束縛,吾人將不存在PrK之情況下產生之sfGFP之低含量歸因於藉由內源性tRNAs解碼及sfGFP
中之UBP保留度損失(表 5
)。然而,含有PrK之sfGFP之相對量(圖 8A
)及所表現之sfGFP之絕對量(圖 8B
及圖 8C
)隨著培養基中之PrK之增加以劑量依賴型方式增加,最終導致PrK之幾乎完全導入,表明AX
C及GX
C密碼子之內源性通讀可以由所填充之PrK-tRNAPyl
(GY
T)或PrK-tRNAPyl
(GY
C)之足夠濃度有效抑制。
圖 9
說明其螢光顯示在圖 3A
中之培養物之細胞生長。資料顯示為平均值±s.d.,n
=4個培養物,各自自個別群落繁殖。
表 4 | 針對圖 1F 及圖 2D 中所描述之實驗之 sfGFP 中位置 151 處之胺基酸之相對豐度。
自表現在存在或不存在tRNA之情況下之分別具有指定位置151密碼子及反密碼子之sfGFP的細胞純化的sfGFP藉由LC-MS/MS分析。針對報導子肽LEYNFNSHNVX151
ITADK (X=PrK或除K或R之外之任何經鑑別之天然胺基酸)及LEYNFNSHNVX151
(若X=K或R)提取之MS1離子強度表達為針對所有可觀測的報導子肽之離子強度之總和之百分比。數值表對應於在sfGFP之位置151處所偵測之所有胺基酸之平均相對豐度及95% CI的,n
=4個純化sfGFP樣品,各自來自自個別群落繁殖之培養物。自圖 1F
及圖 2D
中所展示之資料中排除<0.1% (平均,針對對應的圖中所指示之密碼子)之數值。
表 5 | UBP 保留度。
在sfGFP誘導之前一段時間且在誘導結束時測定在具有sfGFP之指定位置151密碼子及指定tRNA之反密碼子之質體中UBP之保留度,如方法中所描述。報導之數值為在誘導過程內之平均UBP保留度(由在此等兩個時間點時之保留度計算)±95% CI,n
=4個培養物,各自自個別群落繁殖,除了由星號指示之數值,對其n
=3。n/a,不適用(因為相關序列為天然或不存在)。所有質體均自在存在20 mM PrK或5 mMp
AzF (除了Ser解碼實驗)之情況下生長之培養物分離。SerRS表示帶有內源性大腸桿菌合成酶。負號表示具有tRNAPyl
之細胞中不存在PylRS或不存在異位表現之tRNA。由§指示之列中之保留度對應於亦自其純化sfGFP且藉由TAMRA結合sfGFP之LC-MS/MS及/或西方墨點法分析的培養物(參見圖 1F
(Ser)、圖 2D
(PrK)及圖 3B
(p
AzF));具有星號之列對應於圖 7A
至圖 7D
中所分析之培養物。儘管事實係全部四種非天然三磷酸酯經相同運輸蛋白進入細胞且因此競爭性地抑制彼此之輸入,在培養基中存在(+)或不存在(-)NaM
TP及/或TPT3
TP之情況下未觀測到UBP保留度方面之差異。此等資料,及用於具有高保真PrK導入之高sfGFP表現量的兩種非天然核糖三磷酸酯的要求(圖 7A
至圖 7D
),共同地表明YZ3中之Pt
NTT2運輸蛋白之表現量輸入維持UBP複製及轉錄所必需之必需含量之非天然三磷酸酯。
表 6 | 在 Ser 、 Prk 及 p AzF 導入實驗中表現之 sfGFP 蛋白質之產率
。由純化蛋白質之總量及用於純化之培養物之體積計算產率(參見方法)。資料為平均值±s.d. (n
=4個sfGFP樣品,各自由自個別群落繁殖之培養物純化),且自圖 1F
(用於SerRS)及圖 2D
(用於PylRS)中所分析之相同培養物以及對應於圖 3A
(用於p
AzFRS)中之(+)p
AzF樣品之培養物測定。經純化之sfGFP之產率與純化其之培養物之平均總螢光(不正規化至OD600
)相當。螢光值對應於收集細胞用於sfGFP純化之時間點;參見圖 1C
(Ser)、圖 2B
(PrK)及圖 3A
(p
AzF)。
Claims (56)
- 如請求項1之方法,其中該半合成生物體包含微生物。
- 如請求項1或2之方法,其中該半合成生物體包含細菌。
- 如請求項1或2之方法,其中該半合成生物體包含大腸桿菌(Escherichia coli)。
- 如請求項1或2之方法,其中該非天然核苷酸另外包含非天然糖部分。
- 如請求項10之方法,其中該非天然核苷酸之該非天然糖部分選自由以下2'位置處之修飾所組成之群中:OH、經取代之低碳數烷基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3、OCN、Cl、Br、CN、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2F、O-烷基、S-烷基、N-烷基、O-烯基、S-烯基、N-烯基、O-炔基、S-炔基、N-炔基、O-烷基-O-烷基、2'-F、2'-OCH3、2'-O(CH2)2OCH3、-O[(CH2)O]mCH3、-O(CH2)nOCH3、-O(CH2)nNH2、-O(CH2)nCH3、-O(CH2)n-ONH2及-O(CH2)nON[(CH2)nCH3)]2,其中烷基、烯基及炔基可為經取代或未經取代之C1-C10烷基、C2-C10烯基、C2-C10炔基,其中n及m為1至10;及/或在5'位置處之修飾:5'-乙烯基、5'-甲基(R或S);在4'位置處之修飾:4'-S、雜環烷基、雜環烷芳基、胺基烷胺基、聚烷基胺基、經取代矽烷基,以及其任何組合。
- 如請求項1或2之方法,其中該突變反密碼子或該突變密碼子另外包含非天然主鏈。
- 如請求項1或2之方法,其中該突變反密碼子及該突變密碼子另外包含非天然主鏈。
- 如請求項1或2之方法,其中在轉錄期間,非天然核苷酸藉由RNA聚合酶導入該mRNA中,以生成含有該突變密碼子之該突變mRNA。
- 如請求項1或2之方法,其中在轉錄期間,非天然核苷酸藉由RNA聚合酶導入該tRNA中,以生成含有該突變反密碼子之該突變tRNA。
- 如請求項1或2之方法,其中該突變tRNA帶有非天然胺基酸殘基。
- 如請求項1或2之方法,其中該非天然胺基酸係對疊氮-***酸(pAzF)。
- 如請求項1或2之方法,其中該非天然胺基酸係N 6-[(2-丙炔基氧基)羰基]-L-離胺酸(PrK)。
- 如請求項1或2之方法,其中該突變tRNA係來自馬氏甲烷八疊球菌(Methanosarcina mazei)之tRNA突變體。
- 如請求項1或2之方法,其中該異源性胺基醯基tRNA合成酶係吡咯離胺醯基tRNA合成酶。
- 如請求項1或2之方法,其中該異源性胺基醯基tRNA合成酶係酪胺醯基tRNA合成酶。
- 如請求項1或2之方法,其中該異源性胺基醯基tRNA合成酶係來自巴氏甲烷八疊球菌(Methanosarcina barkeri)。
- 如請求項1或2之方法,其中該異源性胺基醯基tRNA合成酶係來自詹氏甲烷球菌(Methanococcus jannaschii)。
- 如請求項24之細胞,其中該細胞另包含編碼該tRNA之寡核苷酸。
- 如請求項24或25之細胞,其中該細胞另包含編碼該異源性胺基醯基tRNA合成酶之寡核苷酸。
- 如請求項24或25之細胞,其中該細胞另包含編碼該mRNA之寡核苷酸。
- 如請求項24或25之細胞,其中該細胞另包含編碼該tRNA及該mRNA之寡核苷酸。
- 如請求項24或25之細胞,其中該細胞另包含編碼該tRNA、該mRNA及該異源性胺基醯基tRNA合成酶之寡核苷酸。
- 如請求項24或25之細胞,其中該tRNA包含一或多個選自GYT及GYC之反密碼子,其中Y為該非天然核鹼基。
- 如請求項30之細胞,其中該tRNA包含該反密碼子GYT。
- 如請求項30之細胞,其中該tRNA包含該反密碼子GYC。
- 如請求項24或25之細胞,其中該mRNA包含一或多個選自AXC及GXC之密碼子,其中X為該非天然核鹼基。
- 如請求項35之細胞,其中該mRNA包含該密碼子AXC。
- 如請求項35之細胞,其中該mRNA包含該密碼子GXC。
- 如請求項24或25之細胞,其中該異源性胺基醯基tRNA合成酶係來自巴氏甲烷八疊球菌(Methanosarcina barkeri)。
- 如請求項24或25之細胞,其中該異源性胺基醯基tRNA合成酶係來自詹氏甲烷球菌(Methanococcus jannaschii)。
- 如請求項24或25之細胞,其中該異源性胺基醯基tRNA合成酶係吡咯離胺醯基tRNA合成酶。
- 如請求項24或25之細胞,其中該異源性胺基醯基tRNA合成酶係酪胺醯基tRNA合成酶。
- 如請求項24或25之細胞,其中該突變tRNA係來自馬氏甲烷八疊球菌(Methanosarcina mazei)之tRNA突變體。
- 如請求項24或25之細胞,其中該細胞係微生物。
- 如請求項24或25之細胞,其中該細胞係細菌。
- 如請求項24或25之細胞,其中該細胞係大腸桿菌(Escherichia coli)。
- 一種在細胞中生產蛋白質之方法,其中該蛋白質包含非天然胺基酸,該方法包含:使用核苷三磷酸運輸蛋白將非天然核苷酸運輸至如請求項24至51中任一項之細胞中;及提供該非天然胺基酸至該細胞,其中在轉錄包含非天然寡核苷酸之雙股寡核苷酸期間,該非天然核苷酸藉由RNA聚合酶導入以生成該突變mRNA及該突變tRNA;及於該突變tRNA轉譯該突變mRNA之期間生成含有該非天然胺基酸之該蛋白質。
- 如請求項52之方法,其中該運輸至該細胞中之非天然核苷酸包含非天然核鹼基。
- 如請求項52至54中任一項之方法,其中該非天然胺基酸係對疊氮-***酸(pAzF)。
- 如請求項52至54中任一項之方法,其中該非天然胺基酸係N 6-[(2-丙炔基氧基)羰基]-L-離胺酸(PrK)。
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US20240043823A1 (en) | 2024-02-08 |
US20190376054A1 (en) | 2019-12-12 |
KR20200029531A (ko) | 2020-03-18 |
CN111051512A (zh) | 2020-04-21 |
EA202090090A1 (ru) | 2020-06-09 |
TW201920681A (zh) | 2019-06-01 |
KR20240038157A (ko) | 2024-03-22 |
AR112756A1 (es) | 2019-12-11 |
JP7325341B2 (ja) | 2023-08-14 |
IL271903A (en) | 2020-02-27 |
MA49578A (fr) | 2021-04-07 |
CA3069321A1 (en) | 2019-01-17 |
KR102649135B1 (ko) | 2024-03-18 |
US20210222147A1 (en) | 2021-07-22 |
SG11202000167SA (en) | 2020-02-27 |
NZ761479A (en) | 2024-03-22 |
EP3652316A4 (en) | 2021-04-07 |
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WO2019014267A1 (en) | 2019-01-17 |
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