CN111051512A - 非天然核苷酸的掺入及其方法 - Google Patents
非天然核苷酸的掺入及其方法 Download PDFInfo
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- CN111051512A CN111051512A CN201880058859.4A CN201880058859A CN111051512A CN 111051512 A CN111051512 A CN 111051512A CN 201880058859 A CN201880058859 A CN 201880058859A CN 111051512 A CN111051512 A CN 111051512A
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- methoxyuracil
- natural
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- trna
- polymerase
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Abstract
本文公开了利用突变tRNA合成包含非天然氨基酸的蛋白质的方法、组合物和试剂盒。
Description
交叉引用
本申请要求于2017年7月11日提交的美国临时专利申请号62/531,325的权益,该临时申请通过引用全文并入本文。
背景技术
寡核苷酸及其应用彻底改变了生物技术。但是,同时包含DNA和RNA的寡核苷酸均仅包括DNA的四种天然核苷酸——腺苷(A)、鸟苷(G)、胞嘧啶(C)、胸腺嘧啶(T)和RNA的四种天然核苷酸——腺苷(A)、鸟苷(G)、胞嘧啶(C)和尿苷(U),这大大限制了寡核苷酸的潜在功能和应用。
例如通过PCR或恒温扩增系统(例如,用T7 RNA聚合酶转录),用聚合酶来序列特异性地合成/扩增寡核苷酸(DNA或RNA)的能力彻底改变了生物技术。除了在纳米技术中的所有潜在应用之外,这还使多种新技术成为可能,例如RNA和DNA适体以及酶通过SELEX(通过指数富集的配体系统进化)的体外进化。例如,参见Oliphant AR,Brandl CJ&Struhl K(1989),Defining the sequence specificity of DNA-binding proteins by selectingbinding sites from random-sequence oligonucleotides:analysis of yeast GCN4proteins,Mol.Cell Biol.,9:2944-2949;Tuerk C&Gold L(1990),Systematic evolutionof ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNApolymerase,Science,249:505–510;Ellington AD&Szostak JW(1990),In vitroselection of RNA molecules that bind specific ligands,Nature,346:818-822。
在一些方面,这些应用受到天然遗传字母表(DNA中的四种天然核苷酸——A、C、G和T,以及RNA中的四种天然核苷酸——A、C、G和U)中存在的有限化学/物理多样性的限制。本文公开了产生含有扩展的遗传字母表的核酸的另一种方法。
发明内容
在某些实施方案中,本文公开了产生含有非天然氨基酸的蛋白质的方法,该方法包括:制备突变tRNA,其中突变tRNA包含选自表1或表2的突变反密码子序列;制备突变mRNA,其中突变mRNA包含选自表1或表2的突变密码子序列;以及利用突变tRNA和突变mRNA合成含有非天然氨基酸的蛋白质。在一些情况下,蛋白质在无细胞翻译系统中合成。在一些情况下,蛋白质在细胞(半合成生物体或SSO)中合成。在一些情况下,半合成生物体包含微生物。在一些情况下,半合成生物体包含细菌。在一些情况下,半合成生物体包含大肠杆菌(Escherichia coli)。在一些情况下,突变tRNA的突变反密码子与选自表1-3的突变密码子配对。在一些情况下,非天然氨基酸包含至少一个非天然核苷酸。在一些情况下,非天然核苷酸包含非天然核碱基。在一些情况下,非天然核苷酸的非天然碱基选自2-氨基腺嘌呤-9-基、2-氨基腺嘌呤、2-F-腺嘌呤、2-硫尿嘧啶、2-硫胸腺嘧啶、2-硫胞嘧啶、腺嘌呤和鸟嘌呤的2-丙基和烷基衍生物、2-氨基-腺嘌呤、2-氨基-丙基-腺嘌呤、2-氨基吡啶、2-吡啶酮、2’-脱氧尿苷、2-氨基-2’-脱氧腺苷、3-脱氮鸟嘌呤、3-脱氮腺嘌呤、4-硫尿嘧啶、4-硫胸腺嘧啶、尿嘧啶-5-基、次黄嘌呤-9-基(I)、5-甲基-胞嘧啶、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、5-溴尿嘧啶和5-三氟甲基尿嘧啶以及5-溴胞嘧啶和5-三氟甲基胞嘧啶;5-卤代尿嘧啶、5-卤代胞嘧啶、5-丙炔基-尿嘧啶、5-丙炔基胞嘧啶、5-尿嘧啶、5-取代的、5-卤代、5-取代的嘧啶、5-羟基胞嘧啶、5-溴胞嘧啶、5-溴尿嘧啶、5-氯胞嘧啶、氯化胞嘧啶、环胞嘧啶、阿糖胞苷、5-氟胞嘧啶、氟嘧啶、氟尿嘧啶、5,6-二氢胞嘧啶、5-碘胞嘧啶、羟基脲、碘尿嘧啶、5-硝基胞嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶和5-碘尿嘧啶、腺嘌呤和鸟嘌呤的6-烷基衍生物、6-氮嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、氮胞嘧啶、6-偶氮胸腺嘧啶、6-硫鸟嘌呤、7-甲基鸟嘌呤、7-甲基腺嘌呤、7-脱氮鸟嘌呤、7-脱氮鸟苷、7-脱氮-腺嘌呤、7-脱氮-8-氮鸟嘌呤、8-氮鸟嘌呤、8-氮腺嘌呤、8-卤代、8-氨基、8-硫醇、8-硫代烷基和8-羟基取代的腺嘌呤和鸟嘌呤;N4-乙基胞嘧啶、N-2取代的嘌呤、N-6取代的嘌呤、O-6取代的嘌呤、增加双链体形成的稳定性的那些、通用核酸、疏水核酸、混杂核酸(promiscuous nucleic acid)、尺寸扩展的核酸、氟化核酸、三环嘧啶、吩噁嗪胞苷([5,4-b][l,4]苯并噁嗪-2(3H)-酮)、吩噻嗪胞苷(1H-嘧啶并[5,4-b][l,4]苯并噻嗪-2(3H)-酮)、G夹(G-clamp)、吩噁嗪胞苷(9-(2-氨基乙氧基)-H-嘧啶并[5,4-b][1,4]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、次黄嘌呤、黄嘌呤、4-乙酰胞嘧啶、5-(羧基羟甲基)尿嘧啶、5-羧甲基氨基甲基-2-硫尿苷、5-羧甲基氨基甲基尿嘧啶、二氢尿嘧啶、β-D-半乳糖基辫苷、肌苷、N6-异戊烯腺嘌呤、1-甲基鸟嘌呤、1-甲基肌苷、2,2-二甲基鸟嘌呤、2-甲基腺嘌呤、2-甲基鸟嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-腺嘌呤、7-甲基鸟嘌呤、5-甲基氨基甲基尿嘧啶、5-甲氧基氨基甲基-2-硫尿嘧啶、β-D-甘露糖基辫苷、5’-甲氧基羧甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-N6-异戊烯腺嘌呤、尿嘧啶-5氧乙酸、怀丁氧苷(wybutoxosine)、假尿嘧啶、辫苷、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-氧乙酸甲酯、尿嘧啶-5-氧乙酸、5-甲基-2-硫尿嘧啶、3-(3-氨基-3-N-2-羧基丙基)尿嘧啶、(acp3)w和2,6-二氨基嘌呤,以及其中嘌呤或嘧啶碱基被杂环取代的那些。在一些情况下,非天然核苷酸选自(仅示出核碱基部分,为清楚起见省略了核糖和磷酸骨架)
在一些情况下,非天然核苷酸选自(仅示出核碱基部分,为清楚起见省略了核糖和磷酸骨架)
在一些情况下,非天然核苷酸进一步包含非天然糖部分。在一些情况下,非天然核苷酸的非天然糖部分选自在2’位置处的下组修饰:OH;取代的低级烷基、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,其中烷基、烯基和炔基可以为取代或未取代的C1-C10烷基、C2-C10烯基、C2-C10炔基、-O[(CH2)nO]mCH3、-O(CH2)nOCH3、-O(CH2)nNH2、-O(CH2)nCH3、-O(CH2)n-ONH2和-O(CH2)nON[(CH2)nCH3)]2,其中n和m为从1至约10;和/或选自在5’位置处的下组修饰:5’-乙烯基、5’-甲基(R或S)、在4’位置处的修饰、4’-S、杂环烷基、杂环烷芳基、氨基烷基氨基、聚烷基氨基、取代的甲硅烷基、RNA剪切基团、报告基团、嵌入剂、用于改善寡核苷酸的药代动力学性质的基团,或用于改善寡核苷酸的药效学性质的基团,及其任意组合。在一些情况下,突变反密码子或突变密码子进一步包含非天然骨架。在一些情况下,突变反密码子和突变密码子进一步包含非天然骨架。在一些情况下,非天然核苷酸被DNA聚合酶、RNA聚合酶或逆转录酶识别。在一些情况下,在转录过程中RNA聚合酶将非天然核苷酸掺入mRNA中,以产生含有突变密码子的突变mRNA。在一些情况下,在转录过程中RNA聚合酶将非天然核苷酸掺入tRNA中,以产生含有突变反密码子的突变tRNA。在一些情况下,在转录过程中RNA聚合酶将非天然核苷酸掺入mRNA中,以产生突变mRNA。在一些情况下,在转录过程中RNA聚合酶将非天然核苷酸掺入tRNA中,以产生突变tRNA。在一些情况下,突变tRNA带有非天然氨基酸残基。在一些情况下,利用突变tRNA和突变mRNA在翻译过程中产生含有非天然氨基酸的蛋白质。
附图说明
本公开内容的各个方面在所附的权利要求书中具体阐述。通过参考以下对利用到本公开内容原理的说明性实施方案加以阐述的详细描述以及附图,将获得对本内开内容的特征和优点的更好的理解。
在这些附图中:
图1A显示了dNaM-dTPT3 UBP和天然dA-dT碱基对的化学结构。
图1B显示了用于表达sfGFP(AXC)151和tRNA(GYT)Ser的基因盒。PT7和TT7分别表示T7RNAP启动子和终止子。在不存在serT的情况下表达sfGFP的对照中,sfGFP T7终止子后的序列不存在。
图1C显示了表达分别具有所示位置151的密码子和反密码子的sfGFP和tRNASer的细胞的荧光图。减号表示表达盒中不存在serT。t=0对应于添加IPTG以诱导T7 RNAP和tRNASer(如果存在)的表达;在t=0.5h时添加aTc以诱导sfGFP的表达。AGT,天然Ser密码子;TAG,琥珀终止密码子;CTA,琥珀抑制基因反密码子。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图1D显示了表达分别具有所示位置151的密码子和反密码子的sfGFP和tRNASer的细胞的生长图。减号表示表达盒中不存在serT。t=0对应于添加IPTG诱导以T7 RNAP和tRNASer(如果存在)的表达;在t=0.5h时添加aTc以诱导sfGFP的表达。AGT,天然Ser密码子;TAG,琥珀终止密码子;CTA,琥珀抑制基因反密码子。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图1E显示了用α-GFP抗体(N端表位)探测的来自在图1C和图1D示出的最后时间点收集的细胞的裂解液(通过OD600标准化)的蛋白质印迹。
图1F显示了如由LC-MS/MS和基于前体离子强度的定量测定的,在从表达sfGFP(AGT)151或sfGFP(AXC)151和tRNASer(GYT)的细胞纯化的sfGFP的位置151处检测到的氨基酸(由图例中的其单字母代码所示)的相对丰度图(检测到<0.1%(对两个密码子均取平均)的氨基酸未示出;详细信息参见方法,检测到的氨基酸的完整列表参见表4)。数据以单独数据点的平均值示出,n=4个纯化的sfGFP样品,各自来自从单独菌落繁殖的培养物并在图1C和图1D示出的最后时间点收集。
图2A显示了在介质中存在(+)或不存在(–)具有同源反密码子的tRNAPyl、PylRS或20mM PrK(N6-[(2-丙炔基氧基)羰基]-L-赖氨酸)的情况下,表达具有所示位置151密码子的sfGFP的细胞的荧光图。在图2B示出的最后时间点测定荧光。星号表示在表达sfGFP(TAC)151的细胞中不存在tRNAPyl。TAC,天然Tyr密码子;TAG,琥珀终止密码子;n.d.,未测定。数据以单独数据点的平均值示出,各自繁殖自单独菌落。
图2B显示了对图2A所示数据的子集的时程分析。加号和减号分别表示介质中存在或不存在20mM PrK。t=0对应于添加IPTG以诱导PylRS、T7 RNAP和tRNAPyl的表达;在t=1h时添加aTc以诱导sfGFP的表达。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图2C显示了在有或没有点击缀合TAMRA和/或向介质添加20mM PrK的情况下,从表达分别具有所示位置151的密码子和反密码子的sfGFP和tRNAPyl的细胞中纯化的sfGFP的蛋白质印迹。表达sfGFP(TAC)151的细胞中不存在(-)tRNAPyl。从图2B示出的最后时间点收集的培养物中纯化sfGFP。用α-GFP抗体探测蛋白质印迹,并成像以检测sfGFP和缀合的TAMRA二者。
图2D显示了如由LC-MS/MS和基于前体离子强度的定量(检测到<0.1%(对所有密码子均取平均)的氨基酸未示出;详细信息参见方法,检测到的氨基酸的完整列表参见表4)测定的,从表达分别具有所示位置151的密码子和同源反密码子的sfGFP(TAC)151或sfGFP和tRNAPyl的细胞(在图2B示出的最后时间点收集)纯化的sfGFP的位置151处的氨基酸(由图例中的其单字母代码所示)的相对丰度图。数据以单独数据点的平均值示出,n=4个纯化的sfGFP样品,各自来自从单独菌落繁殖的培养物。
图3A显示了在介质中存在(+)或不存在(–)5mM pAzF的情况下,表达分别具有所示位置151的密码子和同源反密码子的sfGFP(TAC)151或sfGFP和tRNApAzF的细胞的荧光图。t=0对应于添加IPTG以诱导pAzFRS、T7 RNAP和tRNApAzF的表达;在t=0.5h时添加aTc以诱导sfGFP的表达。TAC,天然Tyr密码子;TAG,琥珀终止密码子。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。在不存在pAzF的情况下,用sfGFP(AXC)151观察到的荧光归因于tRNApAzF(GYT)装载有天然氨基酸(可能是Tyr)。
图3B显示了在有或没有点击缀合TAMRA和/或向介质添加5mM pAzF的情况下,从表达分别具有所示位置151的密码子和反密码子的sfGFP和tRNApAzF的细胞纯化的sfGFP的蛋白质印迹。所示的减号表示在表达sfGFP(TAC)151的细胞中不存在tRNApAzF。从图3A示出的最后时间点收集的培养物中纯化sfGFP。用α-GFP抗体探测蛋白质印迹,并成像以检测sfGFP和缀合的TAMRA。
图4显示了表达在位置151处具有各种密码子的sfGFP的细胞的荧光。使携带具有所示位置151密码子的sfGFP质粒的细胞生长至OD600约0.5,并用IPTG和aTc诱导。诱导3h后进行荧光测量。数据以单独数据点的平均值示出,n=3个培养物,从单独菌落分离并平行生长。
图5A通过在有或没有具有所示反密码子的tRNASer的情况下表达sfGFP(AXC)151的细胞的荧光图显示了用天然近同源反密码子对AXC密码子的解码。如图1C和图1D所述诱导细胞,并且荧光测量值对应于图1C示出的最后时间点。GYT反密码子和不存在tRNASer(–tRNA)的情况的值对应于图1c、d中的相同值。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图5B通过在有或没有具有所示反密码子的tRNASer的情况下表达sfGFP(AXC)151的细胞的生长图显示了用天然近同源反密码子对AXC密码子的解码。如图1C和图1D所述诱导细胞,并且荧光测量值对应于图1C示出的最后时间点。GYT反密码子和不存在tRNASer(–tRNA)的情况的值对应于图1C和图1D中的相同值。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图6A显示了用tRNAPyl解码AXC和GXC密码子的细胞的蛋白质印迹和生长。在存在(+)或不存在(-)具有同源反密码子的tRNAPyl、PylRS或介质中20mM PrK的情况下,来自表达具有所示位置151密码子的sfGFP的细胞的裂解液的蛋白质印迹(通过OD600标准化)。用α-GFP抗体(N-端表位)探测印迹。如图2B所述诱导细胞并在等效时间点进行收集。
图6B显示了在图6A中分析的培养物的生长。当存在氨酰化tRNAPyl所需的所有组分时,在sfGFP诱导(t=1h)与最终时间点之间的OD600倍数变化最大。OD600绝对值的变化是由于T7 RNAP(以及tRNAPyl,如果存在)诱导开始时(t=0)细胞密度的微小变化。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图7A显示了用tRNAPyl解码AXC和GXC密码子以及随添加的非天然核糖三磷酸变化的细胞生长。示出了在介质中存在(+)或不存在(-)每种非天然核糖三磷酸以及具有或没有20mM PrK的情况下,从表达具有所示位置151的密码子/反义密码子的sfGFP和tRNAPyl的细胞中纯化的sfGFP(下排)的荧光。如图2B所述诱导细胞,并在诱导结束时(约3.5h)进行荧光测量,然后收集细胞并纯化sfGFP蛋白,以进行TAMRA的点击缀合和蛋白质印迹。
图7B显示了随添加的非天然核糖三磷酸变化的用tRNAPyl解码AXC和GXC密码子的凝胶图。用α-GFP抗体探测蛋白质印迹并成像以检测sfGFP和缀合的TAMRA二者;所有泳道均对应于从添加PrK的情况下生长的细胞中纯化的sfGFP。数据以单独数据点的平均值示出,n=3个培养物,各自繁殖自单独菌落;n.d.,未测定。
图7C显示了在存在(+)或不存在(-)两种非天然脱氧核糖三磷酸和每种非天然核糖三磷酸的情况下表达sfGFP(TAC)151的细胞的荧光图和生长图。t=0对应于添加IPTG以诱导T7 RNAP的表达;在t=1h时添加aTc以诱导sfGFP的表达。数据以平均值±s.d.示出,n=3个培养物,各自繁殖自单独菌落。在使用的浓度下(请参见“方法”),dNaMTP和dTPT3TP不会抑制细胞生长,而两种非天然核糖三磷酸,特别是TPT3TP,示出一定程度的生长抑制。
图7D显示了对应于添加有PrK(20mM)的培养物的细胞生长图,其荧光如图2B示出。表达具有天然密码子的sfGFP的细胞无需任何非天然三磷酸即可生长,而表达具有非天然密码子的sfGFP的细胞的生长同时需要非天然脱氧核糖三磷酸和核糖三磷酸。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图8A显示了随介质中PrK浓度变化的用tRNAPyl解码AXC和GXC密码子的凝胶。对从表达具有所示位置151的密码子/反密码子的sfGFP和tRNAPyl的细胞中纯化的sfGFP进行蛋白质印迹,点击缀合TAMRA并向介质中添加所示浓度的PrK。从如图2B中所述收集的细胞中诱导并纯化了sfGFP。用α-GFP抗体探测蛋白质印迹并成像,以检测sfGFP和缀合的TAMRA。
图8B显示了随介质中PrK浓度变化的用tRNAPyl解码AXC和GXC密码子的图。表达分别具有所示位置151的密码子和反密码子的sfGFP和tRNAPyl的细胞的荧光(在c中示出的最后时间点测量)随介质中PrK浓度变化。0和20mM PrK的荧光值分别与(–)和(+)PrK值相同,如图2B示出。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
图8C显示了荧光的时程分析。为了清楚起见,对于每个密码子/反密码子对和PrK浓度,仅示出一种代表性培养物。不受理论的束缚,我们将在不存在PrK时产生的sfGFP的低水平归因于内源性tRNA的解码和sfGFP中UBP保留的丧失(表5)。但是,随着介质中PrK的增加,含有PrK的sfGFP的相对量(图8A)和表达的sfGFP的绝对量(图8B和图8C)以剂量依赖的方式增加,最终导致PrK几乎全部掺入,表明可以用足够浓度的有装载的PrK-tRNAPyl(GYT)或PrK-tRNAPyl(GYC)有效地抑制AXC和GXC密码子的内源性连读。
图8D显示了对于图8C示出的实验,在各种浓度的PrK下细胞生长的时程分析。
图9显示了培养物的细胞生长,其荧光示于图3A中。数据以平均值±s.d.示出,n=4个培养物,各自繁殖自单独菌落。
表4|对于图1F和图2D中所述的实验,sfGFP中位置151处的氨基酸的相对丰度。通过LC-MS/MS分析从在具有或不具有tRNA的情况下表达sfGFP的细胞中纯化的sfGFP,该sfGFP和tRNA分别具有所示位置151的密码子和反密码子。报告肽LEYNFNSHNVX151ITADK(X=PrK或除K或R以外的任何鉴定的天然氨基酸)和LEYNFNSHNVX151(如果X=K或R)的提取的MS1离子强度表示为所有可观测报告肽的离子强度总和的百分比。值的表格对应于在sfGFP的位置151处检测到的所有氨基酸的平均相对丰度和95%CI,n=4个纯化的sfGFP样品,各自来自从单独菌落繁殖的培养物。从图1F和图2D所示的数据中排除了<0.1%(对相应的图中所示的密码子取平均)的值。
表5|UBP保留。如方法中所述,在sfGFP诱导之前和诱导结束的时间点测定了具有sfGFP的所示位置151密码子和所示tRNA的反密码子的质粒中的UBP保留。报告值是诱导过程中的平均UBP保留(从这两个时间点的保留来计算)±95%CI,n=4个培养物,各自繁殖自单独菌落,但用星号所示的值除外,其n=3。n/a,不适用(因为相关序列是天然的或不存在的)。从在20mM PrK或5mM pAzF存在下生长的培养物中分离所有质粒(Ser解码实验除外)。SerRS示出装载有内源性大肠杆菌合成酶。减号表示具有tRNAPyl的细胞中不存在PylRS或不存在异位表达的tRNA。用§所示的行中的保留对应于这样的培养物,从其中还纯化了sfGFP并通过LC-MS/MS和/或对TAMRA缀合的sfGFP的蛋白质印迹进行分析(参见图1F(Ser)、图2D(PrK)和图3B(pAzF));带星号的行对应于图7A-D中分析的培养物。尽管所有四种非天然三磷酸通过相同的转运蛋白进入细胞导致竞争性地抑制彼此的导入,但在介质中存在(+)或不存在(-)NaMTP和/或TPT3TP的情况下,UBP保留均未观察到差异。这些数据以及两种非天然核糖三磷酸对具有高保真PrK掺入的高水平sfGFP表达的要求(图7A-D)共同证明,YZ3中PtNTT2转运蛋白的表达水平导入了维持UBP复制和转录必要的必需水平的非天然三磷酸。
表6|在Ser、Prk和pAzF掺入实验中表达的sfGFP蛋白的产量。从纯化的蛋白质总量和用于纯化的培养物体积计算出产量(参见方法)。数据是平均值±s.d.(n=4个sfGFP样品,各自纯化自从单独菌落繁殖的培养物),并且是从图1F(对于SerRS)和图2D(对于PylRS)中所分析的相同培养物以及对应于图3A(对于pAzFRS)中的(+)pAzF样品的培养物中测定。纯化的sfGFP的产量与其纯化自的培养物的平均总荧光(未相对于OD600标准化)相当。荧光值对应于收集细胞用于sfGFP纯化的时间点;参见图1C(Ser)、图2B(PrK)和图3A(pAzF)。
具体实施方式
某些术语
除非另有定义,否则本文所使用的所有技术和科学术语具有与所要求保护的主题所属领域的技术人员通常所理解的相同含义。应该理解,前面的一般描述和下面的详细描述仅是示例性和说明性的,并且不限制所要求保护的任何主题。在本申请中,单数的使用包括复数,除非另有明确说明。必须注意,在说明书和所附权利要求书中使用的单数形式“一个”“一种”和“该”包括复数指示物,除非上下文另有明确规定。在本申请中,除非另有说明,否则“或”的使用表示“和/或”。此外,术语“包括”以及其他形式(例如“包含”“含有”和“具有”)的使用不是限制性的。
如本文所用,范围和量可以表述为“约”特定值或范围。约还包括该确切量。因此,“约5μL”指“约5μL”,也指“5μL”。通常,术语“约”包括预期在实验误差内的量。
在此使用的章节标题仅用于组织目的,而不应被解释为限制所述的主题。
概述
生命信息由四个字母的遗传字母表编码,其通过选择性地形成两种碱基对——(d)G–(d)C和(d)A–dT/U成为可能。在两个合成核苷酸之间形成的第三个非天然碱基对(UBP)扩展了该系统,从而增加了信息储存的潜力,并具有深远的学术和实践意义。在已报道的各种各样的合成核苷酸类似物中,有一些在原本天然的DNA双链体中彼此稳定配对,但不被聚合酶识别,表明控制双链体DNA中稳定配对的力与控制聚合酶介导的复制的力不相同。因此,已经采取了不同的方法来开发可复制的UBP,例如,被设计为通过天然核苷酸不采用的互补氢键键合(H键合)模式进行相互作用的UBP。尽管天然碱基对也通过H键合形成,但没有理由先验地假设H键合是唯一足以支撑遗传信息的储存(或检索)的力。例如,已经证明,大肠杆菌DNA聚合酶I的Klenow片段(Kf)将dA与非天然核苷酸dF配对,该非天然核苷酸dF的二氟甲苯核碱基是胸腺嘧啶的形状模拟物,其无法形成较强的H键合。这支持了DNA复制的“几何选择”机制,并表明除H键合以外的力也有助于复制。
在体外复制、转录和翻译成蛋白质的UBP的开发为自然信息的储存和检索下潜在的力提供了深入理解,并且还实现在化学和合成生物学中的广泛应用。但是,开发UBP的许多努力的最终目标是在体内将其用作半合成生物体(SSO)——一种稳定地储存和检索增加的(非天然的或合成的,即人造的)信息的生物体的基础。此外,这种SSO具有革命性的实际应用,包括用于人类健康的实际应用。最值得注意的是,SSO革新了不断发展的蛋白质治疗剂领域。但是,与传统的小分子治疗剂比,由于从二十种天然氨基酸可得的化学多样性有限,因此蛋白质治疗剂的分子性质受到严重限制。
我们最近报道了一种大肠杆菌SSO的创建,其借助于来自三角褐指藻(Phaeodactylum tricornutum,PtNTT2)的核苷三磷酸转运蛋白导入来自介质的必需的非天然三磷酸,然后使用它们来复制含有UBP dNaM-dTPT3的质粒。自此我们已经示出,含有该UBP的DNA可以通过T7 RNA聚合酶在SSO中转录,并且当将非天然核苷酸掺入mRNA的密码子时,装载有ncAA并在其反密码子中含有同源非天然核苷酸的不同tRNA可以有效地并选择性地解码该非天然密码子。由于UBP可能在不同密码子的不同位置结合,暗示UBP可以用于编码具有多个不同ncAA的蛋白质。
本文在某些实施方案中公开了利用突变tRNA合成包含非天然氨基酸的蛋白质的方法、组合物和试剂盒。在一些情况下,蛋白质在无细胞翻译系统中合成。在一些情况下,蛋白质在细胞或半合成生物体(SSO)中合成。在一些情况下,半合成生物体包含微生物。在一些情况下,半合成生物体包含细菌。在一些情况下,半合成生物体包含大肠杆菌。在一些情况下,突变tRNA含有突变反密码子序列。在一些情况下,突变反密码子序列是表1所示的反密码子序列。在一些情况下,突变反密码子序列是表2所示的反密码子序列。在一些情况下,突变反密码子序列是表3所示的反密码子序列。
表1
GGY | GYG | YGG |
GAY | GYA | YGA |
GCY | GYC | YGC |
GUY | GYU | YGU |
CAY | CYA | YCA |
CGY | CYG | YCG |
CUY | CYU | YCU |
CCY | CYC | YCC |
AAY | AYA | YAA |
AGY | AYG | YAG |
ACY | AYC | YAC |
AUY | AYU | YAU |
UUY | UYU | YUU |
UAY | UYA | YUA |
UGY | UYG | YUG |
UCY | UYC | YUC |
GYY | YGY | YYG |
CYY | YCY | YYC |
AYY | YAY | YYA |
UYY | YUY | YYU |
YYY |
表2
表3
GXY | GYX | XYG |
YXG | XGY | YGX |
AXY | AYX | XYA |
YXA | XAY | YAX |
CXY | CYX | XYC |
YXC | XCY | YCX |
UXY | UYX | XYU |
YXU | XUY | YUX |
XYY | XXY | YXX |
YXX | YXY | XYX |
在一些情况下,突变tRNA的突变反密码子与突变密码子配对。在一些实施方案中,突变密码子是表1所示的突变密码子。在一些实施方案中,突变密码子是表2所示的突变密码子。在一些实施方案中,突变密码子是表3所示的突变密码子。
在一些实施方案中,表1、表2和表3中所示的Y和X代表非天然核苷酸的非天然碱基。在一些实施方案中,非天然碱基选自2-氨基腺嘌呤-9-基、2-氨基腺嘌呤、2-F-腺嘌呤、2-硫尿嘧啶、2-硫胸腺嘧啶、2-硫胞嘧啶、腺嘌呤和鸟嘌呤的2-丙基和烷基衍生物、2-氨基-腺嘌呤、2-氨基-丙基-腺嘌呤、2-氨基吡啶、2-吡啶酮、2’-脱氧尿苷、2-氨基-2’-脱氧腺苷、3-脱氮鸟嘌呤、3-脱氮腺嘌呤、4-硫尿嘧啶、4-硫胸腺嘧啶、尿嘧啶-5-基、次黄嘌呤-9-基(I)、5-甲基-胞嘧啶、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、5-溴尿嘧啶和5-三氟甲基尿嘧啶以及5-溴胞嘧啶和5-三氟甲基胞嘧啶;5-卤代尿嘧啶、5-卤代胞嘧啶、5-丙炔基-尿嘧啶、5-丙炔基胞嘧啶、5-尿嘧啶、5-取代的、5-卤代、5-取代的嘧啶、5-羟基胞嘧啶、5-溴胞嘧啶、5-溴尿嘧啶、5-氯胞嘧啶、氯化胞嘧啶、环胞嘧啶、阿糖胞苷、5-氟胞嘧啶、氟嘧啶、氟尿嘧啶、5,6-二氢胞嘧啶、5-碘胞嘧啶、羟基脲、碘尿嘧啶、5-硝基胞嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶和5-碘尿嘧啶、腺嘌呤和鸟嘌呤的6-烷基衍生物、6-氮嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、氮胞嘧啶、6-偶氮胸腺嘧啶、6-硫鸟嘌呤、7-甲基鸟嘌呤、7-甲基腺嘌呤、7-脱氮鸟嘌呤、7-脱氮鸟苷、7-脱氮-腺嘌呤、7-脱氮-8-氮鸟嘌呤、8-氮鸟嘌呤、8-氮腺嘌呤、8-卤代、8-氨基、8-硫醇、8-硫代烷基和8-羟基取代的腺嘌呤和鸟嘌呤;N4-乙基胞嘧啶、N-2取代的嘌呤、N-6取代的嘌呤、O-6取代的嘌呤、增加双链体形成的稳定性的那些、通用核酸、疏水核酸、混杂核酸、尺寸扩展的核酸、氟化核酸、三环嘧啶、吩噁嗪胞苷([5,4-b][l,4]苯并噁嗪-2(3H)-酮)、吩噻嗪胞苷(1H-嘧啶并[5,4-b][l,4]苯并噻嗪-2(3H)-酮)、G夹、吩噁嗪胞苷(9-(2-氨基乙氧基)-H-嘧啶并[5,4-b][1,4]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、次黄嘌呤、黄嘌呤、4-乙酰胞嘧啶、5-(羧基羟甲基)尿嘧啶、5-羧甲基氨基甲基-2-硫尿苷、5-羧甲基氨基甲基尿嘧啶、二氢尿嘧啶、β-D-半乳糖基辫苷、肌苷、N6-异戊烯腺嘌呤、1-甲基鸟嘌呤、1-甲基肌苷、2,2-二甲基鸟嘌呤、2-甲基腺嘌呤、2-甲基鸟嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-腺嘌呤、7-甲基鸟嘌呤、5-甲基氨基甲基尿嘧啶、5-甲氧基氨基甲基-2-硫尿嘧啶、β-D-甘露糖基辫苷、5’-甲氧基羧甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-N6-异戊烯腺嘌呤、尿嘧啶-5氧乙酸、怀丁氧苷、假尿嘧啶、辫苷、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-氧乙酸甲酯、尿嘧啶-5-氧乙酸、5-甲基-2-硫尿嘧啶、3-(3-氨基-3-N-2-羧基丙基)尿嘧啶、(acp3)w和2,6-二氨基嘌呤,以及其中嘌呤或嘧啶碱基被杂环取代的那些。
在一些情况下,非天然核苷酸选自(仅示出核碱基部分,为清楚起见省略了核糖和磷酸骨架):
在一些情况下,非天然核苷酸选自(仅显示核碱基部分,为清楚起见省略了核糖和磷酸骨架):
在一些情况下,非天然核苷酸进一步包含非天然糖部分。在一些情况下,非天然糖部分选自在2’位置处的下组修饰:OH;取代的低级烷基、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,其中烷基、烯基和炔基可以为取代或未取代的C1-C10烷基、C2-C10烯基、C2-C10炔基、-O[(CH2)nO]mCH3、-O(CH2)nOCH3、-O(CH2)nNH2、-O(CH2)nCH3、-O(CH2)n-ONH2和-O(CH2)nON[(CH2)nCH3)]2,其中n和m为从1至约10;和/或选自在5’位置处的下组修饰:5’-乙烯基、5’-甲基(R或S)、在4’位置处的修饰、4’-S、杂环烷基、杂环烷芳基、氨基烷基氨基、聚烷基氨基、取代的甲硅烷基、RNA剪切基团、报告基团、嵌入剂、用于改善寡核苷酸的药代动力学性质的基团,或用于改善寡核苷酸的药效学性质的基团,及其任意组合。
在一些情况下,突变反密码子或突变密码子进一步包含非天然骨架。在一些情况下,突变反密码子进一步包含非天然骨架。在一些情况下,突变密码子进一步包含非天然骨架。在一些情况下,非天然骨架选自硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基烷基磷酸三酯、C1-C10膦酸酯、3’-亚烷基膦酸酯、手性膦酸酯、次膦酸酯、磷酰胺酯、3’-氨基磷酰胺酯、氨基烷基磷酰胺酯、硫逐磷酰胺酯、硫逐烷基膦酸酯、硫逐烷基磷酸三酯和硼烷磷酸酯。
在一些情况下,非天然核苷酸被聚合酶识别。在一些情况下,聚合酶是DNA聚合酶、RNA聚合酶或逆转录酶。在一些情况下,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、9°NmTM、TherminatorTMDNA聚合酶、Tne、Tma、TfI、Tth、TIi、Stoffel片段、VentTM和DeepVentTMDNA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、大肠杆菌DNA聚合酶I、大肠杆菌DNA聚合酶III、古细菌DP1I/DP2DNA聚合酶II、9°N DNA聚合酶、Taq DNA聚合酶、DNA聚合酶、Pfu DNA聚合酶、SP6 RNA聚合酶、RB69 DNA聚合酶、禽成髓细胞瘤病毒(AMV)逆转录酶、莫洛尼鼠白血病病毒(MMLV)逆转录酶、II逆转录酶和III逆转录酶。
在一些情况下,聚合酶是DNA聚合酶1-Klenow片段、Vent聚合酶、DNA聚合酶、KOD DNA聚合酶、Taq聚合酶、T7DNA聚合酶、T7 RNA聚合酶、TherminatorTMDNA聚合酶、POLB聚合酶、SP6 RNA聚合酶、大肠杆菌DNA聚合酶I、大肠杆菌DNA聚合酶III、禽成髓细胞瘤病毒(AMV)逆转录酶、莫洛尼鼠白血病病毒(MMLV)逆转录酶、II逆转录酶或III逆转录酶。
在一些情况下,在转录过程中聚合酶将非天然核苷酸掺入mRNA中,以产生含有突变密码子的突变mRNA。在一些情况下,在转录过程中聚合酶将非天然核苷酸掺入mRNA中,以产生突变mRNA。
在一些情况下,在转录过程中聚合酶将非天然核苷酸掺入tRNA中,以产生含有突变反密码子的突变tRNA。在一些情况下,在转录过程中聚合酶将非天然核苷酸掺入tRNA中,以产生突变tRNA。
在一些情况下,突变tRNA代表非天然氨基酸残基。在一些情况下,非天然氨基酸残基是非天然氨基酸,诸如Liu C.C.,Schultz,P.G.Annu.Rev.Biochem.2010,79,413中所述的那些。
在一些情况下,利用突变tRNA和突变mRNA在翻译过程中产生含有非天然氨基酸的蛋白质。在一些情况下,含有非天然氨基酸的蛋白质在无细胞翻译系统下产生。在一些情况下,蛋白质在细胞或半合成生物体(SSO)中合成。在一些情况下,半合成生物体包含微生物。在一些情况下,半合成生物体包含细菌。在一些情况下,半合成生物体包含大肠杆菌。
核酸
核酸(例如,在本文中也称为靶核酸、靶核苷酸序列、感兴趣的核酸序列或感兴趣的核酸区域)可以来自任何来源或组合物,诸如DNA、cDNA、gDNA(基因组DNA)、RNA、siRNA(短抑制RNA)、RNAi、tRNA或mRNA,并且可以是任何形式(例如,线性、环状、超螺旋、单链、双链等)。核酸可包含核苷酸、核苷或多核苷酸。核酸可包含天然和非天然核酸。核酸还可以包含非天然核酸,诸如DNA或RNA类似物(例如,含有碱基类似物、糖类似物和/或非天然骨架等)。应当理解,术语“核酸”并不指代或推断出多核苷酸链的具体长度,因此多核苷酸和寡核苷酸也包括在定义中。示例性天然核苷酸包括但不限于ATP、UTP、CTP、GTP、ADP、UDP、CDP、GDP、AMP、UMP、CMP、GMP、dATP、dTTP、dCTP、dGTP、dADP、dTDP、dCDP、dGDP、dAMP、dTMP、dCMP和dGMP。示例性天然脱氧核糖核苷酸包括dATP、dTTP、dCTP、dGTP、dADP、dTDP、dCDP、dGDP、dAMP、dTMP、dCMP和dGMP。示例性天然核糖核苷酸包括ATP、UTP、CTP、GTP、ADP、UDP、CDP、GDP、AMP、UMP、CMP和GMP。对于RNA,尿嘧啶碱基是尿苷。核酸有时是载体、质粒、噬菌体、自主复制序列(ARS)、着丝粒、人工染色体、酵母人工染色体(例如,YAC)或其他能够复制或被复制的核酸。非天然核酸可以是核酸类似物。
非天然核酸
核苷酸类似物或非天然核苷酸包含含有对碱基、糖或磷酸部分的某种类型修饰的核苷酸。修饰可以包含化学修饰。修饰可以是例如3’OH或5’OH基团、骨架、糖组分或核苷酸碱基的修饰。修饰可以包括添加非天然存在的接头分子和/或链间或链内交联。一方面,修饰的核酸包含3’OH或5’OH基团、骨架、糖组分或核苷酸碱基中的一个或多个的修饰,和/或非天然存在的接头分子的添加。一方面,修饰的骨架包含除磷酸二酯骨架以外的骨架。一方面,修饰的糖包含除脱氧核糖以外的糖(在修饰的DNA中)或除核糖以外的糖(修饰的RNA)。一方面,修饰的碱基包含除腺嘌呤、鸟嘌呤、胞嘧啶或胸腺嘧啶以外的碱基(在修饰的DNA中)或除腺嘌呤、鸟嘌呤、胞嘧啶或尿嘧啶以外的碱基(在修饰的RNA中)。
核酸可以包含至少一种修饰的碱基。对碱基部分的修饰将包括A、C、G和T/U以及不同的嘌呤或嘧啶碱基的天然和合成修饰。在一些实施方案中,修饰是腺嘌呤、鸟嘌呤、胞嘧啶或胸腺嘧啶的修饰形式(在修饰的DNA中)或腺嘌呤、鸟嘌呤、胞嘧啶或尿嘧啶的修饰形式(修饰的RNA)。
非天然核酸的修饰的碱基包括但不限于尿嘧啶-5-基、次黄嘌呤-9-基(I)、2-氨基腺嘌呤-9-基、5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、腺嘌呤和鸟嘌呤的6-甲基和其他烷基衍生物、腺嘌呤和鸟嘌呤的2-丙基和其他烷基衍生物、2-硫尿嘧啶、2-硫胸腺嘧啶和2-硫胞嘧啶、5-卤代尿嘧啶和胞嘧啶、5-丙炔基尿嘧啶和胞嘧啶、6-偶氮尿嘧啶、胞嘧啶和胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-卤代、8-氨基、8-硫醇、8-硫代烷基、8-羟基和其他8-取代的腺嘌呤和鸟嘌呤、5-卤代(尤其是5-溴)、5-三氟甲基和其他5-取代的尿嘧啶和胞嘧啶、7-甲基鸟嘌呤和7-甲基腺嘌呤、8-氮鸟嘌呤和8-氮腺嘌呤、7-脱氮鸟嘌呤和7-脱氮腺嘌呤以及3-脱氮鸟嘌呤和3-脱氮腺嘌呤。某些非天然核酸,例如5-取代的嘧啶、6-氮嘧啶和N-2取代的嘌呤、N-6取代的嘌呤、O-6取代的嘌呤、2-氨基丙基腺嘌呤、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、5-甲基胞嘧啶、增加双链体形成的稳定性的那些、通用核酸、疏水核酸、混杂核酸、尺寸扩展的核酸、氟化核酸、5-取代的嘧啶、6-氮嘧啶和N-2、N-6和0-6取代的嘌呤,包括2-氨基丙基腺嘌呤、5-丙炔基尿嘧啶和5-丙炔基胞嘧啶。5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、腺嘌呤和鸟嘌呤的6-甲基、其他烷基衍生物、腺嘌呤和鸟嘌呤的2-丙基和其他烷基衍生物、2-硫尿嘧啶、2-硫胸腺嘧啶和2-硫胞嘧啶、5-卤代尿嘧啶、5-卤代胞嘧啶、5-丙炔基(-C≡C-Cl1/4)尿嘧啶、5-丙炔基胞嘧啶、嘧啶核酸的其他炔基衍生物、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-卤代、8-氨基、8-硫醇、8-硫烷基、8-羟基和其他8-取代的腺嘌呤和鸟嘌呤、5-卤代(尤其是5-溴)、5-三氟甲基、其他5-取代的尿嘧啶和胞嘧啶、7-甲基鸟嘌呤、7-甲基腺嘌呤、2-F-腺嘌呤、2-氨基-腺嘌呤、8-氮鸟嘌呤、8-氮腺嘌呤、7-脱氮鸟嘌呤、7-脱氮腺嘌呤、3-脱氮鸟嘌呤、3-脱氮腺嘌呤、三环嘧啶、吩噁嗪胞苷([5,4-b][l,4]苯并噁嗪-2(3H)-酮)、吩噻嗪胞苷(1H-嘧啶并[5,4-b][1,4]苯并噻嗪-2(3H)-酮)、G夹、吩噁嗪胞苷(例如9-(2-氨基乙氧基)-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)、其中嘌呤或嘧啶碱基被其他杂环取代的那些、7-脱氮-腺嘌呤、7-脱氮鸟苷、2-氨基吡啶、2-吡啶酮、氮胞嘧啶、5-溴胞嘧啶、溴尿嘧啶、5-氯胞嘧啶、氯化胞嘧啶、环胞嘧啶、阿糖胞苷、5-氟胞嘧啶、氟嘧啶、氟尿嘧啶、5,6-二氢胞嘧啶、5-碘胞嘧啶、羟基脲、碘尿嘧啶、5-硝基胞嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶和5-碘尿嘧啶、2-氨基-腺嘌呤、6-硫鸟嘌呤、2-硫胸腺嘧啶、4-硫胸腺嘧啶、5-丙炔基-尿嘧啶、4-硫尿嘧啶、N4-乙基胞嘧啶、7-脱氮鸟嘌呤、7-脱氮-8-氮鸟嘌呤、5-羟基胞嘧啶、2’-脱氧尿苷、2-氨基-2’-脱氧腺苷,以及在下列文件中描述的那些:美国专利号3,687,808;4,845,205;4,910,300;4,948,882;5,093,232;5,130,302;5,134,066;5,175,273;5,367,066;5,432,272;5,457,187;5,459,255;5,484,908;5,502,177;5,525,711;5,552,540;5,587,469;5,594,121;5,596,091;5,614,617;5,645,985;5,681,941;5,750,692;5,763,588;5,830,653和6,005,096;WO 99/62923;Kandimalla等人,(2001)Bioorg.Med.Chem.9:807-813;The Concise Encyclopedia Of Polymer Science And Engineering,Kroschwitz,J.I.著,JohnWiley&Sons,1990,858-859;Englisch等人,Angewandte Chemie,International Edition,1991,30,613;以及Sanghvi,Y.S.,Chapter 15,AntisenseResearch and Applications,Crooke,S.T.和Lebleu,B.著,CRC Press,1993,273-288。其他碱基修饰可以在例如美国专利号3,687,808;Englisch等人,Angewandte Chemie,International Edition,1991,30,613;和Sanghvi,Y.S.,Chapter 15,AntisenseResearch and Applications,pages 289-302,Crooke,S.T.和Lebleu,B.著,CRC Press,1993中找到。
包含各种杂环碱基和各种糖部分(和糖类似物)的非天然核酸是本领域可得到的,并且该核酸可以包括一种或几种不同于天然存在核酸的主要五个碱基组分的杂环碱基。例如,杂环碱基可包括尿嘧啶-5-基、胞嘧啶-5-基、腺嘌呤-7-基、腺嘌呤-8-基、鸟嘌呤-7-基、鸟嘌呤-8-基、4-氨基吡咯并[2.3-d]嘧啶-5-基、2-氨基-4-氧吡咯并[2,3-d]嘧啶-5-基、2-氨基-4-氧吡咯并[2.3-d]嘧啶-3-基基团,其中嘌呤通过9位,嘧啶通过1-位,吡咯并嘧啶通过7-位并且吡唑并嘧啶通过1-位附接到核酸的糖部分。
核苷酸类似物也可以在磷酸部分被修饰。修饰的磷酸部分包括但不限于那些可被修饰成使得两个核苷酸之间的键含有以下各项的部分:硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基烷基磷酸三酯、甲基和其他烷基膦酸酯(包括3’-亚烷基膦酸酯和手性膦酸酯)、次膦酸酯、磷酰胺酯(包括3’-氨基磷酰胺酯和氨基烷基磷酰胺酯)、硫逐磷酰胺酯、硫逐烷基膦酸酯、硫逐烷基磷酸三酯和硼烷磷酸酯。可以理解的是,两个核苷酸之间的这些磷酸酯或经修饰磷酸酯键可以是通过3’-5’键或2’-5’键,并且该键可以含有反转的极性,例如3’-5’至5’-3’或2’-5’至5’-2’。也包括各种盐、混合盐和游离酸形式。许多美国专利教导了如何制备和使用含有修饰的磷酸酯的核苷酸,包括但不限于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;和5,625,050,其每一个通过引用并入本文。
非天然核酸可包括2’,3’-双脱氧-2’,3’-双脱氢核苷(PCT/US2002/006460)、5’-取代的DNA和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;和Mikhailov等人,Nucleosides&Nucleotides,1991,10(1-3),339-343;Leonid等人,1995,14(3-5),901-905;以及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),或制备成具有修饰碱基的单磷酸的5’-取代的单体(Wang等人,Nucleosides Nucleotides&Nucleic Acids,2004,23(1&2),317-337)。
非天然核酸可在糖环的5’-位置和2’-位置包含修饰(PCT/US94/02993),诸如5’-CH2取代的2’-O保护的核苷(Wu等人,Helvetica Chimica Acta,2000,83,1127-1143以及Wu等人,Bioconjugate Chem.1999,10,921-924)。非天然核酸可以包括已准备好掺入寡核苷酸中的酰胺连接的核苷二聚体,其中二聚体中的3’连接的核苷(5’至3’)包含2’-OCH3和5’-(S)-CH3(Mesmaeker等人,Synlett,1997,1287-1290)。非天然核酸可以包括2’-取代的5’-CH2(或O)修饰的核苷(PCT/US92/01020)。非天然核酸可以包括5’亚甲基膦酸DNA和RNA单体,以及二聚体(Bohringer等人,Tet.Lett.,1993,34,2723-2726;Collingwood等人,Synlett,1995,7,703-705;以及Hutter等人,Helvetica Chimica Acta,2002,85,2777-2806)。非天然核酸可以包括具有2’取代的5’-膦酸单体(US 2006/0074035)和其他修饰的5’-膦酸单体(WO 97/35869)。非天然核酸可以包括5’-修饰的亚甲基膦酸单体(EP614907和EP629633)。非天然核酸可以包括在5’和6’位置包含羟基基团的5’或6’-膦酸核糖核苷类似物(Chen等人,Phosphorus,Sulphur 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以及Hampton等人,J.Med.Chem.,1976,19(8),1029-1033)。非天然核酸可以包括5’-膦酸脱氧核糖核苷单体和具有5’-磷酸基团的二聚体(Nawrot等人,Oligonucleotides,2006,16(1),68-82)。非天然核酸可以包括具有6’-膦酸基团的核苷,其中5’或/和6’-位置未被取代或被硫代叔丁基基团(SC(CH3)3)(及其类似物)、亚甲氨基基团(CH2NH2)(及其类似物)或氰基基团(CN)(及其类似物)取代(Fairhurst等人,Synlett,2001,4,467-472;Kappler等人,J.Med.Chem.,1986,29,1030-1038和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以及Hampton等人,J.Am.Chem.Soc,1973,95(13),4404-4414)。
非天然核酸还可以包括糖部分的修饰。本发明的核酸可以任选地含有一种或多种其中糖基团已被修饰的核苷。此类糖修饰的核苷可赋予增强的核酸酶稳定性、增加的结合亲和力或一些其他有益的生物学特性。在某些实施方案中,核酸包含化学修饰的呋喃核糖环部分。化学修饰的呋喃核糖环的实例包括但不限于添加取代基(包括5’和/或2’取代基;桥接两个环原子以形成双环核酸(BNA);用S、N(R)或C(R1)(R2)(R=H、C1-C12烷基或保护基团)替代核糖基环的氧原子;及其组合。化学修饰的糖的实例可以在WO 2008/101157、US2005/0130923和WO 2007/134181中找到。
修饰的核酸可以包含修饰的糖或糖类似物。因此,除了核糖和脱氧核糖之外,糖部分可以是戊糖、脱氧戊糖、己糖、脱氧己糖、葡萄糖、阿拉伯糖、木糖、来苏糖和糖“类似物”环戊基基团。糖可以是吡喃糖基或呋喃糖基形式。糖部分可以是核糖、脱氧核糖、阿拉伯糖或2’-O-烷基核糖的呋喃糖苷,并且糖可以以[α]或[β]异头构型附接至相应的杂环碱基。糖修饰包括但不限于2’-烷氧基-RNA类似物、2’-氨基-RNA类似物、2’-氟代DNA和2’-烷氧基或氨基-RNA/DNA嵌合体。例如,糖修饰可以包括2’-O-甲基尿苷和2’-O-甲基胞苷。糖修饰包括2’-O-烷基取代的脱氧核糖核苷和2’-O-乙二醇样的核糖核苷。这些糖或糖类似物以及其中这些糖或类似物附接至杂环碱基(核酸碱基)的相应“核苷”的制备是已知的。也可以进行糖修饰并将其与其他修饰结合。
对糖部分的修饰包括核糖和脱氧核糖的天然修饰以及非天然修饰。糖修饰包括但不限于在2’位置处的以下修饰: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、烯基和炔基可以是取代或未取代的C1至C10烷基或C2至C10烯基和炔基。2’糖修饰还包括但不限于-O[(CH2)nO]mCH3、-O(CH2)nOCH3、-O(CH2)nNH2、-O(CH2)nCH3、-O(CH2)n-ONH2和-O(CH2)nON[(CH2)nCH3)]2,其中n和m为从1至约10。
在2’位置处的其他修饰包括但不限于:C1-C10低级烷基、取代的低级烷基、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、NH2、杂环烷基、杂环烷芳基、氨基烷基氨基、聚烷基氨基、取代的甲硅烷基、RNA剪切基团、报告基团、嵌入剂、用于改善寡核苷酸的药代动力学性质的基团,或用于改善寡核苷酸的药效学性质的基团,以及具有类似性质的其他取代基。也可以在糖上的其他位置进行类似的修饰,特别是在3’端核苷酸上或2’-5’连接的寡核苷酸中的糖的3’位置和5’端核苷酸的5’位置。修饰的糖还将包括在桥接环的氧处含有修饰例如CH2和S的那些。核苷酸糖类似物也可以具有代替戊呋喃糖基糖的糖模拟物,例如环丁基部分。有许多美国专利教导了此类修饰糖结构的制备,例如4,981,957;5,118,800;5,319,080;5,359,044;5,393,878;5,446,137;5,466,786;5,514,785;5,519,134;5,567,811;5,576,427;5,591,722;5,597,909;5,610,300;5,627,053;5,639,873;5,646,265;5,658,873;5,670,633;4,845,205;5,130,302;5,134,066;5,175,273;5,367,066;5,432,272;5,457,187;5,459,255;5,484,908;5,502,177;5,525,711;5,552,540;5,587,469;5,594,121、5,596,091;5,614,617;5,681,941;和5,700,920,其每一个均通过引用整体并入本文,其详细描述了一系列碱基修饰。这些专利中的每一个均通过引用并入本文。
具有修饰糖部分的核酸的实例包括但不限于包含5’-乙烯基、5’-甲基(R或S)、4’-S、2’-F、2’-OCH3和2’-O(CH2)2OCH3取代基基团的核酸。在2’位置处的取代基也可以选自烯丙基、氨基、叠氮基、巯基、O-烯丙基、O-C C10烷基、OCF3、O(CH2)2SCH3、O(CH2)2-O-N(Rm)(Rn)和O-CH2-C(=O)-N(Rm)(Rn),其中每个Rm和Rn独立地为H或取代或未取代的C1-C10烷基。
在某些实施方案中,本发明的核酸包括一种或多种双环核酸。在某些此类实施方案中,双环核酸包含在4’核糖环原子与2’核糖环原子之间的桥。在某些实施方案中,本文提供的核酸包括一种或多种双环核酸,其中桥包含4’至2’双环核酸。这种4’至2’双环核酸的实例包括但不限于下式之一:4’-(CH2)-O-2’(LNA);4’-(CH2)-S-2’;4’-(CH2)2-O-2’(ENA);4’-CH(CH3)-O-2’和4’-CH(CH2OCH3)-O-2’及其类似物(参见2008年7月15日授权的美国专利7,399,845);4’-C(CH3)(CH3)-O-2’及其类似物(参见WO2009/006478、WO2008/150729、US2004/0171570、美国专利7,427,672、Chattopadhyaya等人,J.Org.Chem.,209,74,118-134以及于2008年12月8日公开的WO2008/154401)。还可参见,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(2007年7月4日);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;美国专利号7,053,207、6,268,490、6,770,748、6,794,499、7,034,133、6,525,191、6,670,461和7,399,845;国际申请WO 2004/106356、WO 1994/14226、WO 2005/021570和WO 2007/134181;美国专利公开号US2004/0171570、US2007/0287831和US2008/0039618;美国专利序列号12/129,154、60/989,574、61/026,995、61/026,998、61/056,564、61/086,231、61/097,787和61/099,844;和PCT国际申请号PCT/US2008/064591、PCT US2008/066154和PCT US2008/068922、PCT/DK98/00393;以及美国专利号4,849,513;5,015,733;5,118,800;和5,118,802。
在某些实施方案中,核酸可以包含相连接的核酸。核酸可以使用任何核酸间键连接在一起。核酸间连接基团的两个主要类别由磷原子的存在或不存在来定义。代表性的含磷的核酸间键包括但不限于磷酸二酯、磷酸三酯、甲基膦酸酯、磷酰胺酯和硫代磷酸酯(P=S)。代表性的不含磷的核酸间连接基团包括但不限于亚甲基甲基亚氨基(-CH2-N(CH3)-O-CH2-)、硫代二酯(-OC(O)-S-)、硫逐氨基甲酸酯(-O-C(O)(NH)-S-);硅氧烷(-O-Si(H)2-O-);和N,N*-二甲基肼(-CH2-N(CH3)-N(CH3)-)。在某些实施方案中,具有手性原子的核酸间键可以制备作为单独的对映异构体(例如,烷基膦酸酯和硫代磷酸酯)的外消旋混合物。非天然核酸可以含有单个修饰。非天然核酸可在一个部分内或不同部分之间含有多个修饰。
对核酸的骨架磷酸修饰包括但不限于甲基膦酸酯、硫代磷酸酯、磷酰胺酯(桥接或非桥接)、磷酸三酯、二硫代磷酸酯(phosphorodithioate)、二硫代磷酸酯(phosphodithioate)和硼烷磷酸酯,并且可以以任何组合使用。也可以使用其他非磷酸酯键。
在一些实施方案中,骨架修饰(例如,甲基膦酸酯、硫代磷酸酯、氨基磷酸酯和二硫代磷酸酯核苷酸间键)可以赋予经修饰核酸以免疫调节活性和/或增强它们在体内的稳定性。
磷衍生物(或修饰的磷酸基团)可以连接至糖或糖类似物部分,并且可以是一磷酸、二磷酸、三磷酸、烷基膦酸酯、硫代磷酸酯、二硫代磷酸酯、磷酰胺酯等。含有修饰的磷酸酯键或非磷酸酯键的示例性多核苷酸可在下列文件中找到:Peyrottes等人,(1996)Nucleic Acids Res.24:1841-1848;Chaturvedi等人,(1996)Nucleic Acids Res.24:2318-2323;和Schultz等人,(1996)Nucleic Acids Res.24:2966-2973;Matteucci(1997)"Oligonucleotide Analogs:an Overview"in Oligonucleotides as TherapeuticAgents,(DJ.Chadwick和G.Cardew著)John Wiley and Sons,NewYork,NY;(Zon(1993)"Oligonucleoside Phosphorothioates"in Protocols for Oligonucleotides andAnalogs,Synthesis and Properties(Agrawal著)Humana Press,pp.165-190);(Miller等人,(1971)JACS 93:6657-6665);(Jager等人,(1988)Biochem.27:7247-7246);(Nelson等人,(1997)JOC 62:7278-7287)(美国专利号5,453,496);Micklefield,J.2001,CurrentMedicinal Chemistry 8:1157-1179。
骨架修饰可包含用替代部分诸如阴离子、中性或阳离子基团代替磷酸二酯键。此类修饰的实例包括:阴离子核苷间键;N3’至P5’磷酰胺酯修饰;硼烷磷酸酯DNA;prooligonucleotide;中性核苷间键诸如甲基膦酸酯;酰胺连接的DNA;亚甲基(甲基亚氨基)键;formacetal和thioformacetal键;含有磺酰基基团的骨架;吗啉代寡核苷酸;肽核酸(PNA);和带正电荷的脱氧核糖核酸胍(DNG)寡核苷酸,Micklefield,J.2001,CurrentMedicinal Chemistry 8:1157-1179。修饰的核酸可以包含嵌合或混合的骨架,该嵌合的或混合的骨架包含一种或多种修饰,例如磷酸酯键的组合,诸如磷酸二酯键和硫代磷酸酯键的组合。
针对磷酸的取代物可以是例如短链烷基或环烷基核苷间键、混合的杂原子和烷基或环烷基核苷间键,或一个或多个短链杂原子或杂环核苷间键。这些包括具有吗啉基键的那些(部分由核苷的糖部分形成);硅氧烷骨架;硫化物、亚砜和砜骨架;甲乙酰基(formacetyl)和硫代甲乙酰基骨架;亚甲基甲乙酰基和硫代甲乙酰基骨架;含烯烃的骨架;氨基磺酸盐骨架;亚甲基亚氨基和亚甲基肼基骨架;磺酸酯和磺酰胺骨架;酰胺骨架;其他具有混合的N、O、S和CH2组成部分的取代物。许多美国专利公开了如何制备和使用这些类型的磷酸替代,包括但不限于5,034,506;5,166,315;5,185,444;5,214,134;5,216,141;5,235,033;5,264,562;5,264,564;5,405,938;5,434,257;5,466,677;5,470,967;5,489,677;5,541,307;5,561,225;5,596,086;5,602,240;5,610,289;5,602,240;5,608,046;5,610,289;5,618,704;5,623,070;5,663,312;5,633,360;5,677,437;和5,677,439,其每一个通过引用并入本文。还应理解,在核苷酸取代物中,核苷酸的糖和磷酸部分都可以被例如酰胺型键(氨基乙基甘氨酸)(PNA)替代。美国专利5,539,082、5,714,331和5,719,262教导了如何制备和使用PNA分子,其每一个均通过引用并入本文。(也参见Nielsen等人,Science,1991,254,1497-1500)。缀合物可以化学连接至核苷酸或核苷酸类似物。这样的缀合物包括但不限于脂质部分,例如胆固醇部分(Letsinger等人,Proc.Natl.Acad.Sci.USA,1989,86,6553-6556),胆酸(Manoharan等人,Bioorg.Med.Chem.Let.,1994,4,1053-1060),硫醚例如己基-S-三苯甲基硫醇(Manoharan等人,Ann.KY.Acad.Sci.,1992,660,306-309;Manoharan等人,Bioorg.Med.Chem.Let.,1993,3,2765-2770),硫代胆固醇(Oberhauser等人,Nucl.Acids Res.,1992,20,533-538),脂族链例如十二烷二醇或十一烷基残基(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),磷脂例如二-十六烷基-外消旋-甘油或三乙铵1-二-O-十六烷基-外消旋-甘油-S-H-膦酸酯(Manoharan等人,Tetrahedron Lett.,1995,36,3651-3654;Shea等人,Nucl.Acids Res.,1990,18,3777-3783),多胺或聚乙二醇链(Manoharan等人,Nucleosides&Nucleotides,1995,14,969-973),或金刚烷乙酸(Manoharan等人,Tetrahedron Lett.,1995,36,3651-3654)、棕榈基部分(Mishra等人,Biochem.Biophys.Acta,1995,1264,229-237),或十八烷基胺或己氨基-羰基-氧胆固醇部分(Crooke等人,J.Pharmacol.Exp.Ther.,1996,277,923-937)。许多美国专利教导了这些缀合物的制备,包括但不限于美国专利号4,828,979;4,948,882;5,218,105;5,525,465;5,541,313;5,545,730;5,552,538;5,578,717,5,580,731;5,580,731;5,591,584;5,109,124;5,118,802;5,138,045;5,414,077;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241,5,391,723;5,416,203,5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587,371;5,595,726;5,597,696;5,599,923;5,599,928和5,688,941,其每一个通过引用并入本文。
聚合酶
聚合酶的特别有用的功能是使用现有的核酸作为模板来催化核酸链的聚合。其他有用功能在本文其他地方描述。有用的聚合酶的实例包括DNA聚合酶和RNA聚合酶。
在例如需要非天然核酸掺入的多种情况(包括扩增、测序、标记、检测、克隆以及多种其他情况)下,非常需要改善聚合酶对非天然核酸的特异性、持续合成性或其他特征的能力。本发明提供了对非天然核酸具有修饰性质的聚合酶、制备这种聚合酶的方法、使用这种聚合酶的方法以及许多其他特征,这些特征在下面的全面评论中将变得显而易见。
在一些情况下,本文公开内容包括例如在DNA扩增期间将非天然核酸掺入生长中的模板拷贝中的聚合酶。在一些实施方案中,可以修饰聚合酶,使得聚合酶的活性位点被修饰成减少非天然核酸进入该活性位点的空间进入阻碍。在一些实施方案中,可以修饰聚合酶以提供与非天然核酸的一种或多种非天然特征的互补性。因此,本发明包括包含异源或重组聚合酶的组合物及其使用方法。
聚合酶可以使用与蛋白质工程化有关的方法来修饰。例如,可以基于晶体结构进行分子建模,以鉴定聚合酶上可以进行突变以修饰靶活性的位置。可以将被鉴定为替代目标的残基替代为使用能量最小化建模、同源性建模和/或保守氨基酸置换所选择的残基,诸如Bordo等人,J Mol Biol 217:721-729(1991)和Hayes等人,Proc Natl Acad Sci,USA99:15926-15931(2002)中所述的。
多种聚合酶中的任何一种均可用于本文阐述的方法或组合物中,包括例如,从生物系统中分离的基于蛋白质的酶及其功能变体。除非另有说明,否则对特定聚合酶的引用,例如以下示例的那些,应理解为包括其功能变体。在一些实施方案中,聚合酶是野生型聚合酶。在一些实施方案中,聚合酶是修饰的或突变的聚合酶。
还可以使用具有用于改善非天然核酸进入活性位点区域的特征以及在活性位点区域中与非天然核苷酸配位的特征的聚合酶。在一些实施方案中,修饰的聚合酶具有修饰的核苷酸结合位点。
在一些实施方案中,修饰的聚合酶对非天然核酸的特异性为野生型聚合酶对该非天然核酸的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的或野生型聚合酶对包含修饰的糖的非天然核酸的特异性为野生型聚合酶对天然核酸和/或不具有该修饰糖的非天然核酸的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的或野生型聚合酶对包含修饰的碱基的非天然核酸的特异性为野生型聚合酶对天然核酸和/或不具有该修饰碱基的非天然核酸的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的或野生型聚合酶对包含三磷酸的非天然核酸的特异性为野生型聚合酶对包含三磷酸的核酸和/或不具有该三磷酸的非天然核酸的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。例如,修饰的或野生型聚合酶对包含三磷酸的非天然核酸的特异性可以为野生型聚合酶对具有二磷酸或一磷酸的非天然核酸,或没有磷酸的非天然核酸或其组合的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。
在一些实施方案中,修饰的或野生型聚合酶对非天然核酸具有放宽的特异性。在一些实施方案中,修饰的或野生型聚合酶对非天然核酸具有特异性,并且对天然核酸的特异性为野生型聚合酶对该天然核酸的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的或野生型聚合酶对包含修饰的糖的非天然核酸具有特异性,并且对天然核酸的特异性为野生型聚合酶对该天然核酸的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的或野生型聚合酶对包含修饰的碱基的非天然核酸具有特异性,并且对天然核酸的特异性为野生型聚合酶对该天然核酸的特异性的至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。
核酸外切酶活性的缺乏可以是野生型特征或由变体或工程化聚合酶赋予的特征。例如,exo-Klenow片段是Klenow片段的突变版本,缺少3’至5’校对核酸外切酶活性。
本发明的方法可以用于扩展任何这样的DNA聚合酶的底物范围,所述DNA聚合酶缺乏固有的3至5’核酸外切酶校对活性或其中3至5’核酸外切酶校对活性已例如通过突变被禁用。DNA聚合酶的实例包括polA、polB(参见例如Parrel&Loeb,Nature Struc Biol2001)、polC、polD、polY、polX和逆转录酶(RT),但最好是能持续合成的高保真聚合酶(PCT/GB2004/004643)。在一些实施方案中,修饰的或野生型聚合酶基本上缺乏3’至5’校对核酸外切酶活性。在一些实施方案中,修饰的或野生型聚合酶对于非天然核酸基本上缺乏3’至5’校对核酸外切酶活性。在一些实施方案中,修饰的或野生型聚合酶具有3’至5’校对核酸外切酶活性。在一些实施方案中,修饰的或野生型聚合酶对于天然核酸具有3’至5’校对核酸外切酶活性,而对于非天然核酸则基本上缺乏3’至5’校对核酸外切酶活性。
在一些实施方案中,修饰的聚合酶的3’至5’校对核酸外切酶活性为野生型聚合酶的校对核酸外切酶活性的至少约60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的聚合酶对非天然核酸的3’至5’校对核酸外切酶活性为野生型聚合酶对天然核酸的校对核酸外切酶活性的至少约60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的聚合酶对非天然核酸具有3’至5’校对核酸外切酶活性,并且对天然核酸的3’至5’校对核酸外切酶活性为野生型聚合酶对天然核酸的校对核酸外切酶活性的至少约60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。在一些实施方案中,修饰的聚合酶对天然核酸的3’至5’校对核酸外切酶活性为野生型聚合酶对该天然核酸的校对核酸外切酶活性的至少约60%、70%、80%、90%、95%、97%、98%、99%、99.5%、99.99%。
在相关方面,本发明提供了制备修饰的聚合酶的方法,该方法包括在结构上建模亲本聚合酶,例如DNA聚合酶;鉴定一种或多种影响复合物稳定性或活性位点中的核苷酸接近或结合的复合物稳定性或核苷酸相互作用特征,或者核苷酸类似物在活性位点的互补性特征;以及使亲本聚合酶突变以包括或去除这些特征。例如,可以使聚合酶突变以改善非天然核苷酸到活性位点的空间接近性,或改善非天然核苷酸与聚合酶之间的电荷-电荷或疏水相互作用。该方法还包括测定与亲本聚合酶相比,所得的修饰的聚合酶是否显示出核苷酸或非天然核苷酸向生长中的核酸拷贝中掺入的增加。
聚合酶可以根据其从核酸的解离速率来表征。在一些实施方案中,聚合酶对于一种或多种天然和非天然核酸具有相对低的解离速率。在一些实施方案中,聚合酶对于一种或多种天然和非天然核酸具有相对高的解离速率。解离速率是聚合酶的活性,其可以被调节以在本文阐述的方法中调整反应速率。
当与特定的天然和/或非天然核酸或天然和/或非天然核酸的集合一起使用时,聚合酶可以根据其保真度来表征。保真度通常是指当制备核酸模板的拷贝时,聚合酶将正确的核酸掺入生长中的核酸链中的准确性。当天然和非天然核酸例如以相等的浓度存在而竞争聚合酶-链-模板核酸二元复合物中同一位点的链合成时,DNA聚合酶保真度可以通过正确和不正确的天然和非天然核酸掺入的比率来衡量。DNA聚合酶保真度可以计算为天然和非天然核酸的(kcat/Km)与不正确的天然和非天然核酸的(kcat/Km)的比率;其中kcat和Km是稳态酶动力学中的Michaelis-Menten参数(Fersht,A.R.(1985)Enzyme Structure andMechanism,第二版,p350,W.H.Freeman&Co.,New York.,通过引用并入本文)。在一些实施方案中,具有或不具有校对活性的聚合酶的保真度值为至少约100、1000、10,000、100,000或1×106。
可以使用检测具有特定结构的非天然核酸的掺入的测定法来筛选来自天然来源的聚合酶或其变体。在一实例中,可以筛选聚合酶的掺入非天然核酸或UBP(例如d5SICSTP、dNaMTP或d5SICSTP-dNaMTP UBP)的能力。可以使用与野生型聚合酶相比对非天然核酸表现出修饰的性质的聚合酶,例如异源聚合酶。例如,修饰的性质可以是,例如,Km、kcat、Vmax、在非天然核酸(或天然存在的核苷酸)存在下的聚合酶持续合成性、在非天然核酸存在下聚合酶的平均模板读取长度、聚合酶对非天然核酸的特异性、非天然核酸的结合速率、产物(焦磷酸、三磷酸等)的释放速率、分支速率或其任意组合。在一个实施方案中,修饰的性质是对于非天然核酸的Km降低和/或对于非天然核酸的kcat/Km或Vmax/Km升高。类似地,与野生型聚合酶相比,聚合酶任选地具有增加的非天然核酸结合速率,增加的产物释放速率和/或降低的分支速率。
同时,聚合酶可将天然核酸,例如A、C、G和T,掺入生长中的核酸拷贝中。例如,任选地,聚合酶显示出的对天然核酸的比活性为对应的野生型聚合酶的比活性的至少约5%(例如,5%、10%、25%、50%、75%、100%或更高),并且显示出的在模板存在下用天然核酸的持续合成性为在天然核酸存在下野生型聚合酶的该持续合成性的至少5%(例如,5%、10%、25%、50%、75%、100%或更高)。任选地,对于天然存在的核苷酸,聚合酶显示出为野生型聚合酶的至少约5%(例如,约5%、10%、25%、50%、75%或100%或更高)的kcat/Km或Vmax/Km。
本文中使用的可以有能力掺入具有特定结构的非天然核酸的聚合酶也可以使用定向进化方法来产生。核酸合成测定法可用于筛选对多种非天然核酸中的任一种具有特异性的聚合酶变体。例如,可以筛选聚合酶变体将非天然核酸或UBP(例如dTPT3、dNaM类似物或dTPT3-dNaM UBP)掺入核酸中的能力。在一些实施方案中,这样的测定是体外测定,例如,使用重组聚合酶变体。此类定向进化技术可用于筛选任何合适聚合酶的变体的针对本文阐述的任何非天然核酸的活性。
所述组合物中的修饰的聚合酶可以任选地是修饰的和/或重组的Φ29型DNA聚合酶。任选地,聚合酶可以是修饰的和/或重组的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。
通常可用于本发明的核酸聚合酶包括DNA聚合酶、RNA聚合酶、逆转录酶及其突变或改变的形式。DNA聚合酶及其性质在DNA Replication第二版,Kornberg和Baker,W.H.Freeman,New York,N.Y.(1991)中以及其他地方有详细描述。可用于本发明的已知常规DNA聚合酶包括但不限于激烈火球菌(Pyrococcus furiosus,Pfu)DNA聚合酶(Lundberg等人,1991,Gene,108:1,Stratagene)、沃氏火球菌(Pyrococcus woesei,Pwo)DNA聚合酶(Hinnisdaels等人,1996,Biotechniques,20:186-8,Boehringer Mannheim)、嗜热栖热菌(Thermus thermophilus,Tth)DNA聚合酶(Myers和Gelfand 1991,Biochemistry30:7661)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)DNA聚合酶(Stenesh和McGowan,1977,Biochim Biophys Acta 475:32)、Thermococcus litoralis(TIi)DNA聚合酶(也称为VentTMDNA聚合酶,Cariello等人,1991,Polynucleotides Res,19:4193,New EnglandBiolabs)、9°NmTMDNA聚合酶(New England Biolabs)、Stoffel片段、Thermo(Amersham Pharmacia Biotech UK)、TherminatorTM(New England Biolabs)、海栖热袍菌(Thermotoga maritima,Tma)DNA聚合酶(Diaz和Sabino,1998Braz J Med.Res,31:1239)、水生栖热菌(Thermus aquaticus,Taq)DNA聚合酶(Chien等人,1976,J.Bacteoriol,127:1550)、DNA聚合酶、Pyrococcus kodakaraensis KOD DNA聚合酶(Takagi等人,1997,Appl.Environ.Microbiol.63:4504)、JDF-3DNA聚合酶(来自热球菌属(thermococcus sp.)JDF-3,专利申请WO 0132887)、火球菌GB-D(PGB-D)DNA聚合酶(也称为Deep VentTMDNA聚合酶,Juncosa-Ginesta等人,1994,Biotechniques,16:820,New England Biolabs)、UlTmaDNA聚合酶(来自嗜热的海栖热袍菌;Diaz和Sabino,1998Braz J.Med.Res,31:1239;PEApplied Biosystems)、Tgo DNA聚合酶(来自thermococcus gorgonarius,RocheMolecular Biochemicals)、大肠杆菌DNA聚合酶I(Lecomte和Doubleday,1983,Polynucleotides Res.11:7505)、T7 DNA聚合酶(Nordstrom等人,1981,J Biol.Chem.256:3112)和古细菌DP1I/DP2 DNA聚合酶II(Cann等人,1998,Proc.Natl.Acad.Sci.USA 95:14250)。嗜温聚合酶和嗜热聚合酶都被考虑。嗜热DNA聚合酶包括但不限于9°NmTM、TherminatorTM、Taq、Tne、Tma、Pfu、TfI、Tth、TIi、Stoffel片段、VentTM和Deep VentTMDNA聚合酶、KOD DNA聚合酶、Tgo、JDF-3及其突变体、变体和衍生物。也可以考虑是3’核酸外切酶缺陷型突变体的聚合酶。可用于本发明的逆转录酶包括但不限于来自HIV、HTLV-1、HTLV-II、FeLV、FIV、SIV、AMV、MMTV、MoMuLV和其他逆转录病毒的逆转录酶(参见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))。聚合酶的其他实例包括但不限于9°NDNA聚合酶、Taq DNA聚合酶、DNA聚合酶、Pfu DNA聚合酶、RB69DNA聚合酶、KODDNA聚合酶和DNA聚合酶(Gardner等人,(2004)"Comparative Kinetics ofNucleotide Analog Incorporation by Vent DNA Polymerase(J.Biol.Chem.,279(12),11834-11842;Gardner和Jack"Determinants of nucleotide sugar recognition in anarchaeon DNA polymerase"Nucleic Acids Research,27(12)2545-2553)。从非嗜热生物体分离的聚合酶可以是能热失活的。实例是来自噬菌体的DNA聚合酶。应该理解,可以修饰来自多种来源中的任一种的聚合酶以提高或降低其对高温条件的耐受性。在一些实施方案中,聚合酶可以是嗜热的。在一些实施方案中,嗜热聚合酶可以是能热失活的。嗜热聚合酶通常用于高温条件或用于热循环条件,诸如聚合酶链反应(PCR)技术所采用的条件。
在一些实施方案中,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、9°NmTM、TherminatorTMDNA聚合酶、Tne、Tma、TfI、Tth、TIi、Stoffel片段、和DeepVentTMDNA聚合酶、KOD DNA聚合酶、Tgo、JDF-3、Pfu、Taq、T7DNA聚合酶、T7 RNA聚合酶、PGB-D、UlTma DNA聚合酶、大肠杆菌DNA聚合酶I、大肠杆菌DNA聚合酶III、古细菌DP1I/DP2DNA聚合酶II、9°N DNA聚合酶、Taq DNA聚合酶、DNA聚合酶、Pfu DNA聚合酶、SP6 RNA聚合酶、RB69 DNA聚合酶、禽成髓细胞瘤病毒(AMV)逆转录酶、莫洛尼鼠白血病病毒(MMLV)逆转录酶、II逆转录酶和III逆转录酶。
在一些实施方案中,聚合酶是DNA聚合酶1-Klenow片段、Vent聚合酶、DNA聚合酶、KOD DNA聚合酶、Taq聚合酶、T7DNA聚合酶、T7 RNA聚合酶、TherminatorTMDNA聚合酶、POLB聚合酶、SP6 RNA聚合酶、大肠杆菌DNA聚合酶I、大肠杆菌DNA聚合酶III、禽成髓细胞瘤病毒(AMV)逆转录酶、莫洛尼鼠白血病病毒(MMLV)逆转录酶、II逆转录酶或III逆转录酶。
另外,此类聚合酶可用于DNA扩增和/或测序应用,包括实时应用,例如,在包括通过聚合酶将非天然核酸残基掺入DNA的扩增或测序的情况下的实时应用。在其他实施方案中,掺入的非天然核酸可以与天然残基相同,例如其中在掺入过程中通过聚合酶的作用除去非天然核酸的标记或其他部分,或者该非天然核酸可以具有一种或多种将其与天然核酸区分开的特征。
至少自从地球上所有生命的最后共同祖先以来,遗传信息就被储存在四字母的字母表中,通过形成两个碱基对而得以繁殖和检索。合成生物学的中心目标是创造新的生命形式和功能,而实现这一目标的最普遍途径是创造半合成生物体(SSO),其DNA具有两个额外的字母来构成第三个非天然碱基对(UBP)。此前,我们产生此类SSO所做的努力最终产生了一个大肠杆菌菌株,该菌株借助于来自三角褐指藻(PtNTT2)的核苷三磷酸转运蛋白从介质中导入必需的非天然三磷酸,然后使用它们来复制含有UBP dNaM-dTPT3的质粒(图1A)。尽管SSO可以储存更多的信息,但它无法检索到信息,这需要UBP在体内转录为mRNA和tRNA,tRNA用非天然氨基酸进行氨酰化,以及最后UBP有效参与在核糖体处的解码。在这里,我们报道了含有dNaM和dTPT3的DNA在体内转录成具有两种不同的非天然密码子的mRNA和具有同源非天然反密码子的tRNA,以及其在核糖体处的有效解码以指导天然或非规范氨基酸(ncAA)的位点特异性掺入,成为超折叠(superfolder)绿色荧光蛋白(sfGFP)。结果证明,除氢键键合以外的相互作用也可以有助于信息储存和检索的每个步骤。所得的SSO可以编码和检索增加的信息,并且应该能作为创建新生命形式和功能的平台。
绿色荧光蛋白和变体如sfGFP已成为使用琥珀抑制系统研究ncAA掺入的模型系统,包括在位置Y151处,其已被示出可以耐受多种天然和ncAA(图4)。为了探索非天然密码子的解码,我们首先关注于sfGFP的位置151处的Ser掺入,因为大肠杆菌丝氨酸氨酰基tRNA合成酶(SerRS)的tRNA氨基酰化不依赖于反密码子识别,从而消除了装载不足的潜在复杂情况。用编码sfGFP和大肠杆菌tRNASer基因(serT)的质粒转化SSO菌株YZ3,其中sfGFP密码子151(TAC)被非天然密码子AXC(sfGFP(AXC)151;X=NaM)替换,并且serT的反密码子被非天然反密码子GYT(tRNASer(GYT);Y=TPT3)替代(图1B)。转化株在介质中生长,该介质补充有dNaMTP和dTPT3TP,然后进一步补充有NaMTP和TPT3TP以及异丙基-β-D-硫代半乳糖苷(IPTG),以诱导T7 RNA聚合酶(T7 RNAP)和tRNASer(GYT)的表达。在短暂的tRNA诱导期后,添加脱水四环素(aTc)以诱导sfGFP(AXC)151的表达。
诱导后,与用编码在位置151处具有天然Ser密码子的sfGFP的质粒转化的细胞(sfGFP(AGT)151;图1C)相比,用编码sfGFP(AXC)151但缺乏tRNASer(GYT)的对照质粒转化的细胞示出显著降低的荧光。此外,诱导sfGFP(AXC)151(图1D)后细胞生长开始进入平台期,这可能是由于核糖体的停滞和隔绝(sequestering)。用抗GFP抗体对这些细胞的裂解物进行蛋白质印迹,结果揭示sfGFP表达显著降低,并且存在在非天然密码子位置处截短的sfGFP(图1E)。相反,用编码sfGFP(AXC)151和tRNASer(GYT)二者的质粒转化的细胞显示的荧光几乎等于表达sfGFP(AGT)151的对照细胞的荧光(图1C),sfGFP(AXC)151诱导后细胞生长没有平台期(图1D),而来自这些细胞的裂解物的蛋白质印迹仅揭示出全长sfGFP蛋白(图1E)。此外,我们评估了以相同方式表达的所有四个天然近同源tRNA(tRNASer(GNT);N=G、C、A或T)解码AXC密码子的能力。在每种情况下,几乎没有观察到荧光并且保持了生长缺陷(图5A和图5B)。这些数据表明,PtNTT2能够导入两个非天然核苷酸的脱氧核糖三磷酸和核糖三磷酸,T7 RNA聚合酶能够在体内转录含有非天然核苷酸的mRNA和tRNA,并且核糖体只能用非天然反密码子有效地解码非天然密码子。
为了评估解码的保真度,我们通过LC/MS-MS和经由峰强度的相对定量分析了从表达sfGFP(AXC)151和tRNASer(GYT)二者的细胞中纯化的蛋白质,揭示了在位置151处Ser的98.5±0.7%(95%CI,n=4)掺入,主要的污染物是Ile/Leu(图1F,表4)。假设sfGFP(AXC)151基因中的UBP保留为98±2%(95%CI,n=4)(表5)并且X→T通常是复制过程中的主要突变(对于AXC,这将是导致产生Ile密码子ATC),我们将大部分在位置151不含有Ser的蛋白质归因于复制过程中UBP的丢失,并得出结论,使用非天然密码子进行翻译的保真度较高。
表4
表5
*对应于图7A-图7D中分析的培养物
为了证明用UBP编码ncAA,我们构建了与上面所使用的质粒类似的质粒,但是用马氏甲烷八叠球菌(Methanosarcina mazei)tRNAPyl(GYT)基因代替了tRNASer基因。tRNAPyl可以被巴氏甲烷八叠球菌(Methanosarcina barkeri)吡咯赖氨酸氨酰基tRNA合成酶(PylRS)选择性地装载ncAA N6-[(2-丙炔基氧基)羰基]-L-赖氨酸(PrK)。除了密码子AXC,我们还分析了密码子GXC和对应的tRNAPyl(GYC)。携带编码IPTG可诱导的PylRS的单独质粒的SSO用所需质粒转化,并在添加或不添加PrK的情况下生长。在不存在PylRS、同源非天然tRNAPyl或PrK的情况下用表达sfGFP(AXC)151或sfGFP(GXC)151的细胞进行的对照实验中,我们仅观察到低细胞荧光(图2A)、sfGFP的截短(图6A和图6B)以及细胞生长的平台期(图6B)。相反,对于具有其同源非天然tRNA的非天然mRNA,当存在PylRS并添加PrK时,我们观察到高荧光(对于AXC和GXC分别为sfGFP(TAC)151的64%和69%)(图2A和图2B)、全长sfGFP的稳健产生(图6A)和正常生长(图6B)。
为了验证PrK的掺入,使用C端Strep-标记II从细胞裂解物中亲和纯化出sfGFP,并对其进行了铜催化的点击化学反应以附接羧基四甲基罗丹明(TAMRA)染料(TAMRA-PEG4-N3),该染料会在SDS-PAGE期间改变sfGFP的电泳迁移率,因此使我们能够通过蛋白质印迹来评估PrK掺入的保真度(图2C)。我们观察到强TAMRA信号,并且当从在补充有PrK的介质中培养的表达sfGFP(AXC)151和tRNAPyl(GYT)或sfGFP(GXC)151和tRNAPyl(GYC)的细胞中纯化时,几乎所有sfGFP均发生了移位(图2C)。相反,当NaMTP、TPT3TP或两者不存在时,几乎观察不到TAMRA信号或移位的sfGFP(图7A和图7B)。最后,在从表达sfGFP(TAC)151以及任一非天然tRNA的细胞中纯化的蛋白质中未观察到TAMRA信号或移位的sfGFP(图2C)。该数据表明,PrK通过通过具有非天然反密码子的tRNA对非天然密码子的解码而特异性地掺入sfGFP中。
在最佳PrK浓度下(图8A-图8D),我们分别对于AXC和GXC密码子纯化了54±4和55±6μg/mL的sfGFP(s.d.,n=4,约sfGFP(TAC)151对照的40%)(表6)。并且根据质谱分析,具有PrK的sfGFP的纯度对于AXC密码子为96.2±0.3%(95%CI,n=4),对于GXC密码子为97.5±0.7%(95%CI,n=4)(图2D)。尽管由于在添加非天然核糖三磷酸后生长适度降低(图7C和图7D),纯化的sfGFP蛋白的产量比琥珀抑制(87±6μg/mL,s.d.,n=4(表6))略低,但当相对于细胞密度进行标准化时(图2A和图2B),两种非天然密码子的解码产生的荧光均高于琥珀抑制,意味着用非天然密码子解码比琥珀抑制更有效。
为了探索UBP对其他ncAA的编码,我们检查了AXC密码子和进化的詹氏甲烷球菌(Methanococcus jannaschii)TyrRS/tRNATyr对(pAzFRS/tRNApAzF)对对叠氮基苯丙氨酸(pAzF)的编码。通过合成酶诱导和向生长介质中添加pAzF,我们观察到了与表达天然sfGFP(TAC)151的细胞产生的荧光相当的稳健荧光以及在sfGFP(AXC)151和tRNApAzF(GYT)情况下的正常生长(图3A,图9)。纯化全长sfGFP(86±6μg/mL,s.d.,n=4;sfGFP(TAC)151对照的68%,表6),并使用二苯并环辛基(DBCO)基团进行无铜点击化学反应以附接TAMRA(TAMRA-PEG4-DBCO)。我们观察到TAMRA稳健地缀合至sfGF,该sfGF分离自在存在pAzF的情况下进行培养并表达sfGFP(AXC)151和tRNApAzF(GYT)的细胞(图3B)。尽管由于叠氮基部分的分解,我们无法准确评估pAzF掺入的保真度,但约93%的sfGFP蛋白发生了移位,这足以媲美通过琥珀抑制产生的约95%的sfGFP移位(图3B)。
表6
至少自从地球上所有生命的最后共同祖先以来,蛋白质的产生是通过解码仅用四核苷酸遗传字母表书写的密码子。现在,我们展示了用扩展的遗传字母表书写的两个新密码子的解码,并使用该新密码子位点特异地将ncAA掺入蛋白质中。我们发现,对于信息储存和检索的每个步骤,显然对天然碱基对具有中心作用的氢键可能至少部分被互补性的堆积和疏水力所代替。非天然密码子尽管具有新颖的解码机制,但仍可以像其完全自然的对应物一样有效地被解码。虽然我们仅研究了两个非天然密码子的解码,但UBP不太可能仅限于这些,并且当与最近报道的可增强UBP保留的Cas9编辑系统结合时,可能会提供比以往所能使用的更多的密码子。因此,报道的SSO可能只是能够获得天然生物体无法得到的广泛形式和功能的第一个新形式的半合成生命。
尽管本文中已经示出和描述了本发明的优选实施方案,但是对于本领域技术人员显而易见的是,这些实施方案仅以示例的方式提供。在不背离本发明的情况下,本领域技术人员现在将想到许多变化、改变和替代。应当理解,本文所述的本发明的实施方案的各种替代方案可以用于实施本发明。意图是所附权利要求限定本发明的范围,并且由此涵盖这些权利要求范围内的方法和结构及其等同物。
Claims (23)
1.一种产生含有非天然氨基酸的蛋白质的方法,所述方法包括:
·制备突变tRNA,其中所述突变tRNA包含选自表1或表2的突变反密码子序列;
·制备突变mRNA,其中所述突变mRNA包含选自表1或表2的突变密码子序列;和
·利用所述突变tRNA和所述突变mRNA合成所述含有非天然氨基酸的蛋白质。
2.根据权利要求1所述的方法,其中所述蛋白质在无细胞翻译系统中合成。
3.根据权利要求1所述的方法,其中所述蛋白质在细胞(半合成生物体或SSO)中合成。
4.根据权利要求3所述的方法,其中所述半合成生物体包含微生物。
5.根据权利要求3或4中任一项所述的方法,其中所述半合成生物体包含细菌。
6.根据权利要求3-5中任一项所述的方法,其中所述半合成生物体包含大肠杆菌。
7.根据权利要求1-6中任一项所述的方法,其中所述突变tRNA的所述突变反密码子与选自表1-3的突变密码子配对。
8.根据权利要求1-7中任一项所述的方法,其中所述非天然氨基酸包含至少一个非天然核苷酸。
9.根据权利要求1-8中任一项所述的方法,其中所述非天然核苷酸包含非天然核碱基。
10.根据权利要求1-9中任一项所述的方法,其中所述非天然核苷酸的所述非天然碱基选自2-氨基腺嘌呤-9-基、2-氨基腺嘌呤、2-F-腺嘌呤、2-硫尿嘧啶、2-硫胸腺嘧啶、2-硫胞嘧啶、腺嘌呤和鸟嘌呤的2-丙基和烷基衍生物、2-氨基-腺嘌呤、2-氨基-丙基-腺嘌呤、2-氨基吡啶、2-吡啶酮、2’-脱氧尿苷、2-氨基-2’-脱氧腺苷、3-脱氮鸟嘌呤、3-脱氮腺嘌呤、4-硫尿嘧啶、4-硫胸腺嘧啶、尿嘧啶-5-基、次黄嘌呤-9-基(I)、5-甲基-胞嘧啶、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、5-溴尿嘧啶和5-三氟甲基尿嘧啶以及5-溴胞嘧啶和5-三氟甲基胞嘧啶;5-卤代尿嘧啶、5-卤代胞嘧啶、5-丙炔基-尿嘧啶、5-丙炔基胞嘧啶、5-尿嘧啶、5-取代的、5-卤代、5-取代的嘧啶、5-羟基胞嘧啶、5-溴胞嘧啶、5-溴尿嘧啶、5-氯胞嘧啶、氯化胞嘧啶、环胞嘧啶、阿糖胞苷、5-氟胞嘧啶、氟嘧啶、氟尿嘧啶、5,6-二氢胞嘧啶、5-碘胞嘧啶、羟基脲、碘尿嘧啶、5-硝基胞嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-氟尿嘧啶和5-碘尿嘧啶、腺嘌呤和鸟嘌呤的6-烷基衍生物、6-氮嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、氮胞嘧啶、6-偶氮胸腺嘧啶、6-硫鸟嘌呤、7-甲基鸟嘌呤、7-甲基腺嘌呤、7-脱氮鸟嘌呤、7-脱氮鸟苷、7-脱氮-腺嘌呤、7-脱氮-8-氮鸟嘌呤、8-氮鸟嘌呤、8-氮腺嘌呤、8-卤代、8-氨基、8-硫醇、8-硫代烷基和8-羟基取代的腺嘌呤和鸟嘌呤;N4-乙基胞嘧啶、N-2取代的嘌呤、N-6取代的嘌呤、O-6取代的嘌呤、增加双链体形成的稳定性的那些、通用核酸、疏水核酸、混杂核酸、尺寸扩展的核酸、氟化核酸、三环嘧啶、吩噁嗪胞苷([5,4-b][l,4]苯并噁嗪-2(3H)-酮)、吩噻嗪胞苷(1H-嘧啶并[5,4-b][l,4]苯并噻嗪-2(3H)-酮)、G夹(G-clamps)、吩噁嗪胞苷(9-(2-氨基乙氧基)-H-嘧啶并[5,4-b][1,4]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、次黄嘌呤、黄嘌呤、4-乙酰胞嘧啶、5-(羧基羟甲基)尿嘧啶、5-羧甲基氨基甲基-2-硫尿苷、5-羧甲基氨基甲基尿嘧啶、二氢尿嘧啶、β-D-半乳糖基辫苷、肌苷、N6-异戊烯腺嘌呤、1-甲基鸟嘌呤、1-甲基肌苷、2,2-二甲基鸟嘌呤、2-甲基腺嘌呤、2-甲基鸟嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-腺嘌呤、7-甲基鸟嘌呤、5-甲基氨基甲基尿嘧啶、5-甲氧基氨基甲基-2-硫尿嘧啶、β-D-甘露糖基辫苷、5’-甲氧基羧甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲硫基-N6-异戊烯腺嘌呤、尿嘧啶-5氧乙酸、怀丁氧苷、假尿嘧啶、辫苷、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-氧乙酸甲酯、尿嘧啶-5-氧乙酸、5-甲基-2-硫尿嘧啶、3-(3-氨基-3-N-2-羧基丙基)尿嘧啶、(acp3)w和2,6-二氨基嘌呤,以及其中嘌呤或嘧啶碱基被杂环取代的那些。
13.根据权利要求1-12中任一项所述的方法,其中所述非天然核苷酸进一步包含非天然糖部分。
14.根据权利要求13所述的方法,其中所述非天然核苷酸的所述非天然糖部分选自在2’位置处的下组修饰:OH;取代的低级烷基、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,其中所述烷基、烯基和炔基可以为取代或未取代的C1-C10烷基、C2-C10烯基、C2-C10炔基、-O[(CH2)nO]mCH3、-O(CH2)nOCH3、-O(CH2)nNH2、-O(CH2)nCH3、-O(CH2)n-ONH2和-O(CH2)nON[(CH2)nCH3)]2,其中n和m为从1至约10;和/或选自在5’位置处的下组修饰:5’-乙烯基、5’-甲基(R或S)、在4’位置处的修饰、4’-S、杂环烷基、杂环烷芳基、氨基烷基氨基、聚烷基氨基、取代的甲硅烷基、RNA剪切基团、报告基团、嵌入剂、用于改善寡核苷酸的药代动力学性质的基团,或用于改善寡核苷酸的药效学性质的基团,及其任意组合。
15.根据权利要求1-14中任一项所述的方法,其中所述突变反密码子或所述突变密码子进一步包含非天然骨架。
16.根据权利要求1-14中任一项所述的方法,其中所述突变反密码子和所述突变密码子进一步包含非天然骨架。
17.根据权利要求1-16中任一项所述的方法,其中所述非天然核苷酸被DNA聚合酶、RNA聚合酶或逆转录酶识别。
18.根据权利要求1-17中任一项所述的方法,其中在转录过程中,所述RNA聚合酶将非天然核苷酸掺入所述mRNA中,以产生含有突变密码子的突变mRNA。
19.根据权利要求1-18中任一项所述的方法,其中在转录过程中,所述RNA聚合酶将非天然核苷酸掺入所述tRNA中,以产生含有突变反密码子的突变tRNA。
20.根据权利要求1-19中任一项所述的方法,其中在转录过程中,所述RNA聚合酶将非天然核苷酸掺入所述mRNA中,以产生突变mRNA。
21.根据权利要求1-20中任一项所述的方法,其中在转录过程中,所述RNA聚合酶将非天然核苷酸掺入所述tRNA中,以产生突变tRNA。
22.根据权利要求1-21中任一项所述的方法,其中所述突变tRNA带有非天然氨基酸残基。
23.根据权利要求1-22中任一项所述的方法,其中利用所述突变tRNA和所述突变mRNA在翻译过程中产生含有非天然氨基酸的蛋白质。
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IL271903A (en) | 2020-02-27 |
CA3069321A1 (en) | 2019-01-17 |
US20200131555A1 (en) | 2020-04-30 |
WO2019014267A1 (en) | 2019-01-17 |
KR102649135B1 (ko) | 2024-03-18 |
AR112756A1 (es) | 2019-12-11 |
KR20200029531A (ko) | 2020-03-18 |
TWI821192B (zh) | 2023-11-11 |
MA49578A (fr) | 2021-04-07 |
US11834689B2 (en) | 2023-12-05 |
AU2018300069A1 (en) | 2020-02-27 |
EA202090090A1 (ru) | 2020-06-09 |
JP2020526197A (ja) | 2020-08-31 |
SG11202000167SA (en) | 2020-02-27 |
KR20240038157A (ko) | 2024-03-22 |
US20210222147A1 (en) | 2021-07-22 |
JP2023175678A (ja) | 2023-12-12 |
EP3652316A1 (en) | 2020-05-20 |
NZ761479A (en) | 2024-03-22 |
EP3652316A4 (en) | 2021-04-07 |
JP7325341B2 (ja) | 2023-08-14 |
US20190376054A1 (en) | 2019-12-12 |
US20240043823A1 (en) | 2024-02-08 |
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