Y639F/H784A T7 RNA聚合酶双突变体在使用非标准NTP合成RNA方面表现出优异特性。
A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs.
作者信息
Padilla Robert, Sousa Rui
机构信息
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
出版信息
Nucleic Acids Res. 2002 Dec 15;30(24):e138. doi: 10.1093/nar/gnf138.
Abstract
A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2'-deoxy, 2'-NH2 and 2'-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2'-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2'-substituents (2'-OMe and 2'-N3), and may also enhance use of NTPs with modifications at other than the 2'-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.
摘要
酪氨酸639突变为苯丙氨酸的T7 RNA聚合酶能够轻易地将2'-脱氧、2'-氨基和2'-氟核糖核苷三磷酸用作底物，并且已被广泛用于合成各种应用的修饰RNA。该突变体不易使用具有较大2'-取代基的核糖核苷三磷酸，也不利于掺入在其他位置具有修饰的核糖核苷三磷酸。在Y639F背景中引入第二个突变（H784A）可显著提高对具有较大2'-取代基（2'-甲氧基和2'-叠氮基）的核糖核苷三磷酸的利用率，并且还可能提高对2'-位以外具有修饰的核糖核苷三磷酸的使用。因此，Y639F/H784A双突变体对于将各种非规范的核糖核苷酸掺入RNA可能格外有用。
相似文献
1
A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs.Y639F/H784A T7 RNA聚合酶双突变体在使用非标准NTP合成RNA方面表现出优异特性。
Nucleic Acids Res. 2002 Dec 15;30(24):e138. doi: 10.1093/nar/gnf138.
2
Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutantT7 RNA polymerase (RNAP).使用突变型T7 RNA聚合酶（RNAP）高效合成用非经典核糖2'-基团高度修饰的核酸。
Nucleic Acids Res. 1999 Mar 15;27(6):1561-3. doi: 10.1093/nar/27.6.1561.
3
A mutant T7 RNA polymerase as a DNA polymerase.一种作为DNA聚合酶的突变型T7 RNA聚合酶。
EMBO J. 1995 Sep 15;14(18):4609-21. doi: 10.1002/j.1460-2075.1995.tb00140.x.
4
Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization.噬菌体T7 RNA聚合酶及其活性位点突变体。动力学、光谱学和量热学表征。
J Mol Biol. 1994 Mar 18;237(1):5-19. doi: 10.1006/jmbi.1994.1205.
5
Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA.能够同时合成RNA和DNA的T7 RNA聚合酶突变体。
FEBS Lett. 1995 Aug 7;369(2-3):165-8. doi: 10.1016/0014-5793(95)00732-o.
6
Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase.突变型T7 RNA聚合酶催化合成核糖/脱氧核糖混合多聚核苷酸
FEBS Lett. 1998 Nov 20;439(3):302-6. doi: 10.1016/s0014-5793(98)01393-3.
7
Interaction of ribonucleotides with T7 RNA polymerase: probable role of GTP in transcription initiation.核糖核苷酸与T7 RNA聚合酶的相互作用：GTP在转录起始中的可能作用。
Biochem Biophys Res Commun. 1993 Sep 15;195(2):616-22. doi: 10.1006/bbrc.1993.2090.
8
Roles of histidine 784 and tyrosine 639 in ribose discrimination by T7 RNA polymerase.组氨酸784和酪氨酸639在T7 RNA聚合酶核糖识别中的作用。
Biochemistry. 2000 Feb 8;39(5):919-23. doi: 10.1021/bi992324+.
9
A combined in vitro/in vivo selection for polymerases with novel promoter specificities.针对具有新型启动子特异性的聚合酶进行的体外/体内联合筛选。
BMC Biotechnol. 2001;1:13. doi: 10.1186/1472-6750-1-13. Epub 2001 Dec 28.
10
T7 transcript length determination using enzymatic RNA sequencing.使用酶促RNA测序确定T7转录本长度
Methods Mol Biol. 1997;74:91-7. doi: 10.1385/0-89603-389-9:91.
引用本文的文献
1
Functional Relevance of CASP16 Nucleic Acid Predictions as Evaluated by Structure Providers.结构提供者评估的CASP16核酸预测的功能相关性。
Proteins. 2025 Sep 4. doi: 10.1002/prot.70043.
2
Effects of Nucleoside Modifications on mRNA Translation: Choosing the Right Modifications.核苷修饰对mRNA翻译的影响：选择合适的修饰
Methods Mol Biol. 2025;2965:127-149. doi: 10.1007/978-1-0716-4742-4_4.
3
Functional relevance of CASP16 nucleic acid predictions as evaluated by structure providers.结构供应商评估的CASP16核酸预测的功能相关性。
bioRxiv. 2025 Apr 18:2025.04.15.649049. doi: 10.1101/2025.04.15.649049.
4
In Vitro Transcription of Plant miRNA for Structural and Processing Analysis.用于结构和加工分析的植物微小RNA的体外转录
Methods Mol Biol. 2025;2900:107-116. doi: 10.1007/978-1-0716-4398-3_6.
5
Bacteriophage RNA polymerases: catalysts for mRNA vaccines and therapeutics.噬菌体RNA聚合酶：mRNA疫苗和治疗药物的催化剂。
Front Mol Biosci. 2024 Nov 21;11:1504876. doi: 10.3389/fmolb.2024.1504876. eCollection 2024.
6
Template-independent synthesis and 3'-end labelling of 2'-modified oligonucleotides with terminal deoxynucleotidyl transferases.无模板合成与末端脱氧核苷酸转移酶的 2'-修饰寡核苷酸 3'-末端标记。
Nucleic Acids Res. 2024 Sep 23;52(17):10085-10101. doi: 10.1093/nar/gkae691.
7
RNA nanostructures for targeted drug delivery and imaging.RNA 纳米结构用于靶向药物递送和成像。
RNA Biol. 2024 Jan;21(1):1-19. doi: 10.1080/15476286.2024.2328440. Epub 2024 Mar 31.
8
Development and Comparison of 4-Thiouridine to Cytidine Base Conversion Reaction.4-硫尿苷向胞苷碱基转化反应的开发与比较
ACS Omega. 2024 Feb 14;9(8):9300-9308. doi: 10.1021/acsomega.3c08516. eCollection 2024 Feb 27.
9
An Aptamer That Rapidly Internalizes into Cancer Cells Utilizes the Transferrin Receptor Pathway.一种能迅速内化进入癌细胞的适体利用转铁蛋白受体途径。
Cancers (Basel). 2023 Apr 14;15(8):2301. doi: 10.3390/cancers15082301.
10
CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.CRISPR-Cas9 对酶促合成碱基修饰核酸的识别。
Nucleic Acids Res. 2023 Feb 28;51(4):1501-1511. doi: 10.1093/nar/gkac1147.
本文引用的文献
1
Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability.停滞的T7 RNA聚合酶延伸复合物的表征揭示了多种有助于稳定性的因素。
J Mol Biol. 2000 Oct 6;302(5):1049-62. doi: 10.1006/jmbi.2000.4114.
2
Chemical probing of RNA by nucleotide analog interference mapping.通过核苷酸类似物干扰图谱对RNA进行化学探测。
Methods Enzymol. 2000;317:92-109. doi: 10.1016/s0076-6879(00)17008-9.
3
Roles of histidine 784 and tyrosine 639 in ribose discrimination by T7 RNA polymerase.组氨酸784和酪氨酸639在T7 RNA聚合酶核糖识别中的作用。
Biochemistry. 2000 Feb 8;39(5):919-23. doi: 10.1021/bi992324+.
4
Structure of a transcribing T7 RNA polymerase initiation complex.转录中的T7 RNA聚合酶起始复合物的结构。
Science. 1999 Dec 17;286(5448):2305-9. doi: 10.1126/science.286.5448.2305.
5
Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutantT7 RNA polymerase (RNAP).使用突变型T7 RNA聚合酶（RNAP）高效合成用非经典核糖2'-基团高度修饰的核酸。
Nucleic Acids Res. 1999 Mar 15;27(6):1561-3. doi: 10.1093/nar/27.6.1561.
6
A nuclease-resistant protein kinase C alpha ribozyme blocks glioma cell growth.一种核酸酶抗性蛋白激酶Cα核酶可阻断胶质瘤细胞生长。
Nat Biotechnol. 1998 Jun;16(6):556-61. doi: 10.1038/nbt0698-556.
7
Mechanism of ribose 2'-group discrimination by an RNA polymerase.RNA聚合酶对核糖2'-基团的识别机制。
Biochemistry. 1997 Jul 8;36(27):8231-42. doi: 10.1021/bi962674l.
8
A mutant T7 RNA polymerase as a DNA polymerase.一种作为DNA聚合酶的突变型T7 RNA聚合酶。
EMBO J. 1995 Sep 15;14(18):4609-21. doi: 10.1002/j.1460-2075.1995.tb00140.x.
9
Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.使用T7 RNA聚合酶和合成DNA模板进行寡核糖核苷酸合成。
Nucleic Acids Res. 1987 Nov 11;15(21):8783-98. doi: 10.1093/nar/15.21.8783.
10
A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.一种用于特异性基因的可控专一性表达的噬菌体T7 RNA聚合酶/启动子系统。
Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074-8. doi: 10.1073/pnas.82.4.1074.
Zotero 插件