In order to introduce pyrimidine C5-propynyl modification into boranophosphate oligodeoxyribonucleotides (BP- ODNs), 5-(1-propynyl)-2'-deoxyuridine 5'-(alpha-P-borano) triphosphate (d5PUTPalphaB) was synthesized. The two diastereomers were separated by reverse-phase HPLC. Kinetic studies showed that the Rp isomer was a slightly better substrate for MMLV reverse transcriptase than thymidine triphosphate or Rp-thymidine 5'-(alpha-P-borano)triphosphate. Using the Rp isomers of d5PUTPalphaB and the other three 5'-(alpha-P-borano) triphosphates, a DNA primer could be extended to the full length of the template.
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http://dx.doi.org/10.1081/ncn-200059307 | DOI Listing |
Vavilovskii Zhurnal Genet Selektsii
February 2022
Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Modern approaches to the detection and analysis of low-copy-number RNAs are often based on the use of RNA-dependent DNA polymerases, for example, in reverse-transcription PCR. The accuracy and eff iciency of cDNA synthesis in the reverse-transcription reaction catalyzed by reverse transcriptase (RNA-dependent DNA polymerase) signif icantly affect the correctness of the results of PCR diagnostic assays and/or RNA sequencing. In this regard, many studies are focused on the optimization of the reverse-transcription reaction, including the search for more perfect primers necessary to obtain a full-length DNA copy of RNA under study.
View Article and Find Full Text PDFPLoS One
October 2020
Department of Chemistry, University of Colorado Denver, Denver, Colorado, United States of America.
Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position.
View Article and Find Full Text PDFChempluschem
May 2020
Department of Chemistry and Biochemistry and the Viral Information Institute, San Diego State University, San Diego, CA, 92182, USA.
We report on the ability of the reverse transcriptases (RTs) from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M-MLV), and human immunodeficiency virus 1 (HIV-1) to generate labeled DNA using the fluorescent tricyclic cytidine analogues d(tC)TP and d( tC)TP as substrates. Michaelis-Menten kinetics for the insertion of these analogues show V /K from 0.0-5 times that of natural dCTP across from G, depending on the polymerase and whether the template is RNA or DNA.
View Article and Find Full Text PDFDNA Res
October 2018
Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
In this study, we investigated CIS reaction (clamping-mediated incorporation of single-stranded DNA with concomitant DNA syntheses) of Moloney murine leukaemia virus reverse transcriptase (MMLV-RT), and established a set of conditions with which single-stranded DNA is ligated to a G-tailed model substrate DNA at efficiencies close to 100%. Prior to the CIS reaction, a target blunt-end DNA was 3' G-tailed by MMLV-RT in the presence of a tailing enhancer, deoxycytidine. In the CIS reaction, the G-tail reacted with a single-stranded DNA carrying a stretch of Cs on its 3' end (termed as GAO for guide adaptor oligonucleotide), and MMLV-RT performed DNA polymerization, starting from the 3' overhang, using the GAO as a template.
View Article and Find Full Text PDFZhong Nan Da Xue Xue Bao Yi Xue Ban
July 2017
Department of Infectious Diseases, Xiangya Hospital, Central South University, Key Laboratory of Viral Hepatitis of Hunan Province, Changsha 410008, China.
To observe the sensitivity of transcription mediated amplification (TMA), and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA). Methods: TMA system was established with TaqMan probes, specific primers, moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase, and reaction substrates. The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro.
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