Decreased Km to dNTPs is an essential M-MuLV reverse transcriptase adoption required to perform efficient cDNA synthesis in One-Step RT-PCR assay

Abstract

Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display technique for in vitro evolution of the Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT) that would be able to perform efficient full-length cDNA synthesis in PCR buffer optimized for Thermus aquaticus DNA polymerase. The most frequent mutations found in a selected library were analyzed. Aside from the mutations, which switch off RNase H activity of RT and are beneficial for the full-length cDNA synthesis, we have identified several mutations in the active center of the enzyme (Q221R and V223A/M), which result in 4-5-fold decrease of Km for dNTPs (<0.2 mM). The selected mutations are in surprising agreement with the natural evolution process because they transformed the active center from the oncoretroviral M-MuLV RT-type to the lenitiviral enzyme-type. We believe that this was the major and essential phenotypic adjustment required to perform fast and efficient cDNA synthesis in PCR buffer at 0.2-mM concentration of each dNTP.

M-MuLV reverse transcriptase: Selected properties and improved mutants

“…Most mutations in these important positions result in the decrease of polymerase activity and fidelity. A rare exception is V223, as V223A/M mutations increase processivity and decrease K m*dNTP [45] , [76] . Another example is mutations of catalytically active residues in the RNAse H domain; the most frequent of them is D524A [33] , [74] , [81] , [87] .…”

“…Residue I597 is located near a positively charged C-helix in the RNAse H domain. However, substitution I597A impairs viral replication and is prone to mutate spontaneously to I597V, and the latter does not affect RNAse H activity [76] .…”

Evaluation of iceA1 Gene Expression of Helicobacter pylori Risk Factor of Gastric Cancer in Transgenic Brinjal

“…cDNA synthesis: cDNA was synthesized as described by Palikša et al, with M-MuLV RT-PCR Kit (Genei). 26 About 2μg of the RNA obtained in the previous section was used as a starting material. The RNA concentration obtained was 40μg/ml, so we used approximately 1.02μl of total RNA was used, along with random primers and 1μL of RT enzyme.…”

Phosphoryl guanidine oligonucleotides as primers for RNA-dependent DNA synthesis using murine leukemia virus reverse transcriptase

“…A popular enzyme for the reverse-transcription reaction is the Moloney murine leukemia virus (MMLV) reverse transcriptase. This reverse transcriptase is capable of (a) catalyzing DNA synthesis from a DNA or RNA template (Palikša et al, 2018;Li R. et al, 2020); (b) template-free synthesis of short DNA fragments (Ohtsubo et al, 2017); (c) DNA synthesis with strand displacement (Kelleher, Champoux, 1998;Malik et al, 2017); (d) switching a template strand (Wulf et al, 2019); and (e) cleavage of RNA as part of a hybrid RNA-DNA complex (Schultz, Champoux, 2008;Li R. et al, 2020). In laboratory studies, a recombinant variant devoid of RNase H activity (MMLV H-) is used.…”

“…In laboratory studies, a recombinant variant devoid of RNase H activity (MMLV H-) is used. An extensive body of research deals with optimizing the reverse-transcription reaction, including a search for more efficient primers based on modified oligonucleotides with enhanced affinity for complementary nucleic acids (Heuverswyn et al, 2016;Menéndez-Arias et al, 2016;Palikša et al, 2018;Li R. et al, 2020). The currently known completely uncharged analogs of oligonucleotides -morpholine oligonucleotides and peptide nucleic acids -cannot serve as a substrate of enzymatic reactions, probably owing to the unusual/ foreign backbone, which is substantially different in structure from the natural sugar-phosphate backbone.…”