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

E S Dyudeeva,I A Pyshnaya

doi:10.18699/vjgb-22-02

Abstract

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. The best-known completely uncharged analogs of oligonucleotides – morpholine oligonucleotides and peptide nucleic acids – cannot be substrates for enzymes that process nucleic acids. The aim of this work was to conduct a pilot study of uncharged phosphoryl guanidine oligodeoxyribonucleotides (PGOs) as primers for mouse leukemia virus reverse transcriptase (MMLV H-). Specif ic features of elongation of partially and completely uncharged PGO primers were investigated. It was demonstrated that PGOs can be elongated eff iciently, e. g., in the presence of a fragment of human ribosomal RNA having complex spatial structure. It was shown that the proportion (%) of abortive elongation products of a PGO primer depends on buffer ionic strength, nucleotide sequence of the primer, and the presence and location of phosphoryl guanidine groups in the primer. The results indicate the suitability of PGOs, including completely electroneutral ones, as primers for reverse-transcription PCR, thereby opening up new prospects for the creation of experimental models for the analysis of highly structured RNA.

Highlights

Among RNA detection methods, the most popular is reverse-transcription polymerase chain reaction (RT-PCR), which consists of two sequential steps: (1) enzymatic synthesis of cDNA from an RNA template using reverse transcriptase and (2) obtaining the corresponding fragment of double-stranded DNA by standard PCR
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
The combination of these factors makes phosphoryl guanidine (PG)-containing oligonucleotides a promising platform for the development of highly specific probes and, possibly, enables their applications in some enzymatic transformations of nucleic acids catalyzed by either reverse transcriptase or DNA-dependent DNA polymerases (Kupryushkin et al, 2017; Chubarov et al, 2020)

Summary

Introduction

The detection of certain RNAs – some of the most important natural biopolymers – is an extremely relevant task in molecular diagnostics because such biomolecules are indicators of the state of the cell or an organism as a whole, of its metabolic status, or the presence of infection. 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). It is reported that spatial structure of duplexes containing PGOs is virtually the same as the structure of the double helix of two native nucleic acids (Lomzov et al, 2019) The combination of these factors makes phosphoryl guanidine (PG)-containing oligonucleotides a promising platform for the development of highly specific probes and, possibly, enables their applications in some enzymatic transformations of nucleic acids catalyzed by either reverse transcriptase or DNA-dependent DNA polymerases (Kupryushkin et al, 2017; Chubarov et al, 2020)

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