vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJGB-22-02vavilov-3250Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYФосфорилгуанидиновые олигонуклеотиды как праймеры для РНК-зависимого ДНК синтеза с помощью обратной транскриптазы вируса лейкемии мышейPhosphoryl guanidine oligonucleotides as primers for RNA-dependent DNA synthesis using murine leukemia virus reverse transcriptaseДюдееваЕ. С.DyudeevaE. S.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-7559-2376ПышнаяИ. А.PyshnayaI. A.

Новосибирск

Novosibirsk

pyshnaya@niboch.nsc.ruИнститут химической биологии и фундаментальной медицины Сибирского отделения Российской академии наукРоссияInstitute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of SciencesRussian Federation202228022022261513Copyright © Дюдеева Е.С., Пышная И.А., 20222022Дюдеева Е.С., Пышная И.А.Dyudeeva E.S., Pyshnaya I.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3250

Современные подходы к обнаружению и анализу низкокопийных РНК часто основываются на использовании РНК-зависимых ДНК-полимераз, например, метод ОТ-ПЦР. Точность и эффективность синтеза кДНК в реакции обратной транскрипции (ОТ), катализируемой ревертазой (РНК-зависимой ДНКполимеразой), значительно влияют на корректность результатов ПЦР-диагностики и/или секвенирования РНК. В связи с этим множество исследований посвящено оптимизации реакции ОТ, в том числе поиску более совершенных праймеров, необходимых для наработки полноразмерной ДНК-копии исследуемой РНК. Наиболее известные полностью незаряженные аналоги олигонуклеотидов – морфолиновые олигонуклеотиды и пептидонуклеиновые кислоты – не могут являться субстратами для ферментов, процессирующих НК. Целью нашей работы было пилотное исследование незаряженных фосфорилгуанидиновых олигодезоксирибонуклеотидов (ФГО) в качестве праймеров для обратной транскриптазы вируса лейкемии мышей (MMLV H-). Изучены особенности превращения частично и полностью незаряженных ФГОпраймеров. Продемонстрировано, что ФГО способны эффективно удлиняться, в том числе в присутствии фрагмента рРНК человека, обладающего сложной пространственной структурой. Доля абортивных продуктов удлинения ФГО-праймера зависит от ионной силы использованного буферного раствора, нуклеотидной последовательности праймера и наличия и расположения в его составе ФГ-групп. Полученные данные показывают возможность использования ФГО, включая полностью электронейтральные, как праймеров в реакциях ОТ-ПЦР, что открывает новые перспективы для создания систем анализа высокоструктурированных РНК.

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 efficiency of cDNA synthesis in the reverse-transcription reaction catalyzed by reverse transcriptase (RNA-dependent DNA polymerase) significantly 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-). Specific features of elongation of partially and completely uncharged PGO primers were investigated. It was demonstrated that PGOs can be elongated efficiently, 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. 

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The authors declare that there are no conflicts of interest present.