Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase

Okano, Hiroyuki; Katano, Yuta; Baba, Misato; Fujiwara, Ayako; Hidese, Ryota; Fujiwara, Shinsuke; Yanagihara, Itaru; Hayashi, Tsukasa; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

doi:10.1016/j.enzmictec.2016.10.003

Cited by 12 publications

(5 citation statements)

References 26 publications

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“…To test whether m6A does in fact ameliorate the stability of the secondary structure of SARS-CoV-2 5’-UTR, we developed a novel assay based on the reverse transcription reaction (Figure 4c). We used a genetically engineered thermostable reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) that, critically, exhibits low strand displacement potential (46), and whose reverse transcription efficiency is thus significantly compromised by secondary structure of template RNAs. By carrying out the RT reaction at denaturing and non-denaturing conditions, we could assay mRNA secondary structure stability based on comparative loss of RT efficiency between the two conditions (Figure 4c).…”

Section: Resultsmentioning

confidence: 99%

N6-Adenosine Methylation of SARS-CoV-2 5’-UTR Regulates Translation

Aly

Scott

Calderon

et al. 2022

Preprint

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The coronavirus disease 2019 (COVID19) continues to spread despite global vaccination efforts. This, alongside the rapid emergence of vaccine resistant variants, creates a need for orthogonal therapeutic strategies targeting more conserved facets of severe acute respiratory syndrome coronavirus (SARS-CoV-2). One conserved feature of all coronaviruses is their ability to undergo discontinuous transcription wherein individual open reading frames fuse with the 5-UTR leader sequence during negative-strand RNA synthesis. As such all viral protein coding genes use the same 5-UTR for translation. Using in vitro reporter assays, we demonstrate that the SARS-CoV-2 5-UTR efficiently initiates protein translation despite its predicted structural complexity. Through a combination of bioinformatic and biochemical assays, we demonstrate that a single METTL3-dependent m6A methylation event in SARS-CoV-2 5-UTR regulates the rate of translation initiation. We show that m6A likely exerts this effect by destabilizing secondary structure in the 5-UTR, thereby facilitating access to the ribosomal pre-initiation complex. This discovery opens new avenues for novel therapeutic strategies aimed at controlling the ability of SARS-CoV-2 to replicate in host cells.

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Section: Resultsmentioning

confidence: 99%

N6-Adenosine Methylation of SARS-CoV-2 5’-UTR Regulates Translation

Aly

Scott

Calderon

et al. 2022

Preprint

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“…Nevertheless, the products of primer elongation vary in their electrophoretic mobility and hence in length: abortive elongation products are present. This finding is probably due to structural complexity of the RNA template because reverse transcriptase, lacking a helicase activity, is unable to overcome RNA hairpins or other secondary structures, which can lead to dissociation of the enzyme from the substrate (DNA-RNA complex) (Okano et al, 2017). As a consequence, expected processivity of the reverse-transcription reaction is lower.…”

Section: Resultsmentioning

confidence: 99%

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

Dyudeeva

Pyshnaya

2022

Vestn. VOGiS

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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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“…In amplification techniques using multiple enzymes such as NASBA (1,30), optimization is more complicated than when using a single enzyme as in the case of PcR. In this case, statistical methods such as Taguchi's method have been successfully used for optimization (31) (Fig. 2).…”

Section: Thermostabilization Of Reverse Transcriptasementioning

confidence: 99%

Alteration of enzymes and their application to nucleic acid amplification (Review)

2020

Self Cite

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Since the discovery of polymerase chain reaction (PcR) in 1985, several methods have been developed to achieve nucleic acid amplification, and are currently used in various fields including clinical diagnosis and life science research. Thus, a wealth of information has accumulated regarding nucleic acid-related enzymes. In this review, some nucleic acid-related enzymes were selected and the recent advances in their modification along with their application to nucleic acid amplification were described. The discussion also focused on optimization of the corresponding reaction conditions. Using newly developed enzymes under well-optimized reaction conditions, the sensitivity, specificity, and fidelity of nucleic acid tests can be improved successfully. Contents 1. Introduction 2. Thermostabilization of reverse transcriptase 3. creation of the reverse transcriptase activity in thermostable dNA polymerase 4. Use of helicase to increase specificity 5. Fidelity evaluation with NGS 6. Use of recombinase and single-strand binding protein for isothermal DNA amplification 7. Other considerable factors involved in nucleic acid amplification 8. conclusions and future perspectives

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Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase

Cited by 12 publications

References 26 publications

N6-Adenosine Methylation of SARS-CoV-2 5’-UTR Regulates Translation

N6-Adenosine Methylation of SARS-CoV-2 5’-UTR Regulates Translation

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

Alteration of enzymes and their application to nucleic acid amplification (Review)

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