Temperature effects on the fidelity of a thermostable <scp>HIV</scp>‐1 reverse transcriptase

Saura, María; Menéndez‐Arias, Luis

doi:10.1111/febs.12605

Cited by 11 publications

(8 citation statements)

References 39 publications

(67 reference statements)

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“…The RT primer base pairing takes place before RT reaction, supporting that there are non-A residues within the body of poly(A) tails. Although RT is very inefficient (2–6 × 10 −4 relative normal rate) to extend on primers with mismatches on the very 3′ end 36 , we cannot exclude the possibility that some of the middle RT primer anchoring events detected here resulted from mispriming at the middle of pure A tails. These data validate that the non-A modifications within poly(A) tails is not likely caused by sequencing or library preparation artifacts.…”

Section: Resultsmentioning

confidence: 81%

Poly(A) inclusive RNA isoform sequencing (PAIso−seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails

et al. 2019

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Message RNA poly(A) tails are vital for their function and regulation. However, the full-length sequence of mRNA isoforms with their poly(A) tails remains undetermined. Here, we develop a method at single-cell level sensitivity that enables quantification of poly(A) tails along with the full-length cDNA while reading non-adenosine residues within poly(A) tails precisely, which we name poly(A) inclusive RNA isoform sequencing (PAIso−seq). Using this method, we can quantify isoform specific poly(A) tail length. More interestingly, we find that 17% of the mRNAs harbor non-A residues within the body of poly(A) tails in mouse GV oocytes. We show that PAIso−seq is sensitive enough to analyze single GV oocytes. These findings will not only provide an accurate and sensitive tool in studying poly(A) tails, but also open a door for the function and regulation of non-adenosine modifications within the body of poly(A) tails.

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Section: Resultsmentioning

confidence: 81%

Poly(A) inclusive RNA isoform sequencing (PAIso−seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails

et al. 2019

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“…The fidelity of DNA-dependent DNA synthesis was determined using the forward mutation assay45, under the previously described conditions406869. Gap filling synthesis reactions were carried out at 37 °C during 60 min, in 25 μl of 25 mM Tris-HCl (pH 8.0) buffer containing 100 mM KCl, 2 mM DTT, 4 mM MgCl 2 , 250 μM of each dNTP and 100 nM RT.…”

Section: Methodsmentioning

confidence: 99%

“…Mutant frequencies were calculated as the ratio of mutant (light blue or colourless) plaques to the total number of plaques screened. Mutant phenotypes were confirmed by nucleotide sequencing of the phage replicative-form DNA using primer 5′-GCTTGCTGCAACTCTCTCAG-3′69. It should be noted that silent mutations and mutations that do not affect the β-galactosidase activity may not be detected in these assays.…”

Section: Methodsmentioning

confidence: 99%

Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases

Saura

Sebastián-Martín

García‐Marquina

et al. 2017

Sci Rep

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Nucleoside reverse transcriptase (RT) inhibitors constitute the backbone of current therapies against human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2, respectively). However, mutational pathways leading to the development of nucleoside analogue resistance are different in both types of HIV. In HIV-2, resistance to all approved nucleoside analogues is conferred by the combination of RT substitutions K65R, Q151M and M184V. Nucleotide incorporation kinetic analyses of mutant and wild-type (WT) HIV-2 RTs show that the triple-mutant has decreased catalytic efficiency due to the presence of M184V. Although similar effects were previously reported for equivalent mutations in HIV-1 RT, the HIV-2 enzymes were catalytically less efficient. Interestingly, in highly divergent HIV-1 RTs, K65R confers several-fold increased accuracy of DNA synthesis. We have determined the intrinsic fidelity of DNA synthesis of WT HIV-2 RT and mutants K65R and K65R/Q151M/M184V. Our results show that those changes in HIV-2 RT have a relatively small impact on nucleotide selectivity. Furthermore, we found that there were less than two-fold differences in error rates obtained with forward mutation assays using mutant and WT HIV-2 RTs. A different conformation of the β3-β4 hairpin loop in HIV-1 and HIV-2 RTs could probably explain the differential effects of K65R.

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“…For each experiment a fresh aliquot was mixed with either 30 pg mouse cerebral RNA (used as a background) mimicking a single-cell or 3 ng mimicking 100-cell bulk sample 25 (details in RNA material , Supp. data 1,2). In total, the ERCC Spike-in accounted for ~13 % of mRNA and ~0.5 % of total RNA, respectively.…”

Section: Templatementioning

confidence: 97%

“…Detailed information on assay optimization and validation is listed in ERCC Spike-in assay validation (Supp. data 1,2). For each assay, three performance metrics were determined: efficiency (E), limit of detection (LOD) and limit of quantification (LOQ) (LOD -LOQ tab, Supp.…”

Section: Quantitative Pcrmentioning

confidence: 99%

Performance comparison of reverse transcriptases for single-cell studies

Žucha

Androvic²,

Kubista

et al. 2019

Preprint

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Background: Recent technical advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on conversion of RNA to cDNA by reverse transcription (RT). However, RT is recognized as highly limiting step due to its inherent variability and suboptimal sensitivity, especially at minute amounts of RNA. Primary factor influencing RT outcome is reverse transcriptase (RTase). Recently, several new RTases with potential to decrease the loss of information during RT have been developed, but the thorough assessment of their performance is missing. Methods:We have compared the performance of 11 RTases in RT-qPCR on single-cell and 100-cell bulk templates using two priming strategies: conventional mixture of random hexamers with oligo(dT)s and reduced concentration of oligo(dT)s mimicking common singlecell RNA-Seq library preparation protocols. Based on the performance, two RTases were further tested in high-throughput single-cell experiment.Results: All RTases tested reverse transcribed low-concentration templates with high accuracy (R 2 > 0.9445) but variable reproducibility (median CVRT = 40.1 %). The most pronounced differences were found in the ability to capture rare transcripts (0 -90% reaction positivity rate) as well as in the rate of RNA conversion to cDNA (7.3 -124.5 % absolute yield). Finally, RTase performance and reproducibility across all tested parameters were compared using Z-scores and validity of obtained results was confirmed in a single-cell model experiment. The better performing RTase provided higher positive reaction rate and expression levels and improved resolution in clustering analysis. Conclusions:We performed a comprehensive comparison of 11 RTases in low RNA concentration range and identified two best-performing enzymes (Maxima H-; SuperScript IV). We found that using better-performing enzyme (Maxima H-) over commonly-used belowaverage performer (SuperScript II) increases the sensitivity of single-cell experiment. Our results provide a reference for the improvement of current single-cell quantification protocols.

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Temperature effects on the fidelity of a thermostable HIV‐1 reverse transcriptase

Cited by 11 publications

References 39 publications

Poly(A) inclusive RNA isoform sequencing (PAIso−seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails

Poly(A) inclusive RNA isoform sequencing (PAIso−seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails

Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases

Performance comparison of reverse transcriptases for single-cell studies

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