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Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.

Publication ,  Journal Article
Kladwang, W; Topkar, VV; Liu, B; Rangan, R; Hodges, TL; Keane, SC; Al-Hashimi, H; Das, R
Published in: Biochemistry
June 16, 2020

Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for the transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes' behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2' hydroxyl acylation, as probed by reverse transcription. Here, reanalysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on (A)12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3' untranslated region requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger an unexpectedly effective reverse transcriptase bypass and that chemical modifications in mRNA poly(A) tails may be generally undercounted.

Duke Scholars

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Published In

Biochemistry

DOI

EISSN

1520-4995

Publication Date

June 16, 2020

Volume

59

Issue

23

Start / End Page

2154 / 2170

Location

United States

Related Subject Headings

  • Reverse Transcription
  • RNA
  • Polymers
  • Magnetic Resonance Spectroscopy
  • Electrophoresis, Capillary
  • Biochemistry & Molecular Biology
  • Adenosine
  • 3404 Medicinal and biomolecular chemistry
  • 3205 Medical biochemistry and metabolomics
  • 3101 Biochemistry and cell biology
 

Citation

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Kladwang, W., Topkar, V. V., Liu, B., Rangan, R., Hodges, T. L., Keane, S. C., … Das, R. (2020). Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches. Biochemistry, 59(23), 2154–2170. https://doi.org/10.1021/acs.biochem.0c00020
Kladwang, Wipapat, Ved V. Topkar, Bei Liu, Ramya Rangan, Tracy L. Hodges, Sarah C. Keane, Hashim Al-Hashimi, and Rhiju Das. “Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.Biochemistry 59, no. 23 (June 16, 2020): 2154–70. https://doi.org/10.1021/acs.biochem.0c00020.
Kladwang W, Topkar VV, Liu B, Rangan R, Hodges TL, Keane SC, et al. Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches. Biochemistry. 2020 Jun 16;59(23):2154–70.
Kladwang, Wipapat, et al. “Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.Biochemistry, vol. 59, no. 23, June 2020, pp. 2154–70. Pubmed, doi:10.1021/acs.biochem.0c00020.
Kladwang W, Topkar VV, Liu B, Rangan R, Hodges TL, Keane SC, Al-Hashimi H, Das R. Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches. Biochemistry. 2020 Jun 16;59(23):2154–2170.
Journal cover image

Published In

Biochemistry

DOI

EISSN

1520-4995

Publication Date

June 16, 2020

Volume

59

Issue

23

Start / End Page

2154 / 2170

Location

United States

Related Subject Headings

  • Reverse Transcription
  • RNA
  • Polymers
  • Magnetic Resonance Spectroscopy
  • Electrophoresis, Capillary
  • Biochemistry & Molecular Biology
  • Adenosine
  • 3404 Medicinal and biomolecular chemistry
  • 3205 Medical biochemistry and metabolomics
  • 3101 Biochemistry and cell biology