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| Status |
Public on Apr 22, 2020 |
| Title |
Anomalous reverse transcription through chemical modifications in polyadenosine stretches |
| Organisms |
Human immunodeficiency virus; synthetic construct |
| Experiment type |
Other
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| Summary |
Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for 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, re-analysis 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 RNA requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger unexpectedly effective reverse transcriptase bypass and that chemical modifications in poly(A) mRNA tails may be generally undercounted.
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| Overall design |
Sequencing cDNA truncations/mutations caused by chemical modifications in RNA (in vitro transcribed or chemically synthesized)
There are three overarching samples. Two are of RNA libraries comprised of enzymatically synthesized RNA. The third is of chemically synthesized RNA. For all three, “polyA length” and “reactivity” analyses have been carried out.
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| Contributor(s) |
Kladwang W, Topkar VV, Liu B, Rangan R, Hodges TL, Keane SC, al-Hashimi H, Das R |
| Citation(s) |
32407625 |
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https://doi.org/10.1101/2020.01.07.897843
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| Submission date |
Apr 21, 2020 |
| Last update date |
May 19, 2021 |
| Contact name |
Rhiju Das |
| E-mail(s) |
rhiju@stanford.edu
|
| Organization name |
Stanford University School of Medicin
|
| Department |
Biochemistry
|
| Lab |
Das
|
| Street address |
279 Campus Dr, B419 Beckman Center
|
| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platforms (2) |
| GPL17769 |
Illumina MiSeq (synthetic construct) |
| GPL28440 |
Illumina MiSeq (Human immunodeficiency virus) |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA627227 |
| SRA |
SRP257782 |
