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| Status |
Public on Mar 15, 2023 |
| Title |
Detection of Ψ in RNA oligonucleotides and in tRNAs from S. cerevisiae strains by reverse transcription using RT-KTq I614Y |
| Organisms |
Saccharomyces cerevisiae; synthetic construct |
| Experiment type |
Other
|
| Summary |
We have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT‑signatures specific for pseudouridine (Ψ) without prior chemical treatment of the RNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish U from Ψ in RNA oligonucleotides (modified or unmodified) used in the previous polymerase screening and oligonucleotides which are designed from the human 18S rRNA at the position around 1445. Finally, RT-KTq I614Y was used to detect Ψ55 in tRNAGly(GCC) from Saccharomyces cerevisiae wildtype compared to data from tRNAGly(GCC) from S. cerevisiae pus4Δ.
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| Overall design |
RNA oligonucleotides (modified or unmodified) used in the enzyme screening and oligonucleotides (modified or unmodified) designed from the human 18S rRNA were reverse transcribed by RT-KTq I614Y. Respective cDNA was used to prepare DNA amplicon libraries which were analyzed by NGS. Furthermore, purified total tRNA fractions from S. cerevisiae wt and pus4Δ deletion strain were employed to detect Ψ55.
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| Contributor(s) |
Huber LB, Marx A, Motorin Y, Marchand V |
| Citation(s) |
36971106 |
| |
| Submission date |
Nov 03, 2022 |
| Last update date |
Jun 14, 2023 |
| Contact name |
Luisa Barbara Huber |
| E-mail(s) |
[email protected]
|
| Organization name |
Universität Konstanz
|
| Department |
Department of Chemistry
|
| Lab |
Andreas Marx Lab
|
| Street address |
Universitätsstraße 10
|
| City |
Konstanz |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
78464 |
| Country |
Germany |
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| Platforms (2) |
| GPL31112 |
NextSeq 2000 (Saccharomyces cerevisiae) |
| GPL32628 |
NextSeq 2000 (synthetic construct) |
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| Samples (14)
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| GSM6706480 |
unmodified RNA oligo used in screening - RT-KTq - 100µMdATP |
| GSM6706481 |
Ψ-modified RNA oligo used in screening - RT-KTq - 100µMdATP |
| GSM6706482 |
unmodified RNA oligo used in screening - RT-KTq I614Y - 100µMdATP |
| GSM6706483 |
Ψ-modified RNA oligo used in screening - RT-KTq I614Y - 100µMdATP |
| GSM6706484 |
unmodified RNA oligo used in screening - RT-KTq - 2µMdATP |
| GSM6706485 |
Ψ-modified RNA oligo used in screening - RT-KTq - 2µMdATP |
| GSM6706486 |
unmodified RNA oligo used in screening - RT-KTq I614Y - 2µMdATP |
| GSM6706487 |
Ψ-modified RNA oligo used in screening - RT-KTq I614Y - 2µMdATP |
| GSM6706488 |
unmodified RNA oligo designed from human 18S rRNA - RT-KTq - 2µMdATP |
| GSM6706489 |
Ψ-modified RNA oligo designed from human 18S rRNA - RT-KTq - 2µMdATP |
| GSM6706490 |
unmodified RNA oligo designed from human 18S rRNA - RT-KTq I614Y - 2µMdATP |
| GSM6706491 |
Ψ-modified RNA oligo designed from human 18S rRNA - RT-KTq I614Y - 2µMdATP |
| GSM6706492 |
S. cerevisiae tRNAGly(GCC) wt |
| GSM6706493 |
S. cerevisiae tRNAGly(GCC) pus4Δ |
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| Relations |
| BioProject |
PRJNA897897 |
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