Angewandte Chemie International Edition
10.1002/anie.202001300
COMMUNICATION
Gel images show Cy5 channel. For reference, the input RNA is shown to the
left (stained with Sybr gold). c) Orthogonal labeling of a 41-nt RNA transcript
with FJ1 and FH14 ribozymes, and analysis by cleavage with an 8-17
deoxyribozyme). The multi-channel image shows Cy5-labeled RNA in red,
fluorescein-labeled RNA in green, and the double-labeled RNA in yellow. d)
Orthogonal labeling of E.coli 16S and 23S rRNA with FJ1 and FH14. Lanes 1
and 4 contain matched ribozymes and substrates, lanes 2 and 3 are negative
controls with mismatched ribozyme - substrate combination showing orthogon-
ality, and lane 5 contains both ribozymes and both substrates. e) Primer
extension assay confirms labelling sites of both FJ1 and FH14. Exemplarily
shown for A272 and A1572 on 23S rRNA. Additional sites in Figure S13.
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In summary, we have established RNA-catalyzed labeling of
RNA using the antiviral nucleoside analog tenofovir as scaffold for
bioorthogonal ribozyme substrates. This work also demonstrates
the reliability of the nucleotide transferase in vitro selection
strategy and the design of the structured RNA pool for addressing
a predetermined nucleotide in the target RNA for covalent labeling.
Moreover, the analysis of the enriched library by Illumina
sequencing revealed additional ribozyme variants with greater
generality that would have been missed by traditional Sanger
sequencing. Importantly, the resulting phosphonate linkages
proved more stable towards yeast debranching enzyme (Dbr1)
compared to the previously used natural phosphate linkages.
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Other chemical modification such as thiophosphates or
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In
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[17] Deep sequencing libraries were prepared from cDNA of rounds 7 and 12
by PCR using an 8-nt UMI as described in the supporting information.
Illumina sequencing was performed at the Core Unit Systems Medicine
at the University of Würzburg. Analysis of the sequencing data was
performed using fastaptamer perl scripts: K. K. Alam, J. L. Chang, D. H.
Burke, Mol. Ther. Nucleic Acids 2015, 4, e230.
Acknowledgements
[
18] FJ is our laboratory nomenclature of the tenofovir transferase ribozyme
selection, and the numbers 1 and 8 relate to arbitrary clone numbers for
Sanger sequencing. C in FJC refers to sequence cluster identified in
NGS data; C9 is the 9th cluster in the output generated by
fastaptamer_cluster. See supporting information for more details.
This work was supported by the ERC-CoG illumizymes (682586
to C.H.). A. Hoskins (University of Wisconsin) is gratefully
acknowledged for providing recombinant Dbr1 enzyme.
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Keywords: ribozyme • in vitro selection • site-specific RNA
[20] The tenofovir analogs synthesized in this study were used as racemic
6
labeling • antiviral nucleoside analog • tenofovir
mixtures. The comparison with commercially available N -unmodified
(
R)-tenofovir diphosphate revealed similar activity of FJ1 with
enantiomerically pure and racemic tenofovir diphosphate substrate
Figure S15).
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(
[
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