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Duplex stem replacement with bPNA+ triplex hybrid stems enables reporting on tertiary interactions of internal RNA domains

preprint
revised on 30.03.2019 and posted on 02.04.2019 by Dennis Bong, Shiqin Miao, Yufeng Liang, Jie Mao, Ila Marathe

We report herein the synthesis and DNA/RNA binding properties of bPNA+, a new variant of bifacial peptide nucleic acid (bPNA) that binds oligo T/U nucleic acids to form triplex hybrids. By virtue of a new bivalent sidechain on bPNA+, similar DNA affinity and hybrid thermostability can be obtained with half the molecular footprint of previously reported bPNA. Lysine derivatives bearing two melamine bases (K2M) can be prepared on multigram scale by double reductive alkylation with melamine acetaldehyde, resulting in a tertiary amine sidechain that affords both peptide solubility and selective base-triple formation with 4 T/U bases; the Fmoc-K2M derivative can be used directly in solid phase peptide synthesis, rendering bPNA+ conveniently accessible. A compact bPNA+ binding site of two U6 domains can be genetically encoded to replace existing 6 bp stem elements at virtually any location within an RNA transcript. We thus replaced internal 6 bp RNA stems that supported loop regions with 6 base-triple hybrid stems, using fluorophore-labeled bPNA+. As the loop regions engaged in RNA tertiary interactions, the labeled hybrid stems provided a fluorescent readout; bPNA+ enabled this readout without covalent chemical modification or introduction of new structural elements. This strategy was demonstrated to be effective for reporting on widely observed RNA tertiary interactions such as intermolecular RNA-RNA kissing loop dimerization, RNA-protein binding as well as intramolecular RNA tetraloop-tetraloop receptor binding, illustrating the potential general utility of this method. The modest 6 bp stem binding footprint of bPNA+ makes the hybrid stem replacement method practical for noncovalent installation of synthetic probes of RNA interactions. We anticipate that bPNA+ structural probes will be useful for the study of tertiary interactions in long noncoding RNAs.


Funding

GM111995-01A1

GRT00044810

DMR 1802432

GM120582

History

Email Address of Submitting Author

bong.6@osu.edu

Institution

The Ohio State University

Country

USA

ORCID For Submitting Author

0000-0003-3778-9183

Declaration of Conflict of Interest

no conflict

Version Notes

version one

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