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Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains.

ABSTRACT: 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 side chain 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 side chain 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, and 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.

SUBMITTER: Miao S

PROVIDER: S-EPMC7043357 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains.

Miao Shiqin S Liang Yufeng Y Marathe Ila I Mao Jie J DeSantis Chris C Bong Dennis D

Journal of the American Chemical Society 20190529 23

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 side chain 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 (K<sup>2M</sup>) can be prepared on multigram scale by double reductive alkylation with mela ...[more]

PMID: 31094510

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Project description:DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2'-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10x faster than the thymidine derivative when incorporated at the 5'-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.

Dataset Information

Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains.

Publications

Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains.

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