Journal
NUCLEIC ACIDS RESEARCH
Volume 46, Issue 15, Pages 7506-7521Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gky631
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Funding
- Singapore Ministry of Education (MOE) [RGT3/13, RG42/15, RG152/17]
- MOE [MOE2013-T2-2-024, MOE2015-T2-1-028]
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Double-stranded RNA (dsRNA) structures form triplexes and RNA-protein complexes through binding to single-stranded RNA (ssRNA) regions and proteins, respectively, for diverse biological functions. Hence, targeting dsRNAs through major-groove triplex formation is a promising strategy for the development of chemical probes and potential therapeutics. Short (e.g., 6-10 mer) chemically-modified Peptide Nucleic Acids (PNAs) have been developed that bind to dsRNAs sequence specifically at physiological conditions. For example, a PNA incorporating a modified base thio-pseudoisocytosine (L) has an enhanced recognition of a G-C pair in an RNA duplex through major-groove L.G-C base triple formation at physiological pH, with reduced pH dependence as observed for C+.G-C base triple formation. Currently, an unmodified T base is often incorporated into PNAs to recognize a Watson-Crick A-U pair through major-groove T.A-U base triple formation. A substitution of the 5-methyl group in T by hydrogen and halogen atoms (F, Cl, Br, and I) causes a decrease of the pK(a) of N3 nitrogen atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions. Here, we synthesized a series of PNAs incorporating uracil and halouracils, followed by binding studies by non-denaturing polyacrylamide gel electrophoresis, circular dichroism, and thermal melting. Our results suggest that replacing T with uracil and halouracils may enhance the recognition of an A-U pair by PNA.RNA(2) triplex formation in a sequence-dependent manner, underscoring the importance of local stacking interactions. Incorporating bromouracils and chlorouracils into a PNA results in a significantly reduced pH dependence of triplex formation even for PNAs containing C bases, likely due to an upshift of the apparent pKa of N3 atoms of C bases. Thus, halogenation and other chemical modifications may be utilized to enhance hydrogen bonding of the adjacent base triples and thus triplex formation. Furthermore, our experimental and computational modelling data suggest that PNA.RNA(2) triplexes may be stabilized by incorporating a (UL)-U-Br step but not an (LU)-U-Br step, in dsRNA-binding PNAs.
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