Journal
BIOCHEMISTRY
Volume 58, Issue 32, Pages 3444-3453Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.9b00521
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Funding
- Singapore Ministry of Education (MOE) [RG152/17]
- MOE [MOE2015-T2-1-028]
- JSPS [17K20145]
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Chemically modified short peptide nucleic acids (PNAs) recognize RNA duplexes under near physiological conditions by major-groove PNA.RNA-RNA triplex formation and show great promise for the development of RNA-targeting probes and therapeutics. Thymine (T) and uracil (U) are often incorporated into PNAs to recognize A-U pairs through major-groove T.A-U and U-A-U base triple formation. Incorporation of a modified nucleobase, 2-thiouracil (s(2)U), into triplex forming oligonucleotides stabilizes both DNA and RNA triplexes. Thiolation of uracil causes a decrease in the dehydration energy penalty for triplex formation as well as a decrease in the pK(a) of the N3 atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions, similar to the previously reported thiolation effect of pseudoisocytosine (J to L substitution). Here, we incorporated s(2)U into short PNAs, followed by binding studies of a series of s(2)U-modified PNAs. We demonstrated by nondenaturing polyacrylamide gel electrophoresis and thermal melting experiments that s(2)U and L incorporated into dsRNA-binding PNAs (dbPNAs) enhance the recognition of A-U and G-C pairs, respectively, in RNA duplexes in a position-independent manner, with no appreciable binding to the DNA duplex. Combining s(2)U and L modifications in dbPNAs facilitates enhanced recognition of dsRNAs and maintains selective binding to dsRNAs over ssRNAs. We further demonstrated through a cell-free assay the application of the s(2)U- and L-modified dbPNAs (8-mer, with a molecular mass of similar to 2.3 kDa) in the inhibition of the pre-microRNA-198 maturation in a substrate-specific manner. Thus, s(2)U-modified dbPNAs may be generally useful for the enhanced and selective recognition of RNA duplexes and for the regulation of RNA functions.
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