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Title: Sequence- and structure-specific probing of RNAs by short nucleobase-modified dsRNA-binding PNAs incorporating a fluorescent light-up uracil analog
Authors: Krishna, Manchugondanahalli Shivakumar
Toh, Desiree-Faye Kaixin
Meng, Zhenyu
Ong, Alan Ann Lerk
Wang, Zhenzhang
Lu, Yunpeng
Xia, Kelin
Prabakaran, Mookkan
Chen, Gang
Keywords: Science::Chemistry
Issue Date: 2019
Source: Krishna, M. S., Toh, D. K., Meng, Z., Ong, A. A. L., Wang, Z., Lu, Y., Xia, K., Prabakaran, M. & Chen, G. (2019). Sequence- and structure-specific probing of RNAs by short nucleobase-modified dsRNA-binding PNAs incorporating a fluorescent light-up uracil analog. Analytical Chemistry, 91(8), 5331-5338.
Project: RGT3/13
Journal: Analytical Chemistry 
Abstract: RNAs are emerging as important biomarkers and therapeutic targets. The strategy of directly targeting double-stranded RNA (dsRNA) by triplex-formation is relatively underexplored mainly due to the weak binding at physiological conditions for the traditional triplex-forming oligonucleotides (TFOs). Compared to DNA and RNA, peptide nucleic acids (PNAs) are chemically stable and have a neutral peptide-like backbone, and thus, they show significantly enhanced binding to natural nucleic acids. We have successfully developed nucleobase-modified dsRNA-binding PNAs (dbPNAs) to facilitate structure-specific and selective recognition of dsRNA over single-stranded RNA (ssRNA) and dsDNA regions at near-physiological conditions. The triplex formation strategy facilitates the targeting of not only the sequence but also the secondary structure of RNA. Here, we report the development of novel dbPNA-based fluorescent light-up probes through the incorporation of A-U pair-recognizing 5-benzothiophene uracil (btU). The incorporation of btU into dbPNAs does not affect the binding affinity toward dsRNAs significantly, in most cases, as evidenced by our nondenaturing gel shift assay data. The blue fluorescence emission intensity of btU-modified dbPNAs is sequence- and structure-specifically enhanced by dsRNAs, including the influenza viral RNA panhandle duplex and HIV-1–1 ribosomal frameshift-inducing RNA hairpin, but not ssRNAs or DNAs, at 200 mM NaCl, pH 7.5. Thus, dbPNAs incorporating btU-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.
ISSN: 0003-2700
DOI: 10.1021/acs.analchem.9b00280
Rights: © 2019 American Chemical Society. All rights reserved.
Fulltext Permission: none
Fulltext Availability: No Fulltext
Appears in Collections:SPMS Journal Articles

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