BUNSEKI KAGAKU
Print ISSN : 0525-1931
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Triplex-Forming Peptide Nucleic Acid Probes Having Cyanine Base Surrogates for Fluorogenic Sensing of Double-Stranded RNA
Seiichi NISHIZAWATakaya SATOEn Ting Tabitha LEENaonari SAKAMOTOToshiki CHIBATakaaki TANABEYukina YOSHINOYuki TAKAHASHIYusuke SATO
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2022 Volume 71 Issue 3 Pages 133-144

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Abstract

Here we report on triplex-forming peptide nucleic acid (PNA) probes having thiazole orange (TO) as a base surrogate for fluorogenic sensing of double stranded RNA (dsRNA). Following parent forced intercalation of thiazole orange (FIT) probe for single-stranded RNA or DNA, our PNA probe, named triplex-forming FIT (tFIT) probe, forms the very stable triplex with the target dsRNA at acidic pH (dissociation constant Kd = 23±0.5 nM; pH 5.5, 25°C), and the binding is accompanied by the remarkable light-up response of the TO unit (λem = 542 nm; I/I0 = 120-fold). The association kinetics of tFIT probe is very fast (association rate constant kon = 3.8 × 105 M−1 s−1), and the association phase of the PNA-dsRNA triplex formation comprises a non-directional nucleation-zipping mechanism that is coupled with the conformational transition of the unbound PNA. Importantly, the TO base surrogate functions as a universal base for any base-pair opposite the TO unit, allowing its binding to pyrimidine bases (C and U) in the target dsRNA. Red-emissive cyanine dyes, including quinolone blue (QB: λem = 608 nm; I/I0 = 146-fold) and benzo[c,d]indole-quinoline (BIQ: λem = 663 nm; I/I0 = 41-fold), are also available as the fluorogenic universal base, which will further enhance the practicability for analytical applications. We also demonstrate that the conjugation of small molecule with tFIT probe having natural nucleobases does promote the effective triplex-formation of PNA even at a neutral pH, resulting in markedly strong binding with the bacterial ribosomal RNA A-site (Kd = 190±72 nM; pH 7.0, 25°C). These promising binding and signaling functions of tFIT probes are discussed as a rational basis for the advanced design of triplex-forming PNA that overcomes both the pH and sequence limitations.

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© 2022 The Japan Society for Analytical Chemistry
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