Fluorescence Sensing of the Panhandle Structure of the Influenza A Virus RNA Promoter by Thiazole Orange Base Surrogate-Carrying Peptide Nucleic Acid Conjugated with Small Molecule

Abstract

We have developed a new class of triplex-forming peptide nucleic acid (PNA)-based fluorogenic probes for sensing of the panhandle structure of the influenza A virus (IAV) RNA promoter region. Here, a small molecule (DPQ) capable of selectively binding to the internal loop structure was conjugated with triplex-forming forced intercalation of the thiazole orange (tFIT) probe with natural PNA nucleobases. The resulting conjugate, tFIT-DPQ, showed a significant light-up response (83-fold) upon strong (K d = 107 nM) and structure-selective binding to the IAV RNA promoter region under physiological conditions (pH 7.0, 100 mM NaCl). We demonstrated the conjugation of these two units through the suitable spacer was key to show useful binding and fluorogenic signaling functions. tFIT-DPQ facilitated the sensitive and selective detection of IAV RNA based on its binding to the promoter region. Furthermore, we found that tFIT-DPQ could work as a sensitive indicator for screening of test compounds targeting the IAV RNA promoter region in the fluorescence indicator displacement assay.

Kinetic analysis of highly effective triplex formation between a small molecule–peptide nucleic acid conjugate probe and the influenza A virus RNA promoter region at neutral pH

“…It was established that the TO base surrogate connected through the propyl linker in the tFIT probes was the best. Probe 2 was indeed found to work as a stronger binder than any other tFIT-DPQ probe, 25 and the binding affinity (Kd) reached 52  16 nM. UV melting experiments confirm that the binding is not based on duplex-or triplex-invasion of tFIT-DPQ probes when binding to the panhandle structure of IAV RNA.…”

“…1C). One is the previously reported tFIT-DPQ (Lys) (NH2-TC(TO-C1)TCTTT-Lys-Lys(DPQ)-CONH2) with Kd of 185  31 nM, 25 here we call probe 1. Another is a newly designed one (named probe 2), in which the TO base surrogate was attached to the PNA backbone with a propyl linker (H2N-TC(TO-C3)TCTTT-Lys-Lys(DPQ)-CONH2).…”

One-step synthesis of triethanolamine-capped Pt nanoparticle for colorimetric and electrochemiluminescent immunoassay of SARS-CoV spike proteins

“…Medical diagnosis of early form of disease relies on the effective quantitative detection of patient biomarkers, so various testing technologies, such as enzyme-linked immunosorbent assay (ELISA) [12] , [13] , surface plasmon resonance (SPR) [14] , [15] and fluorescence analysis [16] , have been widely employed in disease diagnosis. Among numerous analytic methods, electrochemiluminescent (ECL) [17] , [18] , [19] and colorimetric [20] , [21] methods hold promise for achieving sensitive, widespread and facile disease diagnosis due to their high selectivity and high noise-to-signal.…”

Classical thiazole orange and its regioisomer as fluorogenic probes for nucleolar RNA imaging in living cells

“…RNA-selective small molecular weight probes have been of great interest as a tool for the regulation, inhibition, and/or study of biological RNA functions. 1–3 Following our previous studies on fluorescent probes targeting biologically important RNAs, 4–7 our particular interest herein is the design of fluorogenic dyes for the imaging of nucleolar RNA in living cells based on the considerable attention on the role of nucleolus in major physiological functions, such as stress response, development and aging, and canceration. 8 In contrast to well-established DNA-selective dyes, such as Hoechst 33342, RNA-selective dyes have been less developed because of the challenge to make small molecules have RNA selectivity over DNA.…”