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.

In order to overcome the pH limitations of triplex-forming peptide nucleic acid (PNA) in binding to double-stranded RNA (dsRNA), we have recently proposed a new design of triplex-forming PNA-based fluorogenic probes that work at neutral pH for sensing the panhandle structure of the influenza A virus (IAV) RNA promoter region. Our strategy is based on the conjugation of a small molecule (DPQ) capable of selectively binding to the internal loop structure with the triplex-forming forced intercalation of thiazole orange (tFIT) probe with natural PNA nucleobases. In this work, the triplex formation of tFIT-DPQ conjugate probes with IAV target RNA at neutral pH was examined by means of a stopped-flow technique UV melting and fluorescence titration experiments. The obtained results revealed that (i) the conjugation strategy is responsible for the observed strong binding affinity due to a very fast association rate constant and a slow dissociation rate constant; (ii) the binding follows a pattern of the DPQ unit binding first to the internal loop region, followed by the tFIT unit binding to the complementary dsRNA region. Our results emphasize the importance of both the tFIT and the DPQ components of the conjugate probe design and revealed an association mechanism for the tFIT-DPQ probe-dsRNA triplex formation towards the IAV RNA at neutral pH.

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.

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 (Kd = 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.