Deep-Red Light-up Signaling of Benzo[c,d]indole–Quinoline Monomethine Cyanine for Imaging of Nucleolar RNA in Living Cells and for Sequence-Selective RNA Analysis

Abstract

RNA-binding small probes with deep-red emission are promising for RNA analysis in biological media without suffering from background fluorescence. Here benzo­[c,d]­indole–quinoline (BIQ), an asymmetric monomethine cyanine analogue, was newly developed as a novel RNA-selective probe with light-up signaling ability in the deep-red spectral range. BIQ features a significant light-up response (105-fold) with an emission maximum at 657 nm as well as improved photostability over the commercially available RNA-selective probe, SYTO RNA select. BIQ was successfully applied to the fluorescence imaging of nucleolar RNAs in living cells with negligible cytotoxicity. Furthermore, we found the useful ability of BIQ as a base surrogate integrated in peptide nucleic acid (PNA) oligonucleotides for RNA sequence analysis. BIQ base surrogate functioned as a deep-red light-up base surrogate in forced intercalation (FIT) and triplex-forming FIT (tFIT) systems for the sequence-selective detection of single-stranded and double-stranded RNAs, respectively.

Deep-red fluorogenic cyanine dyes carrying an amino group-terminated side chain for improved RNA detection and nucleolar RNA imaging

“…7 Meanwhile, it is highly likely that the uorescence signal in the nucleolus arises from the binding of BIQ-NH 2 to RNAs that were abundant in the nucleolus, such as ribosomal RNAs (rRNAs). 3,4 This was supported by the examination of nuclease digestion in the xed-permeabilized cells (Fig. 5).…”

“…These BIQ derivatives had much superior photostability compared to SYTO RNA select whose emission decreased as much as 52%. 4 Taken together, both BIQ-N + Me 3 and BIQ-NH 2 are promising probes for the robust and improved sensitivity for RNA sensing in solutions.…”

Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS)

“…12,13 The need to achieve more costeffective, faster analysis and higher single nucleotide resolution continues to drive technological developments. 14 Recent examples include amendments to existing technologies such as molecular beacons, 15,16 melting analysis, 17,18 environmentally sensitive uorescent nucleobases, [19][20][21][22][23][24] and strand displacement probes 25,26 or new technologies such as polymerase-amplied release of ATP (POLARA) 27 or graphene-based biosensors for real-time kinetic monitoring of hybridization. 28 The analysis of SNVs requires technologies with the highest nucleotide resolution to ascertain the polymorphism or variation.…”

Nucleic Acids Analysis

“…Furthermore, simultaneous detection of multiple targets can be achieved in the same sample by using different fluorochromes that can be spectrally resolved [189,190]. With these features, a variety of strategies have been designed for imaging and detecting nucleic acids with superb sensitivity, high selectivity and good biocompatibility in living cells and in vivo [190][191][192][193][194].…”