A Lucifer‐Based Environment‐Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs

Abstract

Fluorescence‐based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment‐sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore (1,8‐naphthalimide) at the 5‐position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells—poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.

Peptide nucleic acid (PNA) and its applications in chemical biology, diagnostics, and therapeutics

“…Effort to design nucleobase-dyes hybrids that are environmentally sensitive have also been reported. [59][60][61] Probes that have leveraged this strategy have also been combined with a GO adsorption/quenching approach for a sensitive detection of human telomeric repeats.…”

Clickable PNA Probes for Imaging Human Telomeres and Poly(A) RNAs

“…As a positive control, cells were incubated with Cy5-(dT) 30 , a commonly used DNA ON probe for poly­(A) RNA imaging. , The DNA probe produced a staining pattern similar to that of the PNA probe (Figure B). The binding of PNA–DNA probes to poly­(A) RNAs was further confirmed by RNase A treatment . Cells treated with RNase A and then hybridized with the probes revealed no detectable fluorescence signal from the cells (Figure ).…”

“…The binding of PNA–DNA probes to poly(A) RNAs was further confirmed by RNase A treatment. 85 Cells treated with RNase A and then hybridized with the probes revealed no detectable fluorescence signal from the cells ( Figure 7 ).…”

Short Tandem Repeat DNA Profiling Using Perylene-Oligonucleotide Fluorescence Assay

“…STR4.3’s emission maximum moved further to 538 nm, and the intensity increased by 1.6-fold upon hybridization. This points to the high sensitivity of the probes’ optical properties to both the type of modified nucleoside and to its positioning within the probe. − …”