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

Area of Science:

• Molecular Biology
• Biochemistry
• Chemical Biology

Background:

• Fluorescence-based oligonucleotide (ON) hybridization probes are crucial for RNA detection and profiling in cells.
• Cellular RNA's complex structures limit accessibility and hybridization efficiency, hindering probe application.
• Existing probes face challenges in target accessibility and hybridization efficiency within cellular environments.

Purpose of the Study:

• To develop a robust hybridization probe for effective RNA imaging within cellular environments.
• To overcome limitations of conventional oligonucleotide probes in detecting structured RNA targets.
• To create a sensitive and specific probe for visualizing polyadenylated [poly(A)] RNAs.

Main Methods:

• Development of a microenvironment-sensitive fluorescent nucleobase analogue (Lucifer chromophore attached to uracil).
• Integration of the fluorescent analogue into peptide nucleic acid (PNA) oligomers for stable RNA hybridization.
• Utilizing the PNA probe's fluorescence response to neighboring bases for signal detection.

Main Results:

• The PNA-based fluorescent probe demonstrated high responsiveness to its neighboring base environment.
• A notable fluorescence enhancement was observed when the PNA probe encountered an adenine repeat in RNA.
• A poly(T) PNA probe successfully visualized polyadenylated [poly(A)] RNAs in cellular imaging experiments.

Conclusions:

• Responsive fluorescent nucleobase analogues can be incorporated into PNA oligomers to create effective hybridization probes.
• The developed PNA probes offer a robust method for detecting and imaging nucleic acid sequences in cells.
• This approach provides a valuable tool for studying RNA motifs like poly(A) tails, crucial for RNA lifecycle.