Simple protocol for sequence-specific detection of mixed-base nucleic acids using a smart probe with NABs

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Fluorescent smart probes (SP) offer sensitive detection of nucleic acid sequences.
• Existing SPs exhibit limited discrimination between target and mismatch sequences.
• Enhanced specificity is crucial for reliable nucleic acid detection in diagnostics.

Purpose of the Study:

• To improve the sequence-specificity of a fluorescent smart probe for ribonucleic acids detection.
• To develop a simple, enzyme-free method for enhanced nucleic acid sequence analysis.
• To enable routine detection and quantification of nucleic acids for genetic and biomedical applications.

Main Methods:

• Utilized a fluorescent smart probe (SP) for detecting a mixed-base ribonucleic acids sequence.
• Introduced nucleic acid blockers (NABs) – unlabeled, non-fluorescent hairpin oligonucleotides.
• NABs were designed to be complementary to mismatch sequences, preventing probe binding.

Main Results:

• The SP demonstrated high sensitivity for the target ribonucleic acids sequence.
• Nucleic acid blockers significantly enhanced sequence-specificity, improving discrimination against mismatch sequences.
• The combined approach achieved precise detection without amplification enzymes.

Conclusions:

• The developed method using SPs and NABs offers a simple and effective way to enhance nucleic acid sequence detection.
• This approach is suitable for room-temperature applications, including genetic testing and biomedical diagnostics.
• The enhanced specificity facilitates routine nucleic acid quantification and analysis.