Specific fluorescence detection strategy for single-stranded nucleic acids by dual-toehold branch migration

Area of Science:

• Molecular Biology
• Biochemistry
• Nucleic Acid Chemistry

Background:

• Accurate detection of single-stranded nucleic acids (ssDNA and RNA) is crucial for understanding disease mechanisms.
• Existing detection methods often lack specificity for single-stranded targets.
• Advancements in nucleic acid detection are needed to improve disease diagnostics.

Purpose of the Study:

• To develop a highly specific fluorescence detection strategy for single-stranded DNA (ssDNA) and microRNA (miRNA).
• To utilize dual-toehold branch migration (DTBM) for enhanced specificity in nucleic acid detection.
• To establish a user-friendly and efficient method for ssDNA and miRNA detection.

Main Methods:

• Developed a DTBM-based fluorescence detection strategy for ssDNA and miRNA.
• Employed KF polymerase to convert ssDNA L1 into a double-stranded probe for DTBM initiation.
• Integrated strand displacement amplification (SDA) to generate ssDNA L2 for SDA-triggered DTBM in miRNA let-7a detection.

Main Results:

• Achieved high specificity for detecting ssDNA L1 and miRNA let-7a.
• Demonstrated average discrimination factors (DFs) of 24.49 for ssDNA L1 and 30.59 for miRNA let-7a.
• Exhibited excellent analytical performance and user-friendliness, requiring no frequent temperature changes or sample additions.

Conclusions:

• The DTBM strategy offers a specific and efficient method for ssDNA and miRNA detection.
• This approach shows significant potential for practical applications in diagnostics and research.
• The user-friendly nature of the assay simplifies complex nucleic acid detection procedures.