Programmable Nucleic Acid Sensing in Human Cells Using Circularizable ssDNA

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Nucleic acid detection and programmable gene expression are crucial for biotechnology and medicine.
• Existing technologies face limitations in sensitivity, specificity, or programmability within living cells.

Purpose of the Study:

• To develop a versatile platform, SONAR (Sensing Of Nucleic acids using ASOs and Reverse-transcriptases), for detecting specific DNA and RNA sequences in human cells.
• To enable target-dependent, programmable gene expression and cell manipulation based on nucleic acid detection.

Main Methods:

• SONAR utilizes circularizable single-stranded DNA (ssDNA) sensors that undergo ligation upon hybridization with target DNA or reverse-transcribed RNA.
• Antisense oligonucleotides (ASOs) prime reverse transcription for RNA detection, generating DNA that facilitates ssDNA circularization.
• Cellular ligases mediate the ligation step, initiating the expression of linked genetic payloads.

Main Results:

• Demonstrated detection of single-stranded DNA (ssDNA), exogenous RNA, and endogenous RNA.
• Successfully coupled nucleic acid sensing to the programmable expression of various protein payloads, including reporters, recombinases, and genome editors.
• Enabled enrichment and clonal recovery of target-positive cells from mixed populations.

Conclusions:

• SONAR provides a versatile framework for targeted nucleic acid detection and inducible gene expression within living cells.
• The platform has broad potential applications in diagnostics, therapeutics, and synthetic biology.
• SONAR represents a significant advancement in programmable cellular control based on nucleic acid signatures.