DNA Flap-Mediated Control of Transcription for Programmable RNA Synthesis

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• RNA-based therapeutics require precise and programmable RNA synthesis.
• Current T7 RNA polymerase (T7RP) regulation often involves auxiliary proteins or chemical agents.
• Need for nucleic acid sequence-based regulatory mechanisms for T7RP.

Purpose of the Study:

• To investigate sequence-specific regulation of T7RP activity using DNA flap sequences.
• To develop novel, protein-free transcription control platforms based on observed sequence preferences.
• To establish a new framework for programmable RNA synthesis.

Main Methods:

• Appending single-stranded DNA flap sequences to the 3' end of the T7 promoter's non-template strand.
• Evaluating the impact of flap sequence composition on T7RP-mediated in vitro transcription efficiency.
• Developing D-FIT (DNAzyme-mediated Flap promoter Induced Transcription control) and M-FIT (MNAzyme-mediated Flap promoter Induced Transcription control) platforms.

Main Results:

• A sequence-dependent inhibitory effect of DNA flaps on T7RP transcription was observed.
• Flaps enriched in pyrimidines (cytosine and thymine) significantly suppressed transcription.
• Successfully developed D-FIT and M-FIT platforms for precise T7RP regulation without auxiliary factors.

Conclusions:

• Uncovered a novel, sequence-specific regulatory mechanism for T7 RNA polymerase.
• Established DNA flap promoters as a tool for programmable RNA synthesis.
• Demonstrated the potential of D-FIT and M-FIT for applications in RNA therapeutics and diagnostics.