Circular single-stranded synthetic DNA delivery vectors for microRNA

Area of Science:

• Molecular Biology
• Biotechnology
• RNA Therapeutics

Background:

• Single-stranded circular oligodeoxynucleotides undergo rolling circle transcription (RCT) by RNA polymerases (RNAPs) into repetitive RNA multimers.
• Redesigning these circular DNA molecules to encode primary microRNA (miRNA) mimics is a novel approach for intracellular RNA production.

Purpose of the Study:

• To develop an improved method for circularizing synthetic DNA for RCT using a thermostable RNA ligase.
• To assess the feasibility of transcribing miRNA mimics from circular DNA templates via RCT.
• To evaluate the potential for endogenous processing of resulting RNA transcripts in human cells.

Main Methods:

• Improved circularization of single-stranded synthetic DNA using a splint-independent thermostable RNA ligase.
• In vitro transcription (RCT) of four miRNA-encoding circular DNA templates using bacterial and phage RNAPs.
• In vitro processing assay of a typical primary-miRNA rolling circle transcript using human Drosha immunoprecipitate.

Main Results:

• The improved circularization method efficiently produced templates for RCT.
• Secondary structures within miRNA-encoding vectors did not impede RCT by RNAPs.
• A representative primary-miRNA rolling circle transcript was accurately processed by human Drosha, indicating compatibility with endogenous miRNA pathways.

Conclusions:

• Circular oligonucleotides are viable candidate vectors for small RNA delivery.
• The developed method facilitates the production of miRNA mimics via RCT.
• Successful in vitro processing suggests potential for intracellular miRNA maturation and function in human cells expressing related RNAPs.