Engineering DNA Assembly in Vivo and Knock Down of Gene Expression Using the Reverse Transcriptase Activity

Technology

A synthetic pathway was developed in E. coli to perform reverse transcription. Reverse transcriptase and a functional template, a conjugate of a eukaryotic t-RNALys with a non­coding targeted RNA, are expressed in the cell.  The functional template serves as a reconstituted replacement for a fundamental missing element in the activation of a reverse transcriptase, such as the HIV reverse transcriptase, process in bacteria. This strategy produces oligonucleotides, which optionally may be further manipulated to cause nucleic acid single strand cross­over assembly for the formation of various DNA nanostructures. Additional internal and/or external stimuli can fine tune the in vivo synthesis of ssDNAs resulting in the dynamic control of the nanostructure shape and/or size and/or constitution. Once the ssDNA oligonucleotide is produced in the cell it may be used in the cell or isolated from the cell for therapeutic applications.  

Problem Addressed

Functional DNA nanostructures have a number of applications, including, but not limited to, scaffolds for the organization of materials, synthesis of organic elements, control of biomineralization, drug delivery, and intracellular sensors.  In vivo implementation has been largely limited as it is costly to generate such elements and current method’s DNA assembly information remains outside of the genetic record of the cell causing it to lose structural information upon division. Further, it remains difficult to build structures using long single stranded DNA (ssDNA). This invention is a method to develop genetic circuits for the self-assembly of nanostructures in vivo using reverse transcription to produce ssDNA. This technology has already been demonstrated through engineering E. coli to reverse transcribe ssDNA for the successful knockdown of genes.  

Advantages     

• Self-assembly of different DNA nanostructures in vivo
• Control of shape/size/constitution