In Vivo ssDNA Synthesis via Reverse Transcriptase and tRNA Templates
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Solution Overview
Problem
Current DNA nanotechnology faces challenges in in-vivo applications and bioengineering of synthetic pathways for self-assembly of single-stranded DNA (ssDNA) within E. coli, particularly due to the lack of essential elements for reverse transcriptase activity in bacteria.
Innovation Solution
The method involves expressing reverse transcriptase and a functional template with a non-coding tRNA structure in bacterial cells, such as E. coli, to produce ssDNA oligonucleotides, utilizing eukaryotic t-RNALys to initiate the polymerase process and enable efficient in vivo DNA production, allowing for the synthesis of ssDNA oligonucleotides that can be used for DNA-directed self-assembly and nanostructure formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If reverse transcriptase is expressed in bacterial cells to produce ssDNA oligonucleotides, then productivity of ssDNA synthesis is improved, but device complexity increases due to the need for eukaryotic tRNA structure and non-coding RNA sequence
Solution Approach 1:
The functional template is segmented into distinct modules: a non-coding RNA sequence that recruits reverse transcriptase and a coding RNA sequence that serves as the template for ssDNA synthesis. This modular design allows independent optimization of each function while maintaining overall system efficiency.
Solution Approach 2:
A non-coding RNA sequence acts as an intermediary element that bridges the eukaryotic reverse transcriptase and the bacterial cellular machinery. This intermediary contains a tRNA-like structure that the reverse transcriptase recognizes, enabling the enzyme to function in bacterial cells without direct modification of the enzyme itself.
2Manufacturing precision
If eukaryotic t-RNALys structure is used to initiate reverse transcriptase activity in bacteria, then manufacturing precision of ssDNA assembly is improved, but ease of manufacture deteriorates due to cross-species biological component integration
Solution Approach 1:
The tRNA-like structure serves multiple functions: it recruits reverse transcriptase to the functional template, positions the enzyme correctly for initiation, and provides a universal recognition signal that works across species boundaries. This multi-functionality reduces the need for separate control mechanisms.
Solution Approach 2:
The secondary structure of the non-coding RNA is designed to mimic the three-dimensional structure of tRNA, creating a structural parameter match that enables recognition by reverse transcriptase. This structural parameter change allows the system to function despite species differences in primary sequence.
3Adaptability or versatility
If in vivo ssDNA synthesis pathway is engineered in E. coli, then adaptability of DNA nanotechnology applications is improved, but loss of information occurs due to lack of intracellular exonuclease activity affecting ssDNA stability
Solution Approach 1:
The system incorporates protective elements in advance: the ssDNA is synthesized with a 5' phosphate group and 3' hydroxyl group configuration that is resistant to degradation, and the functional template is designed to be transcribed and processed quickly to minimize exposure to intracellular nucleases before the ssDNA can be exported or assembled.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables precise self-assembly of DNA nanostructures and overcomes limitations in ssDNA production, allowing for the formation of complex DNA nanostructures and dynamic control of their shape and size, facilitating applications in nanotechnology and biomedicine.
Implementation Method 1
a reverse transcriptase and a functional template, wherein the non-coding RNA sequence is capable of recruiting the reverse transcriptase and the coding RNA sequence is a template for synthesis of the ssDNA oligonucleotide
Implementation Method 2
the non-coding tRNA structure is capable of initiating transcription of the coding RNA sequence using the reverse transcriptase to produce the ssDNA oligonucleotide in the cell
Implementation Method 3
subjecting the set of ssDNA oligonucleotides to conditions to promote DNA-directed self-assembly, wherein the DNA-directed self-assembly produces a nucleic acid nanostructure
Data Source
AI summary
Cells that can synthesize oligonucleotides in vivo to produce a nucleic acid nanostructure are described. Methods for producing oligonucleotide nanostructures for use in regulating gene expression and altering biological pathways are provided. Methods of performing multiplex automated genome editing (MAGE) are also provided.