In Vivo Nucleic Acid Diversity via Error-Prone Reverse Transcriptase
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Solution Overview
Problem
Current methods for targeted nucleic acid diversity generation in vivo are limited by low mutagenesis rates and the inability to precisely control mutation positions, often resulting in nonsense mutations and inefficient exploration of protein sequence diversity.
Innovation Solution
A system utilizing a mutagenic reverse transcriptase from Diversity Generating Retroelements (DGRs) to produce mutagenized cDNA oligos, which are then recombined into a target region using oligo recombineering, enabling precise and targeted mutagenesis within a compact plasmid-borne system in E. coli, with the potential for widespread application in directed evolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If targeted mutagenesis is performed in vitro through molecular biology techniques, then mutation precision is improved, but process complexity and time consumption increase
Solution Approach 1:
The patent replaces in vitro molecular biology techniques with an in vivo system where a mutagenic reverse transcriptase complex naturally performs mutagenesis within the cell. This substitution eliminates the need for complex external molecular biology procedures while maintaining precision through the targeted action of the reverse transcriptase on the specific DNA sequence.
Solution Approach 2:
The system enables the cell to perform mutagenesis autonomously through the expression of the reverse transcriptase complex, which self-assembles and acts on the target sequence without requiring external intervention. The mutagenic cDNA synthesis occurs naturally within the cellular environment, eliminating the need for complex in vitro processing steps.
2Quantity of substance
If error-prone reverse transcription is used to generate diversity, then sequence diversity is improved, but nonsense mutation rate increases
Solution Approach 1:
The patent applies local quality by restricting the mutagenic activity to a specific target sequence region through the design of the reverse transcriptase binding site and template RNA. The error-prone reverse transcription occurs locally at the target locus while the rest of the genome remains unaffected, allowing diversity generation without propagating nonsense mutations throughout the entire genome.
Solution Approach 2:
The system performs preliminary action by using the template RNA to guide the reverse transcriptase to the correct target sequence before mutagenesis occurs. This pre-positioning ensures that error-prone reverse transcription happens only at the intended location, preventing nonsense mutations in other genomic regions.
3Productivity
If DGR system is used for targeted mutagenesis, then mutagenesis rate is improved, but requirement for recognition sequence limits versatility
Solution Approach 1:
The patent segments the DGR system into its core functional components (reverse transcriptase, template RNA, accessory proteins) and separates the recognition sequence requirement from the mutagenesis function. This segmentation allows the mutagenesis machinery to be applied to any target sequence by simply changing the template RNA, while the reverse transcriptase complex remains a reusable component.
Solution Approach 2:
The system achieves universality by designing the reverse transcriptase complex to function with any target sequence through programmable template RNA. Once the complex is expressed, it can be directed to mutagenize different genes and sequences by changing the template RNA, eliminating the need for different recognition sequences for different targets.
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 significantly increases the in vivo mutagenic potential of target sequences, allowing for the production of a vast number of protein sequence variants in a highly targeted manner, while minimizing nonsense mutations and enabling flexible mutagenesis profiles.
Implementation Method 1
a variable region within the genome will be overwritten by a DNA fragment produced from a near repeat template region in a process involving transcription, error-prone reverse transcription of the template and recombination
Implementation Method 2
The error-prone reverse transcription ensures the introduction of genetic diversity at the variable region
Implementation Method 3
recombining the mutagenized cDNA with the homologous DNA sequence in the recombinant cell
Data Source
AI summary
Provided are methods comprising expressing in a recombinant cell a recombinant error-prone reverse transcriptase (RT) and recombinant spacer RNA comprising a target sequence; making a mutagenized cDNA polynucleotide homologous to a DNA sequence in the recombinant cell; expressing a recombinant recombineering system in the recombinant cell; and recombining the mutagenized cDNA with the homologous DNA sequence in the recombinant cell. Also provided are recombinant cells comprising recombinant coding sequences for a recombinant error-prone reverse transcriptase (RT), recombinant spacer RNA comprising a target sequence, and recombinant recombineering system.