RecA Ref Protein Targeted DNA Cleavage
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Solution Overview
Problem
Current methods for cleaving double-stranded DNA at specific locations are limited by the availability of restriction enzymes, which require engineering of recognition sites and can result in non-specific cleavage, and the use of zinc finger endonucleases is cumbersome due to the need for generating new enzymes for each target.
Innovation Solution
The combination of RecA and Ref proteins with a single-stranded DNA targeting oligonucleotide allows for specific cleavage of double-stranded DNA at desired sequences, forming a D-loop and activating Ref to cut both strands, thereby acting as a designer nuclease without the need for engineered recognition sites or new enzyme generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If restriction enzymes are used to cleave DNA at specific sequences, then cleavage can be achieved at recognition sites, but the locations are limited to specific nucleotide sequences and engineering of recognition sites is required
Solution Approach 1:
The patent introduces a single-stranded DNA oligonucleotide as an intermediary component that directs the RecA-Ref complex to the target sequence. This intermediary allows the system to target any DNA sequence without requiring engineered recognition sites, as the oligonucleotide can be designed to match any desired target sequence while the RecA-Ref complex performs the cleavage function
Solution Approach 2:
The RecA-Ref nuclease system serves as a universal platform that can cleave any DNA sequence when directed by an appropriate single-stranded oligonucleotide. Unlike restriction enzymes that are limited to specific recognition sites, this system combines the DNA-binding capability of RecA with the nuclease activity of Ref, creating a multi-functional system adaptable to any target through oligonucleotide design
2Reliability
If restriction enzymes are used for DNA cleavage, then cleavage occurs at recognition sites, but non-specific cleavage at multiple locations may occur
Solution Approach 1:
The system achieves local specificity by using a single-stranded oligonucleotide that is complementary only to the desired target sequence. The RecA protein forms a nucleoprotein filament with this oligonucleotide, and the Ref nuclease is activated only at this specific location where the oligonucleotide hybridizes to the target DNA, preventing non-specific cleavage at other sites
Solution Approach 2:
The single-stranded oligonucleotide acts as a mediator that provides sequence-specific targeting while the RecA-Ref complex provides the cleavage function. This separation of targeting and cleavage functions allows for high specificity, as the oligonucleotide can be designed to match only the desired target sequence, preventing off-target cleavage events
3Manufacturing precision
If zinc finger endonucleases are used for targeted DNA cleavage, then specific chromosomal loci can be targeted, but new enzymes must be generated for each target which is difficult and expensive
Solution Approach 1:
The system segments the DNA targeting function from the nuclease function. The single-stranded oligonucleotide handles the targeting function and can be easily synthesized to match any desired sequence, while the RecA-Ref complex handles the nuclease function. This segmentation allows for easy reconfiguration of targeting specificity without requiring development of new enzyme proteins, unlike zinc finger endonucleases where the entire protein must be redesigned for each target
Solution Approach 2:
Instead of creating new zinc finger endonuclease proteins for each target, the system uses simple single-stranded oligonucleotide copies that can be rapidly synthesized to match any desired target sequence. These oligonucleotide 'copies' direct the existing RecA-Ref nuclease to the target location, avoiding the need to generate new complex enzyme proteins for each application
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 and efficient cleavage of double-stranded DNA at targeted sequences, overcoming limitations of existing technologies by using RecA and Ref proteins to form a nucleoprotein complex that directs specific DNA cleavage, enhancing the precision and efficiency of DNA manipulation.
Implementation Method 1
RecA forms an activated nucleoprotein filament on single-stranded DNA (ssDNA) in the presence of an adenosine nucleotide cofactor (Yu and Egelman, 1992). A RecA filament bound to an oligonucleotide can invade and pair with a homologous duplex DNA, resulting in a displacement loop (D-loop).
Implementation Method 2
In the presence of the RecA-bound and paired DNA targeting fragment, Ref will cleave both strands of the targeted double-stranded DNA molecule at the desired target sequence, within the D-loop.
Data Source
AI summary
Kits and a method for cleaving double-stranded DNA using Ref and RecA protein and variants thereof at a site having a DNA sequence homologous to the sequence on a single-stranded DNA targeting fragment are disclosed.