SARP-Seq Analysis of V(D)J Recombination Sequence Specificity
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Solution Overview
Problem
Existing methods for analyzing DNA sequence specificity in V(D)J recombination are limited by the need for in vitro purification and are not optimal for studying enzyme interactions, and they lack unbiased, high-throughput analysis of endogenous RSSs in diverse chromatin environments.
Innovation Solution
The development of SARP-seq, a method that uses a plasmid recombination assay with degenerate base pairs to investigate RAG-RSS interactions, allowing for high-throughput analysis of DNA sequence specificity by selective amplification of recombination products and next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Bind-n-Seq method is used to identify DNA sequences recognized by proteins, then sequence-specific DNA binding activity can be determined, but the method requires in vitro purification and is not optimal for analyzing enzyme interactions
Solution Approach 1:
The patent uses a plasmid library containing copied RSS sequences with degenerate base pairs instead of requiring purification of protein-DNA complexes. The RAG proteins act on copied RSS sequences in plasmids transfected into cells, and the recombination products are analyzed through sequencing, eliminating the need for in vitro purification steps while maintaining measurement precision
Solution Approach 2:
The patent replaces the mechanical separation and purification steps of Bind-n-Seq with a biological system approach. Instead of physically separating bound from unbound DNA in vitro, the method uses cellular transcription of plasmid DNA and subsequent sequencing to identify RAG recognition sequences, substituting mechanical purification with biological expression and molecular sequencing
2Loss of information
If existing methods are used to analyze DNA sequence specificity, then some sequence information can be obtained, but they lack unbiased, high-throughput analysis of endogenous RSSs in diverse chromatin environments
Solution Approach 1:
The plasmid library system serves multiple functions: it contains diverse RSS sequences with degenerate base pairs representing thousands to millions of potential sequences, enables high-throughput analysis through cellular transcription and next-generation sequencing, and maintains unbiased representation of endogenous RSS variants in diverse chromatin environments. This universal platform simultaneously achieves information completeness and high productivity
Solution Approach 2:
The patent introduces degenerate base pairs at specific positions within RSS sequences, creating a library where each plasmid contains variant RSS sequences. This parameter change in DNA sequence diversity allows unbiased sampling of thousands to millions of potential RSS variants, enabling comprehensive high-throughput analysis of RAG recognition specificity across diverse sequences and chromatin contexts
3Productivity
If plasmid library with degenerate base pairs is used for SARP-seq, then unbiased high-throughput analysis of thousands to millions of sequences is enabled, but the device and method complexity increases
Solution Approach 1:
The patent segments the complex analysis task into distinct modular components: (1) plasmid library construction with degenerate RSS sequences, (2) cellular transfection and RAG expression, (3) V(D)J recombination event occurrence, (4) plasmid DNA recovery and sequencing library preparation, and (5) next-generation sequencing and data analysis. This segmentation enables high-throughput productivity while managing complexity through standardized, reproducible modules
Solution Approach 2:
The patent uses cellular machinery as an intermediary system between the plasmid library and the sequencing analysis. Cells serve as the medium where RAG proteins are expressed, plasmids are transcribed, and recombination events occur naturally. This intermediary biological system simplifies the overall complexity by leveraging evolved cellular processes rather than requiring artificial in vitro systems for each step
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
SARP-seq provides an unbiased, high-throughput analysis of DNA sequence specificity in V(D)J recombination, enabling the evaluation of thousands to millions of potential sequences and identifying motifs used by the V(D)J recombinase, as well as analyzing recombination side-products.
Implementation Method 1
RAG-mediated DNA DSBs occur in the context of a paired complex (PC), with the RAG proteins simultaneously bound to a 12-RSS and a 23-RSS with the intervening DNA looped out
Implementation Method 2
Following RAG-mediated DNA cleavage, the appropriate DNA ends are joined by the action of ubiquitous DNA repair factors that function in nonhomologous DNA end-joining (NHEJ)
Implementation Method 3
NGS methods have been developed to identify rearranged genomic products or DNA cleavage sites that form during V(D)J recombination
Data Source
AI summary
Compositions, kits, systems, and methods are disclosed for use in analysis of DNA sequence-specificity in V(D)J recombination or other types of recombination. In particular (but not by way of limitation), high throughput methods of analyzing DNA sequence-specificity in a V(D)J recombination assay are disclosed.