Multicycle Encoding Assay for Spacer Length Control
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Solution Overview
Problem
Current methods for high-throughput macromolecule characterization, such as those using nucleic acid-encoded binding agents, suffer from rapid loss of encoding efficiency during multiple cycles due to non-templated nucleotide addition by DNA polymerases, leading to increased spacer length and background noise.
Innovation Solution
Employ engineered polymerases with reduced 3′-to-5′ exonuclease activity and template-independent nucleotide addition, use of universal base analogues like 5-Nitroindole, and optimized reaction conditions to control primer extension, ensuring accurate and efficient multi-cycle encoding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA polymerase is used for primer extension reaction, then nucleotide transfer efficiency is improved, but non-templated nucleotide addition increases spacer length and background noise
Solution Approach 1:
The patent extracts and removes the problematic 3′-to-5′ exonuclease activity from the DNA polymerase through site-directed mutagenesis. By eliminating this specific enzymatic function while retaining the 5′-to-3′ polymerization activity, the polymerase can perform primer extension without adding non-templated nucleotides, thus resolving the contradiction between transfer efficiency and spacer length control.
Solution Approach 2:
The patent changes the biochemical parameters of the DNA polymerase by introducing specific mutations (e.g., E. coli Pol I Klenow fragment mutations) that alter its enzymatic properties. This modifies the polymerase's behavior to prevent template-independent nucleotide addition, thereby maintaining precise spacer length control while preserving nucleotide transfer efficiency.
2Reliability
If multiple cycles of encoding are performed, then macromolecule characterization completeness is improved, but encoding efficiency is lost due to spacer length increase
Solution Approach 1:
By removing the 3′-to-5′ exonuclease activity from the polymerase, the patent prevents the accumulation of non-templated nucleotides across multiple encoding cycles. This extraction of the harmful enzymatic function ensures that spacer length remains constant, maintaining encoding efficiency even after multiple cycles of macromolecule characterization.
Solution Approach 2:
The patent performs a preliminary modification of the polymerase enzyme before the multi-cycle encoding process. By pre-engineering the polymerase to lack 3′-to-5′ exonuclease activity, the system is prepared in advance to maintain consistent spacer lengths throughout multiple encoding cycles, preventing encoding efficiency loss before it occurs.
3Measurement precision
If universal base analogues like 5-Nitroindole are used, then primer extension accuracy is improved, but reaction complexity increases
Solution Approach 1:
The patent introduces universal base analogues such as 5-Nitroindole as intermediary molecules in the primer extension reaction. These analogues act as mediators that can base-pair with multiple nucleotides (A, T, G, C), thereby improving primer extension accuracy by ensuring complete and accurate nucleotide transfer from the recording tag to the coding tag, even when sequence information is being decoded.
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
Achieves stable and predictable spacer lengths, reducing background noise and maintaining high signal-to-background ratio, enabling reliable multi-cycle encoding for macromolecule analysis.
Implementation Method 1
a DNA polymerase having 5′-to-3′ polymerization activity
Implementation Method 2
contacting the macromolecule analyte with a first binding agent capable of binding to the macromolecule analyte
Implementation Method 3
transferred identifying information regarding the first binding agent from the first nucleic acid coding tag to the nucleic acid recording tag
Data Source
AI summary
The present disclosure relates to methods and kits for analyzing a macromolecule analyte in a cyclic manner that incorporate basic steps of the ProteoCode™ protein sequencing assay and in addition incorporate approaches that allow to control template independent nucleotide addition by the DNA polymerase, and thus ensure successful multi-cycling encoding. Three different approaches are proposed herein to prevent heterogeneity in the spacer length of the extended recording tag created during the primer extension reaction. The disclosed methods allow for highly-parallelized, accurate and sensitive macromolecule characterization.