Nucleic Acid Sequencing with Unlabeled Terminators and Affinity Detection
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Solution Overview
Problem
Current sequencing-by-synthesis (SBS) methods require expensive reversibly terminated dNTPs with labels that leave chemical scars, reduce incorporation efficiency, and cause quenching and signal reduction, necessitating improvements for cost-effective and efficient nucleic acid sequencing.
Innovation Solution
The use of non-labeled reversible terminator nucleotides (NLRTs) in SBS, identified by affinity reagents that recognize the nucleobase and reversible blocking group, eliminating the need for labeled nucleotides and their cleavable linkers, allowing for efficient sequencing without chemical scars and improved signal detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labeled reversible terminator nucleotides are used for sequencing, then the identity of incorporated nucleotides can be detected by fluorescent color, but chemical scars are left on incorporated bases after label cleavage and incorporation efficiency is reduced
Solution Approach 1:
The patent extracts and removes the fluorescent label and cleavable linker from the reversible terminator nucleotide structure. By using unlabeled dNTPs instead of labeled RTs, the harmful components (chemical scars from linker cleavage) are completely eliminated while maintaining the core sequencing function through alternative detection methods using affinity reagents that recognize the blocking group.
Solution Approach 2:
The patent employs inexpensive, disposable unlabeled dNTPs with removable blocking groups instead of expensive labeled RTs. The blocking groups serve their temporary purpose of controlling single-base incorporation and are then completely removed without leaving any chemical residue, making the process more cost-effective and efficient.
2Measurement precision
If labeled reversible terminator nucleotides with cleavable linkers are used, then nucleotide incorporation can be monitored, but expensive reagents are required and chemical scars remain after label removal
Solution Approach 1:
The patent removes the expensive fluorescent label and cleavable linker components from the sequencing system. By detecting incorporation through affinity reagents that bind to the blocking group itself, the system eliminates the need for costly labeled nucleotides while maintaining accurate incorporation monitoring.
Solution Approach 2:
The patent uses affinity reagents (antibodies, aptamers, or affimers) that recognize and bind to the blocking group structure. This creates a detectable signal through the binding event itself rather than requiring fluorescent labels, providing a cost-effective alternative that maintains measurement precision.
3Measurement precision
If fluorescent labels with cleavable linkers are used on nucleotides, then base identity can be identified, but quenching and excited dye induced termination occur reducing signal
Solution Approach 1:
The patent completely extracts and removes the fluorescent label from the nucleotide structure. By detecting base identity through affinity reagents that recognize the blocking group, the system eliminates all photodamage-related issues including quenching and excited dye induced termination, providing a cleaner, more accurate signal.
Solution Approach 2:
The patent replaces the optical detection system (fluorescent labels and cameras) with a biochemical detection system using affinity reagents. This substitution eliminates the harmful effects of light excitation and quenching while maintaining the ability to identify base identity through specific molecular recognition and detectable binding events.
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 reduces costs, enhances incorporation efficiency, minimizes photodamage, and enables longer and more accurate reads, providing a more economical and effective nucleic acid sequencing method.
Implementation Method 1
binding of an affinity reagent (e.g., antibody, aptamer, affimer, knottin, etc.) that recognizes the base and a cleavable blocking group and optionally the sugar in the last incorporated nucleotide
Implementation Method 2
a blocking group that ensures that only a single base can be added by a DNA polymerase enzyme to the 3' end of a growing DNA copy strand
Implementation Method 3
TCEP (tris(2-carboxyethyl)phosphine) is introduced to cleave the linker and release the fluorophores and to remove the 3'-O-azidomethyl group, regenerating a 3'-OH
Data Source
Figure 1
Figure 2
Figure 3A~3E
AI summary
The invention provides compositions and methods for sequencing nucleic acids and other applications. In sequencing by synthesis, unlabeled reversible terminators are incorporated by a polymerase in each cycle, then labeled after incorporation by binding to the reversible terminator a directly or indirectly labeled antibody or other affinity reagent.