3′-O-NH2 Nucleotide Blocking for Accurate CRT Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing technologies using cyclic reversible terminator (CRT) processes face inefficiencies in blocking and deblocking steps, leading to inaccuracies, reduced read length, and increased noise due to inefficient replacement of protecting groups, which can damage DNA and result in phasing defects.
Innovation Solution
The use of nucleotides with a 3'-O-oxime moiety and a reagent to convert it to a 3'-O-NH2 blocking group, which is resistant to enzymatic modification and can be efficiently deblocked, reducing DNA damage and phasing defects, thereby improving sequencing accuracy and read length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional deblocking reagents are used in CRT processes, then the reversible terminator moiety can be removed, but the DNA can be damaged and sequencing accuracy deteriorates
Solution Approach 1:
The patent changes the chemical parameters of the blocking group by using a 3'-oxime moiety instead of conventional blocking groups. This parameter change allows for milder deblocking conditions that remove the blocking group without damaging the DNA backbone, thereby resolving the contradiction between effective deblocking and DNA integrity
Solution Approach 2:
The patent employs a disposable protecting group strategy where the 3'-oxime moiety is designed to be easily removed after serving its temporary blocking function. This allows the blocking group to be discarded after use without leaving harmful residues that would damage the DNA or affect subsequent cycles
2Productivity
If conventional protecting groups are used in CRT processes, then the 3' position can be blocked, but the replacement efficiency is low and phasing defects increase
Solution Approach 1:
The patent changes the chemical parameters of the blocking group to a 3'-oxime moiety with specific properties: it maintains sufficient blocking efficiency to prevent premature extension while allowing near-quantitative replacement during the deblocking step. This parameter optimization resolves the contradiction between blocking effectiveness and replacement efficiency
Solution Approach 2:
The patent applies preliminary protection using the 3'-oxime moiety before the nucleotide incorporation step. This preliminary blocking action ensures that only properly incorporated nucleotides remain blocked, while unincorporated nucleotides can be removed, thereby preventing phasing defects and improving overall extension efficiency
3Duration of action of moving object
If multiple CRT cycles are performed to increase read length, then more sequence information is obtained, but inefficiencies accumulate and accuracy decreases
Solution Approach 1:
The patent applies preliminary optimization of the blocking and deblocking chemistry before entering the cyclic sequencing process. By pre-selecting the 3'-oxime moiety and matching deblocking reagents, the system establishes high-fidelity chemistry that maintains accuracy over extended read lengths, preventing error accumulation across multiple cycles
Solution Approach 2:
The patent changes the chemical parameters of the blocking group to enable more than 90% replacement efficiency during deblocking. This high replacement efficiency parameter ensures that blocking group removal is nearly complete and consistent across cycles, preventing signal decay and maintaining accuracy over long reads
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
The method provides high-purity, reversibly terminated nucleotides that can be directly added to sequencing reactions, enhancing sequencing accuracy and reducing the need for purification, thus improving sequencing efficiency and cost-effectiveness.
Implementation Method 1
reacting a nucleotide having a protecting group such as a 3'-O-oxime moiety with a reagent to remove the protecting group and deliver to the protecting group's position a blocking group, such as a reagent having the structure R2-ONH2
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3'-O-oxime moiety with a reagent having the structure R2-ONH2 to produce a nucleotide having a 3'-O-NH2 moiety, wherein R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted variant thereof.