3′-Blocked Nucleotides for In-Pore Deblocking and DNA Synthesis
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Solution Overview
Problem
Existing polynucleotide sequencing methods using nanopores are not sufficiently robust, reproducible, or sensitive for practical applications like genome sequencing, requiring improved compositions, systems, and methods for sequencing and synthesizing polynucleotides.
Innovation Solution
The use of 3′-blocked nucleotides that can be selectively deblocked within a nanopore aperture using an initiator, allowing for the incorporation and extension of nucleotides into a growing polynucleotide strand without separate fluidic cycles, utilizing various initiators such as reducing agents and enzymes to remove the 3′-blocking group and activate triggers for degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional nanopore sequencing methods are used, then sequencing can be performed, but the methods are not sufficiently robust, reproducible, or sensitive for practical applications
Solution Approach 1:
The nucleotide is segmented into distinct functional components: a 3′-blocking group that prevents premature extension, a trigger moiety that responds to specific stimuli, and an initiator molecule that activates the deblocking process. This segmentation allows for controlled, stepwise nucleotide incorporation and deblocking, improving reliability while maintaining throughput through automated cycling
Solution Approach 2:
The 3′-blocking group is pre-installed on the nucleotide before incorporation into the polynucleotide chain. This preliminary blocking prevents unwanted extension reactions until the blocking group is selectively removed by the initiator-trigger system, ensuring reproducible and reliable sequencing results
2Measurement precision
If charge blockade labels are used on nucleotide analogs, then nucleotide identity can be detected, but the system lacks sufficient sensitivity and throughput for demanding commercial applications
Solution Approach 1:
The system uses charge blockade labels that create detectable electrical signal changes (analogous to color changes in optical systems) when nucleotides are incorporated into the polynucleotide chain. The nanopore detects these signal changes to determine nucleotide identity with high precision while maintaining throughput through rapid cycling of incorporation and deblocking steps
3Manufacturing precision
If multiple fluidic cycles are used for nucleotide incorporation and deblocking, then complete sequencing can be achieved, but the process complexity and time increase
Solution Approach 1:
The system merges the nucleotide incorporation step and the deblocking step into a unified cyclic process that occurs within the same reaction environment. The 3′-blocking group remains attached during incorporation, then is removed by the initiator-trigger system without requiring separate fluidic exchanges, reducing device complexity while maintaining complete sequencing capability
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
Enables efficient and controlled synthesis of polynucleotides by selectively deblocking nucleotides within the nanopore, enhancing sequencing accuracy and throughput without additional fluidic steps, thus improving the robustness and sensitivity of nanopore-based sequencing.
Implementation Method 1
selectively activating the trigger using an initiator. The activated trigger may be used to remove 3′-blocking group from the nucleotide
Implementation Method 2
the dwell time has been measured for complexes of DNA with the Klenow fragment (KF) of DNA polymerase I atop a nanopore in an applied electric field
Implementation Method 3
a current or flux-measuring sensor has been used in experiments involving DNA captured in an α-hemolysin nanopore
Data Source
AI summary
3′-blocked nucleotides, methods of deblocking the same, and methods of synthesizing polynucleotides using the same are provided herein. In some examples, a nucleotide is disposed within the aperture on the first side of a nanopore. The nucleotide may be coupled to a 3′-blocking group including a trigger. The trigger may be selectively activated using an initiator. The activated trigger may be used to remove the 3′-blocking group from the nucleotide.