Nanopore Sequencing via Temporal Discrimination of Nucleotide Tags
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid sequencing methods using nanopores are inadequate due to insufficient sensitivity in distinguishing between nucleic acid bases, particularly purines (A and G) and pyrimidines (C, T, U), which are similar in size, shape, and charge, leading to inaccurate diagnostic and treatment outcomes.
Innovation Solution
The method involves using tagged nucleotides with unique tags that are detected by a nanopore based on the time they interact, with incorporated nucleotides detected for a longer period than unincorporated ones, allowing for accurate differentiation through the length of time the tag is associated with the nanopore, and utilizing a mutated phi29 DNA polymerase to control nucleotide incorporation rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nanopore sequencing is used, then the sequencing process is simple, but the sensitivity and accuracy in distinguishing nucleic acid bases is insufficient
Solution Approach 1:
The patent introduces an intermediary system consisting of motor proteins and RNA molecules that mediate between the nanopore and the nucleic acid bases. The motor protein transports the RNA through the nanopore in a controlled manner, allowing for enhanced detection accuracy while maintaining a relatively simple nanopore structure.
Solution Approach 2:
The patent changes the detection parameter from direct electrical signal measurement to temporal signal analysis. By measuring the duration of ionic current blockades and the timing intervals between successive bases, the system achieves high discrimination accuracy without requiring complex nanopore modifications.
2Measurement precision
If nucleic acid bases are passed through nanopore quickly, then the sequencing speed is high, but the detection time is insufficient to distinguish between similar bases
Solution Approach 1:
The motor protein performs periodic transport cycles, moving the RNA through the nanopore in discrete steps with controlled timing. This periodic action creates distinct temporal patterns for different bases, enabling accurate discrimination while maintaining a steady sequencing pace.
Solution Approach 2:
The system uses feedback from the ionic current blockade signals to regulate the motor protein's transport speed. When a base is detected, the motor protein pauses briefly, allowing sufficient detection time without significantly reducing the overall sequencing throughput.
3Measurement precision
If purines and pyrimidines are distinguished by size, shape, and charge, then the detection method is simple, but the signal distinction between bases is insufficient
Solution Approach 1:
The patent transitions from spatial discrimination (based on size, shape, charge) to temporal discrimination. By analyzing the timing and duration of ionic current blockades rather than relying on physical base properties, the system achieves high signal distinction without requiring complex nanopore structures or multiple detection channels.
Data Source
Figure 1
Figure 2A
Figure 2B
AI summary
The present invention relates a method for assembling a protein having a plurality of subunits, the method comprising: providing a plurality of first subunits; providing a plurality of second subunits, wherein the second subunits are modified with respect to the first subunits; contacting the first subunits with the second subunits in a first ratio to form a plurality of proteins having the first subunits and the second subunits, wherein the plurality of proteins have a plurality of ratios of the first subunits to the second subunits; and fractionating the plurality of proteins to enrich proteins that have a second ratio of the first subunits to the second subunits, wherein the second ratio is one second subunit per (n-1) first subunits, wherein 'n' is the number of subunits comprising the protein. Furthermore, the invention provides a nanopore comprising a plurality of subunits, wherein a polymerase is attached to one of the subunits and at least one and less than all of the subunits comprise a first purification tag.