Motor Protein Loading on Polynucleotide Adapters
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Solution Overview
Problem
Current methods for loading motor proteins onto polynucleotide adapters for nanopore sensing face challenges in controlling motor protein movement and reducing futile turnover, leading to inefficient polynucleotide characterization.
Innovation Solution
A method involving a polynucleotide adapter with a spacer and a blocking moiety that prevents the motor protein from moving off the spacer, reducing futile turnover by binding a blocking moiety to the adapter, thereby controlling motor protein movement and enhancing characterization efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If a motor protein is used to control polynucleotide movement through the nanopore, then the control of polymer movement is improved, but the motor protein may begin processing the polynucleotide before characterisation starts, leading to loss of information about the processed portion
Solution Approach 1:
The motor protein is pre-loaded onto the adapter in a stalled state before the polynucleotide is introduced. The blocking moiety is attached to prevent the motor protein from moving off the spacer, ensuring it is positioned correctly and ready to control polynucleotide movement only when characterisation begins, without having processed any polynucleotide sequence beforehand.
Solution Approach 2:
A blocking moiety is introduced as an intermediary element that prevents the motor protein from moving off the spacer. This blocking moiety acts as a temporary constraint that keeps the motor protein in the correct position during loading, and can be removed or inactivated when characterisation begins, allowing the motor protein to then control polynucleotide movement without having processed the sequence prematurely.
2Measurement precision
If a spacer is used to stall the motor protein on the adapter, then the motor protein positioning is improved, but the motor protein continues to turnover fuel molecules while stalled, leading to loss of energy
Solution Approach 1:
The motor protein is extracted from its normal processing cycle and placed into a stalled state on the spacer, where it is prevented from moving off. The blocking moiety further extracts or removes the ability of the motor protein to perform futile movements, thereby stopping the turnover of fuel molecules while maintaining the positioning benefit of the spacer.
Solution Approach 2:
The blocking moiety applies a preliminary counter-action to prevent the motor protein from moving off the spacer in futile cycles. By blocking the motor protein's movement in the opposite direction of its processing direction, it counteracts the tendency to perform futile turnover, thereby conserving energy while maintaining precise positioning.
3Productivity
If the motor protein is allowed to move freely onto the polynucleotide, then the processing efficiency is improved, but the movement control becomes uncontrolled, preventing accurate characterisation
Solution Approach 1:
The system transitions from a static blocked state to a dynamic controlled state. The blocking moiety prevents motor protein movement during loading, but when characterisation begins, the block is removed or inactivated, allowing the motor protein to dynamically process the polynucleotide at controlled speeds through the nanopore, achieving both positioning precision and processing efficiency.
Solution Approach 2:
The motor protein is pre-positioned on the adapter in a controlled, stalled state with the blocking moiety in place. This preliminary positioning ensures correct orientation and location before the polynucleotide is introduced. Once characterisation begins, the blocking moiety is removed, allowing the motor protein to proceed with controlled processing of the polynucleotide through the nanopore.
Data Source
AI summary
Provided herein is a method of loading a motor protein onto a polynucleotide adapter. Also provided are polynucleotide adapters and kits comprising such adapters. The adapters find use in characterising analytes such as polynucleotides in methods in which the polynucleotide moves in respect of a nanopore.