Cholesterol-Linked DNA Adapter for tRNA Nanopore Sequencing
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Solution Overview
Problem
Current methods for sequencing and analyzing tRNA molecules are hindered by their complex secondary structure and protein association, leading to challenges such as truncated cDNA formation during reverse transcription and difficulties in mechanical unfolding necessary for nanopore sequencing.
Innovation Solution
A cholesterol-linked DNA or RNA adapter oligonucleotide is enzymatically ligated to tRNA, facilitating mechanical unfolding and threading through a nanopore, allowing for the detection and sequencing of individual tRNA molecules by modulating the translocation process with a non-catalytic protein brake.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If reverse transcription is used to sequence tRNA, then sequencing can be performed, but the complex secondary structure and nucleotide modifications cause truncated cDNA formation
Solution Approach 1:
The patent extracts the problematic secondary structure and modifications from the sequencing process by using nanopore technology to read tRNA directly without reverse transcription, eliminating the source of truncated cDNA while maintaining sequencing capability
Solution Approach 2:
The patent introduces an adapter molecule as an intermediary that binds to tRNA and facilitates its threading through the nanopore, enabling direct sequencing without requiring the tRNA to be converted to cDNA through reverse transcription
2Ease of manufacture
If nanopore sequencing is used for direct tRNA analysis, then sequencing without PCR amplification is achieved, but mechanical unfolding of tRNA is required which is difficult
Solution Approach 1:
The adapter acts as a mechanical lever that applies force to the tRNA molecule, facilitating its unfolding and threading through the nanopore without requiring complex chemical denaturation procedures
Solution Approach 2:
The adapter is pre-bound to the tRNA molecule before nanopore insertion, and its structure is designed to automatically facilitate unfolding as the complex is pulled through the pore, eliminating the need for separate unfolding steps
3Productivity
If traditional RNAseq methods are used, then high throughput sequencing is achieved, but extensive library preparation including PCR amplification is required
Solution Approach 1:
The patent extracts and eliminates the PCR amplification step from the sequencing workflow by using nanopore technology to sequence tRNA molecules directly, reducing library preparation complexity while maintaining the ability to process multiple samples
Solution Approach 2:
The adapter serves as a universal intermediary that can be attached to different tRNA molecules, enabling direct nanopore sequencing without requiring sample-specific primer design or PCR amplification for each tRNA type
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 enables the efficient denaturation and sequencing of tRNA molecules, providing distinct ionic current signals that differentiate between tRNA species, demonstrating the feasibility of direct sequence analysis of tRNA using nanopore technology.
Implementation Method 1
when a molecule such as a nucleotide passes through (or near) a nanopore, it creates a characteristic perturbation of the current signature passing between two sides of the nanopore
Implementation Method 2
The two strands of the adapter act to locally concentrate adapted tRNA at the bilayer
Implementation Method 3
promote their mechanical unfolding
Data Source
AI summary
The invention also encompasses novel structures and methods comprising providing a molecular adapter for capture and manipulation of transfer RNA. The adaptor is bound to a tRNA molecule. The adaptor may be a cholesterol-linked DNA adapter oligonucleotide. The invention is useful in sequencing, identification, manipulation and modification of tRNA.