Direct tRNA Sequencing With Splinted Oligos for Higher Read Yield

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Existing methods for quantifying tRNA modifications and abundances, such as Mass Spectrometry-based and NGS, are inefficient and do not provide positional information, while direct RNA nanopore sequencing methods like those described in Thomas N K et al. suffer from low read yield, fragmentation, and lack of sample multiplexing capabilities.

Innovation Solution

A method involving pre-annealed splinted oligonucleotides and adapter DNA oligonucleotides for tRNA ligation, followed by reverse transcription and nanopore sequencing, which simplifies library preparation and enhances read yield and multiplexing capabilities.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If gel purification is used to select tRNA molecules, then sequencing accuracy is improved, but tRNA fragmentation increases and RNA recovery decreases

Engineering Contradiction:

Improvesequencing accuracyVSAvoidRNA recovery

Core Design Contradiction:

Measurement precisionVSLoss of substance

Solution Approach 1:

The patent extracts only the essential function of gel purification (tRNA selection) and replaces it with a gentler method using splinted oligonucleotides that hybridize to tRNA molecules, allowing selection without the harsh conditions and fragmentation caused by gel electrophoresis

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces splinted oligonucleotides as intermediaries that mediate the selection and ligation of tRNA molecules. These oligonucleotides serve as bridges between tRNA and the sequencing library preparation workflow, enabling accurate selection without direct gel purification of tRNA

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If gel purification is used for tRNA selection, then sequencing accuracy is improved, but library preparation complexity and time increase

Engineering Contradiction:

Improvesequencing accuracyVSAvoidlibrary preparation complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent merges the tRNA selection, enrichment, and library preparation steps into a single streamlined workflow using splinted oligonucleotides. The oligonucleotides perform multiple functions simultaneously: they select tRNA molecules, serve as primers for reverse transcription, and provide sequencing adapters, eliminating the need for separate gel purification steps

Inventive Principle:

Principle #5Merging (Combining)

3Productivity

If incompatible oligonucleotide design is used for tRNA ligation, then ligation efficiency is improved, but sequencing yield decreases due to pore clogging

Engineering Contradiction:

Improveligation efficiencyVSAvoidsequencing yield

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The patent applies local quality by designing oligonucleotides with specific properties at different regions: the splinted oligonucleotides have hybridization regions that match tRNA sequences locally, while the adapter regions are designed with appropriate lengths and sequences to prevent pore clogging. This localized optimization of oligonucleotide properties resolves the contradiction between ligation efficiency and sequencing yield

Inventive Principle:

Principle #3Local quality

4Loss of information

If previous nanopore sequencing method is used, then direct RNA sequencing is achieved, but read yield is extremely low

Engineering Contradiction:

Improvedirect RNA sequencing capabilityVSAvoidread yield

Core Design Contradiction:

Loss of informationVSProductivity

Solution Approach 1:

The patent performs preliminary actions by pre-annealing splinted oligonucleotides to tRNA molecules before library preparation. This pre-hybridization step ensures efficient capture and enrichment of tRNA molecules, significantly increasing the number of readable molecules that pass through the nanopore during sequencing

Inventive Principle:

Principle #10Preliminary action

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 achieves accurate and fast quantification of tRNA abundances and modifications with significantly higher read counts, improving sequencing efficiency and enabling sample multiplexing, thereby overcoming the limitations of previous methods.

Implementation Method 1

loading the product of the previous step to a flow cell which contains a membrane in which is present a nanopore that provides a channel through said membrane, coupled to a current intensity, wherein the product of the previous step passes through the nanopore, causes disruptions in the current intensity

Methodology Applied

Scientific EffectNanopore sequencing: Nanopore

Implementation Method 2

a pre-annealed splinted double-stranded oligonucleotide comprising: a first splinted oligonucleotide, comprising a second splinted oligonucleotide hybridization region and a tRNA hybridization region

Methodology Applied

Scientific EffectHybridization:

Implementation Method 3

performing reverse transcription to linearize the product of step b) to obtain a library

Methodology Applied

Scientific EffectReverse transcription:

Data Source

PatentUS20260085349A1METHOD TO ANALYZE tRNA USING DIRECT SEQUENCING

Publication Date: 2026.03.26 FUNDACIO CENTRE DE REGULACIO GEN MICA

AI summary

The present invention discloses a method to quantify tRNA abundance and tRNA modifications in an RNA sample that comprises contacting RNA with oligonucleotides in the presence of a ligating agent and performing nanopore direct sequencing. It also discloses a kit to perform said method.