Oxidative Adapter Ligation for Native 5′ Cap RNA Sequencing
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Solution Overview
Problem
Existing methods for sequencing and characterizing RNA caps, particularly native 5′ caps, are inadequate, as they either replace the native cap with a non-native cap or fail to accurately sequence the 5′-ends of RNA, leading to loss of cap type differentiation and lack of single-molecule resolution in cap structure prediction.
Innovation Solution
A method involving oxidation of the vicinal diol of the native 5′ cap, followed by ligation of a polynucleotide adapter via a linker, allowing for sequencing of the native cap along with the RNA, including both canonical and non-canonical caps, using nanopore sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If motor protein is used to feed RNA through nanopore, then translocation speed is controlled, but 5′-end including cap passes through too fast for accurate sequencing
Solution Approach 1:
The RNA molecule is segmented into two parts: the 5′-end with cap structure and the 3′-end with poly-A tail. The motor protein is specifically attached to the 3′-end, creating differential translocation behavior where the 3′-end moves slowly under motor control while the 5′-end moves faster, allowing the cap to pass through the nanopore at a speed suitable for sequencing.
Solution Approach 2:
Instead of attaching the motor protein to the 5′-end to control cap translocation, the invention inverts the approach by attaching the motor protein to the 3′-end. This reversal allows the 5′-end to move faster through the pore while the 3′-end provides controlled translocation, solving the speed-precision contradiction for cap sequencing.
2Measurement precision
If adapter is ligated to 5′-end of RNA, then full-length sequencing is enabled, but native cap information is lost
Solution Approach 1:
The invention extracts the motor protein attachment function from the 5′-end cap region and relocates it to the 3′-end poly-A tail. This extraction preserves the native cap structure at the 5′-end intact for information retention, while the 3′-end motor attachment enables full-length sequencing without requiring 5′-end modification.
Solution Approach 2:
The poly-A tail serves as an intermediary element that mediates between the need for motor protein attachment (for controlled translocation) and the need to preserve native cap information. By placing the motor attachment site at the 3′-end rather than modifying the 5′-end cap, the poly-A tail fulfills the motor attachment function while leaving the cap intact.
3Measurement precision
If 5′-end of RNA is sequenced without motor protein, then cap structure can be characterized, but translocation is too fast for accurate basecalling
Solution Approach 1:
Different parts of the RNA molecule are given different translocation qualities: the 3′-end with poly-A tail and motor protein attachment translocates slowly under motor control, while the 5′-end with cap structure translocates faster but still within measurable range. This local differentiation allows cap characterization with sufficient measurement time without requiring the entire molecule to move slowly.
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 accurate sequencing and characterization of native RNA caps at a single-molecule level, preserving cap type information and providing transcript-level specificity, suitable for diagnosing genetic conditions and understanding cap structure roles in RNA fate.
Implementation Method 1
A voltage applied across the membrane sends a current through the pore that a translocating RNA strand can disrupt
Implementation Method 2
i) oxidation of the vicinal diol of the native 5′ cap of the capped RNA
Data Source
AI summary
The present invention provides a method of characterizing a capped ribonucleic acid (RNA) using sequencing, wherein the capped RNA is a ribonucleic acid (RNA) with its native 5′ cap, the method comprising the steps of: (i) oxidation of the vicinal diol of the native 5′ cap of the capped RNA; (ii) ligation of a polynucleotide adapter via a linker to the oxidized diol of the native 5′ cap providing an extended polynucleotide construct and (iii) sequencing at least a portion of the extended polynucleotide construct, wherein said portion includes the native cap. The present invention further provides a method of identifying whether a genetic marker specific for a condition is present in a sample which utilises the method of the invention; as well as kits for use in the methods of the invention. The invention further provides a method of characterising an RNA with a native 5′ cap, which method comprises sequencing at least a portion of a polynucleotide construct comprising said capped RNA and a polynucleotide adapter ligated via a linker to said cap, wherein the linking moiety is formed from the vicinal diol of the native 5′ cap, and wherein said portion includes the native 5′ cap.