Stem-loop Donor DNA for Low Bias ssDNA Ligation

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Solution Overview

Problem

Current methods for intermolecular single-stranded DNA (ssDNA) ligation suffer from nucleotide bias, leading to inefficiencies and misinterpretation of gene expression levels, with limited protocols available and significant bias in existing ligation methods such as Circligase I and T4 RNA ligase I.

Innovation Solution

A hybridization-based strategy involving a donor nucleic acid molecule with a stem-loop structure and 3' terminal overhang, which hybridizes with an acceptor molecule to form a stable nick or gap, allowing for efficient ligation using T4 DNA ligase, reducing sequence bias and improving ligation efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If traditional ligation methods (Circligase I, T4 RNA ligase I) are used for intermolecular ssDNA ligation, then ligation can be performed, but nucleotide bias occurs leading to misinterpretation of gene expression levels

Engineering Contradiction:

Improveaccuracy of gene expression analysisVSAvoidsequence bias in ligation

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent introduces a stem-loop structured DNA molecule as an intermediary component. The stem-loop structure with its specific geometry and base composition acts as a mediator that facilitates ligation between ssDNA molecules while reducing nucleotide bias. The structured intermediary provides a controlled platform that equalizes the ligation probability across different nucleotide sequences.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the structural parameters of the ligation system by using a stem-loop configuration instead of linear ssDNA. This structural parameter change (from linear to folded conformation) fundamentally alters the ligation kinetics and reduces sequence-dependent bias, thereby improving measurement accuracy.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If current ssDNA ligation protocols are used, then ligation reactions can proceed, but ligation efficiency is hampered by nucleotide preferences and biases

Engineering Contradiction:

Improveligation efficiencyVSAvoidnucleotide preference bias

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The stem-loop structured DNA molecule serves as an intermediary that enhances ligation efficiency while minimizing nucleotide preferences. The structured intermediary provides a controlled platform that equalizes the ligation probability across different nucleotide sequences, thereby improving both productivity and reliability.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The stem-loop structure is pre-formed before the ligation reaction, creating a predetermined geometric configuration that facilitates efficient binding and ligation. This preliminary structural organization reduces the entropic barrier to ligation and minimizes sequence-dependent variations in reaction efficiency.

Inventive Principle:

Principle #10Preliminary action

3Device complexity

If simple linear ssDNA molecules are used for ligation, then the system is simple, but sequence bias significantly hampers ligation performance

Engineering Contradiction:

Improvesimplicity of DNA structureVSAvoidsequence bias in ligation

Core Design Contradiction:

Device complexityVSReliability

Solution Approach 1:

The DNA molecule is segmented into distinct functional regions: a stem region with complementary base pairs and a loop region with specific sequence. This segmentation creates a structured configuration that reduces sequence bias while maintaining relative simplicity. The stem-loop structure divides the molecule into functional domains that work together to improve ligation reliability.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent changes the conformational parameter of the DNA from linear to stem-loop structure. This parameter change (structural configuration) introduces geometric constraints that reduce sequence bias without significantly increasing overall complexity, thereby improving ligation reliability.

Inventive Principle:

Principle #35Parameter changes

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 fast, efficient, and low-sequence bias ligation of ssDNA, enhancing the accuracy of gene expression analysis and compatibility with downstream protocols like LMPCR and next-generation sequencing technologies.

Implementation Method 1

hybridizing the single stranded 3' terminal overhang region of the donor nucleic acid molecule to the acceptor molecule thereby forming an acceptor-donor hybrid molecule

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

ligating the 5' end of the donor nucleic acid molecule to the 3' end of the acceptor nucleic acid molecule thereby generating a ligated product

Methodology Applied

Scientific EffectEnzymatic ligation: Enzyme

Data Source

PatentUS9816120B2Low sequence bias single-stranded DNA ligation

Publication Date: 2017.11.14 THE PENN STATE RES FOUND INC

AI summary

The invention provides compositions and methods for ligating single stranded nucleic acids wherein the ligation is based on fast, efficient, and low-sequence bias hybridization of an acceptor molecule with a donor molecule. In one embodiment, the structure of the donor molecule comprises a stem-loop intramolecular nucleotide base pairing (i.e., hairpin) and a 3′-overhang region such that the overhang is able to hybridize to nucleotides present in the 3′ end of the acceptor molecule.