Hairpin Splint ssDNA for Stable Monomeric Cyclization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for preparing small single-stranded DNA (ssDNA) circles are inefficient, particularly for DNA sizes below 30 bases, as they often result in oligomerization due to instability of the monomeric ssDNA-splint complex, leading to low yields and high costs with specialized ligases like CircLigase™.
Innovation Solution
A splint ssDNA design incorporating a linear splint sequence and differing hairpin sequences on each side is used, which forms a stable monomeric complex with ligating linear ssDNA, allowing for efficient cyclization using T4 DNA ligase at higher concentrations, thereby producing ssDNA circles as short as 16 nucleotides with high yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional splint ssDNA is used to prepare small ssDNA circles, then the method works well for long ssDNA (>30 bases), but the monomeric ssDNA-splint complex becomes strained and unstable for short ssDNA, leading to oligomerization
Solution Approach 1:
The splint ssDNA is segmented into three functional regions: a linear splint sequence (10-12 contiguous nucleotides) that hybridizes with the ssDNA ends, and two hairpin sequences on opposite sides that provide structural stability. This segmentation allows each region to optimize its function - the linear portion enables binding while the hairpin portions prevent oligomerization by sterically blocking intermolecular interactions.
Solution Approach 2:
The hairpin sequences act as intermediary elements that mediate between the linear splint sequence and the ssDNA ends. These hairpin structures serve as protective intermediaries that prevent direct interaction between multiple ssDNA-splint complexes, thereby preventing oligomerization while maintaining the stability needed for efficient intramolecular cyclization.
2Reliability
If extremely low ssDNA concentration is used to suppress intermolecular oligomerization, then intramolecular cyclization is promoted, but only very small quantities of ssDNA circles are produced
Solution Approach 1:
The invention changes the critical parameter of splint ssDNA structure from a simple linear sequence to a complex structure with hairpin sequences. This parameter change fundamentally alters the concentration-dependent behavior: the hairpin structures enable high-yield cyclization at physiological concentrations (1-100 μM) by preventing oligomerization through steric blocking, thereby decoupling selectivity from concentration requirements.
3Reliability
If specialized ligase CircLigase is used for small ssDNA cyclization, then ssDNA circles can be prepared, but the enzyme is extremely expensive and has poor ligation efficiency
Solution Approach 1:
The invention replaces the expensive specialized CircLigase enzyme with a conventional, inexpensive T4 DNA ligase. The costly function of cyclizing small ssDNA is achieved not by using a specialized expensive enzyme, but by using a cheap conventional enzyme in conjunction with a specially designed splint structure that makes the reaction efficient and selective.
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 nearly quantitative conversion of linear ssDNA to circular ssDNA, enhancing stability and production efficiency while avoiding oligomerization, even at high DNA concentrations, and is applicable to DNA lengths as short as 16 nucleotides.
Implementation Method 1
The linear splint sequence comprises at least 10 contiguous nucleotides (nts) (such as 10 contiguous nts), half of which hybridize with one end of a ligating linear ssDNA and the other half of which hybridize with the other end of a ligating linear ssDNA
Implementation Method 2
use T4 ligase to join covalently the two ends of the ssDNA
Data Source
AI summary
A splint single-stranded DNA (ssDNA), which comprises a linear splint sequence and a hairpin sequence on each side of the linear splint sequence; a splint ssDNA-ligating linear ssDNA monomeric complex; a method of producing a ssDNA circle; and a kit comprising the splint ssDNA and instructions for using the splint ssDNA to produce a ssDNA circle.