Single-Stranded DNA Ligase Composition Without Ribotailing
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Solution Overview
Problem
Current methods for ligating single-stranded DNA (ssDNA) are inefficient and often require the addition of ribotails, which can be disadvantageous in certain applications.
Innovation Solution
A composition and kit using a protein with an amino acid sequence represented by SEQ ID NO: 2 or 4, or a protein with 70% identity and single-stranded DNA ligation activity, to ligate ssDNA without ribotailing, optionally with PEG, for efficient sequencing library preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If TACS ligation method is used to enhance ssDNA ligation activity, then ligation efficiency is improved, but RNA insertion is required which is disadvantageous for certain applications
Solution Approach 1:
The invention extracts and eliminates the RNA tailing step from the TACS ligation method. By using a novel ssDNA ligase that directly ligates ssDNA without requiring ribotail formation, the harmful RNA insertion is removed while maintaining high ligation efficiency for sequencing library preparation.
Solution Approach 2:
The invention substitutes the enzymatic mechanism of TdT (terminal deoxynucleotidyl transferase) that adds RNA tails with a direct ssDNA ligase mechanism. This replacement eliminates the need for RNA insertion while achieving the same ligation function, resolving the contradiction between efficiency and harmful RNA insertion.
2Reliability
If conventional ssDNA ligation methods are used, then RNA ribotail can be introduced for enhanced ligation activity, but the process complexity increases
Solution Approach 1:
The invention segments the ligation process into a single direct step using novel ssDNA ligase, eliminating the need for the multi-step TACS process (RNA tailing followed by ligation). This segmentation reduces process complexity while maintaining reliable ligation activity for ssDNA sequencing applications.
Solution Approach 2:
The novel ssDNA ligase performs self-service by directly ligating ssDNA molecules without requiring the additional TdT enzyme step for RNA tailing. This self-sufficient mechanism simplifies the overall process while ensuring reliable ligation activity.
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 highly efficient ligation of ssDNAs as donor and acceptor, facilitating effective sequencing library preparation without ribotailing, suitable for sequencing short ssDNAs from degraded or cell-free DNA sources.
Implementation Method 1
a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4, and/or (b) a protein which comprises an amino acid sequence having 70% or higher identity to the amino acid sequence represented by SEQ ID NO: 2 or 4 and has single-stranded DNA ligation activity
Data Source
Figure 1A~1C
Figure 2A~2B
Figure 3A~3D
AI summary
Provided is, for example, a novel enzyme composition having efficient single-stranded DNA ligation activity. The present invention relates to, for example, a composition for ligating single-stranded DNA, used for ligating at least one single-stranded DNA as an acceptor to at least one single-stranded DNA as a donor, the composition comprising (a) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4, and/or (b) a protein which comprises an amino acid sequence having 70% or higher identity to the amino acid sequence represented by SEQ ID NO: 2 or 4 and has single-stranded DNA ligation activity.