Cyclic DNA Molecule for Long Hairpin RNA Library Construction
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Solution Overview
Problem
Current methods for constructing hairpin RNA libraries are inefficient and limited to short hairpin structures, particularly those with stem lengths above 40bp, resulting in low silencing efficiency and poor gene silencing effects compared to long hairpin RNA.
Innovation Solution
A DNA molecule composed of four single-strand DNA fragments linked in specific orders, forming a closed single-strand cyclic molecule with stem-loop structures, recognized by different restriction endonucleases, is used to create a gene silencing vector through enzymatic cleavage and rolling circle replication, enabling the construction of long hairpin RNA libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional two-step method or Gateway recombinant technology is used to construct hpRNA, then the construction process is relatively simple for single genes, but the efficiency is low and it cannot construct gene silencing library
Solution Approach 1:
The DNA molecule is divided into four specific fragments (A, B, C, D) with defined functions: fragment A contains target sequence, fragment B is reverse complement of A, fragments C and D contain stem-loop structures with restriction sites. This segmentation enables systematic assembly of multiple genes into library format while maintaining individual gene integrity and enabling high-throughput processing
Solution Approach 2:
The DNA molecule design incorporates universal elements including restriction endonuclease recognition sequences in stem-loop structures, T7 promoter regions, and polyA signals that can accommodate multiple different target sequences. This universal framework allows the same basic structure to be used for constructing silencing libraries across diverse genes while maintaining consistent expression and processing characteristics
2Reliability
If short hairpin RNA (shRNA) library is constructed using SPEED, REGS, or EPRIL technology, then the construction is feasible, but the stem length is limited to about 20bp resulting in low silencing efficiency
Solution Approach 1:
The invention changes the stem length parameter from the conventional 20bp (shRNA) to significantly longer stems (40-80bp or more), creating long hairpin RNA (lhRNA). This parameter change is achieved through the specific design where fragment A and its reverse complement B anneal to form extended stem structures. The patent demonstrates that longer stems produce higher silencing efficiency while maintaining structural stability through proper design of the stem-loop architecture
3Reliability
If long hairpin RNA (lhRNA) with stem length above 40bp is constructed, then the silencing efficiency is improved, but there is no reported method for construction
Solution Approach 1:
The DNA fragments are pre-designed with incorporated restriction endonuclease recognition sequences within the stem-loop structures before assembly. This preliminary action allows subsequent enzymatic processing to automatically generate the correct long hairpin structure. The T7 promoter and polyA signals are also pre-positioned to ensure proper transcription and processing of the long hairpin RNA, making the complex structure achievable through standardized molecular biology techniques
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
This method allows for the rapid, high-throughput, and cost-effective construction of gene silencing vectors with long hairpin RNA, significantly improving silencing efficiency and enabling the creation of libraries for various biological sources.
Implementation Method 1
The closed single-strand cyclic DNA molecule is synthesized to the double-strand DNA molecule under the action of DNA polymerase with the strand displacement ability to form the double-strand DNA molecule with inverted repeat structure
Implementation Method 2
C and D are DNA fragments with the stem-loop structure, and said C and D fragments meet the requirement of 3) or 4) as follows: 3) The nucleotide sequence of said fragment C or D comprises at least one sequence e, said sequence e forms a recognition sequence R of restriction endonuclease with the complementary sequence thereof
Data Source
Figure 1
Figure 2~3
Figure 4A~5B
AI summary
DNA molecule for expressing hairpin RNA, the constructing method and the use thereof are provided. The DNA molecule is a closed single-strand cyclic DNA molecule which is constructed by linking four fragments A, B, C and D as following oeder: A-C-B-D or A-D-B-C, wherein A is a sense strand or anti-sense strand fragment of the target double-strand DNA; B is the fragment which is reversely complementary to A; C and D are DNA fragments with stem-loop structure and they meet one of the following requirements: 1) the nucleotide sequence of fragment C or D comprises at least one sequence e; 2) the nucleotide sequence of fragment C comprises at least one sequence e, the nucleotide sequence of fragment D comprises at least one sequence f, wherein sequences e and f individually are one strand of different restriction endonuclease recognition sequences. The DNA molecule for expressing hairpin RNA and its constructing method are useful in constructing 1hRNA library.