shRNA 타겟 디자인
VectorBuilder의 shRNA 타겟 디자인 툴로 녹다운 점수가 높은 shRNA를 디자인하면, 목적 유전자(GOIs)를 효과적으로 녹다운 할 수 있습니다.
VectorBuilder는 RNAi 컨소시엄(TRC)에서 사용하는 규칙과 유사한 규칙을 적용하여 shRNA를 설계하고 점수를 매깁니다. 주어진 각 RefSeq 전사체에 대해 후보 대상 사이트로 간주되는 모든 가능한 21mer를 검색합니다. 녹다운 효율성/특이성 또는 복제 가능성을 감소시키는 특성인 4 base이상의 동일 염기를 포함한 경우, 7개 이상의 G 또는 C, <25% 또는 >60% 이상의 GC 함량 및 5’말단의 AA를 지닌 후보는 제외됩니다. 녹다운 점수는 내부 stem-loop, 3’말단쪽 높은 GC 함량, 알려진 miRNA seed 서열, 또는 다른 유전자에 대해 표적 외 일치가 되는 후보에 대해 불이익을 줍니다. 대체 전사체가 있는 유전자의 경우 모든 전사체에 존재하는 타겟 사이트에 더 높은 점수가 부여됩니다.
모든 점수는 ≥0이고 평균은~5, 표준편차는~5, 그리고 점수의 95%는 ≤15입니다. 녹다운 점수가 약 15인 shRNA는 최고의 녹다운 성능과 클로닝 용이성을 갖고 있다고 여겨집니다. 반면 녹다운 점수가 0인 shRNA는 녹다운 성능이 가장 나쁘거나 복제하기 어렵습니다.
RNA interference (RNAi) has been a widely utilized method of gene modulation for many decades. Short RNA sequences (approximately 21-23 nucleotides) complementary to the RNA of a gene of interest are introduced into target cells. The exogenous RNA strand binds to the complementary endogenous mRNA strand. The cell degrades the double-stranded RNA and translation does not occur, knocking down the performance of the gene. It is important to note that this approach does not completely knock out the gene, as some mRNAs will not be bound and will produce functional protein.
One common RNAi approach utilizes short hairpin RNAs (shRNAs). Here the sequence is designed such that a single transcript folds back on itself and hybridizes, forming a hairpin. This double-stranded RNA complex with its internal loop is transported into the cytoplasm, where it is processed by Dicer and is loaded into the RISC complex. The RISC complex facilitates binding between the silencing RNA and the target mRNA, at which point the mRNA will be cleaved or degraded (Figure 1).
Figure 1. Production and function of forms of RNAi.
Why choose shRNA?
The formation of a hairpin from a single transcript allows for customization in introduction, duration, and regulation of silencing. The plasmid coding for the shRNA can be transfected (e.g. through electroporation or microinjection) or transduced (using viruses such as lentivirus or adenovirus). Because of the option to use viral transduction, shRNA vectors can be tagged and/or integrated into the genome using virus machinery and can be introduced to a wide range of cell types.
VectorBuilder’s shRNA Design tool allows you to input your sequence and receive a list of all possible shRNA sequences in order of knockdown score. Our algorithm takes each 21mer (every sequence of 21 base pairs) and determines (1) its clonability and (2) its specificity. Clonability is influenced by the order and distribution of nucleotides. Repeats, GC content that is too high or too low, and formation of stem loops within the shRNA sequence decrease stability and therefore knockdown score. Specificity ensures that only the target gene is affected, so each candidate sequence is compared to the host genome. If there are regions outside of the gene of interest where the shRNA may bind, the knockdown score is decreased. High knockdown scores theoretically reflect high performance of the shRNA, but some unpredicted interactions can occur, reducing the experimental efficiency. Because there is not a guarantee that the theoretical outcomes completely match the experimental outcomes, journals typically require confirmation of shRNA knockdown using two different shRNA sequences. To maximize the changes of having two shRNA sequences with high targeting efficiency, we offer a 3+1 service, where three shRNA sequences and one control (scramble) sequence is provided.
- Sequences in both GenBank and FASTA formats can be recognized.
- Some returns may target the 3’ UTR, but this region can be as effective in knockdown as the coding region.
- Knockdown scores should be validated experimentally, as actual efficiency could depart significantly from what the scores predict. For this reason, it is recommended to test at least three shRNAs to increase the likelihood of success for effective knockdown using two sequences.