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[IDT]RNAi 의 효율 극대화_Dicer Substrate siRNA duplexes
  • 글쓴이 관리자
  • 작성일 2019-08-05 00:11:14
  • 조회수 39019
 전통적인 21-mer siRNAs는 Dicer cleavage과정이 없이 지나치게 됩니다.
그러나 최근에 Dicer는 siRNA duplexes가 RISC로 들어가는데 관계한다는 것을 알려지고 있기 때문에 Dicer와의 interaction에 필수적인
스텝으로 보입니다.

참고논문
 
IDT사와 Beckman Research Institute of the City of Hope National Medical Center의 김동호 박사와 John Rossi박사의 공동연구로 개발된
Dicer-substrate RNAs는 합성된 27-mer duplex RNAs로서 Dicer에 의해 21-mer siRNAs로 만들어지게 됩니다.
이렇게 새롭게 디자인 된 RNAiduplex는 같은 사이트에 대하여 기존의 21mer siRNA보다 10배까지 증가된 효율을 나타냅니다.
IDT는 효율을 극대화하는 dicing 디자인 툴을 개발했으며, 오직 IDT에서만 이용 가능합니다
 
길이제한: Dicer Substrate RNAi oligos는 strand 당 24-30 RNA bases와 3 DNA bases까지 가능합니다.


  * 기존 siRNA VS DsiRNA   /  DsiRNA 작용기전








Figure 1. 27mer DsiRNAs (27+0) are more potent effectors of RNAi than a 21mer siRNA (21+2). Double-stranded RNA (dsRNA) names: number of duplexed bases + number of 3′ overhanging bases or – number of 5′ overhanging bases. Each graph point represents the average of 3 independent measurements. (A–D) EGFP expression levels were determined after cotransfection of HEK293 cells with a fixed amount of EGFP expression plasmid and various concentrations of dsRNAs of varying length. Transfections were performed using (A) 50 nM, (B) 200 pM, and (C) 50 pM of the indicated dsRNAs. Error bars indicate the standard deviation. (D) Dose-response testing of dsRNAs. (E) Left: Dose-response curve of longer dsRNAs transfected into NIH3T3 cells that stably express EGFP. Right: Using an in vitro Dicer cleavage assay to analyze Dicer processing of longer dsRNAs. DsiRNAs and cleavage products are shown in this 15% nondenaturing polyacrylamide gel. [Nat Biotechnol, 23(2):222–6.]




Figure 2. Enhanced duration of RNAi at lower concentrations when comparing 27mer DsiRNA (27+0) to 21mer siRNA (21+2). Double-stranded RNA (dsRNA) names: number of duplexed bases + number of 3′ overhanging bases. (A) Enhanced duration of RNAi by DsiRNAs (up to 10 days) compared to siRNA (approximately 4 days): 5 nM of DsiRNA or siRNA were transfected into NIH3T3 cells stably expressing EGFP. Duplicate samples were taken on the indicated days, and EGFP expression was determined by fluorometry. (B) DsiRNAs can elicit RNAi at low concentrations compared to siRNAs. EGFP expression was determined after dsRNAs were transfected along with the EGFP reporter construct. Target names: site-2 is EGFP-S2 and site-3 is EGFP-S3, which were both targets known to be refractory to RNAi using siRNA. (C, D) Comparison of DsiRNA and siRNA in downregulation of endogenous transcripts (that is, hnRNP H mRNA or La mRNA). (C) hnRNP H knockdown was assayed by western blot and (D) La knockdown by northern blot analyses. The dsRNAs were used at the indicated concentrations. β-Actin was used as an internal specificity and loading standard. [Nat Biotechnol 23(2):222–226.]


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