By Dr Vibhor Mishra
Short Interfering RNA (siRNA) has come of age to unleash its full potential as a potent therapeutic. The onset of a series of FDA approvals of the siRNA medication targeting rare, previously untreatable genetic disorders and several other promising drug candidates in clinical trials have established the RNA Interference (RNAi) technology in the therapeutic realms. Designing a highly effective siRNA is one of the critical challenges of its downstream usage as a potential drug.
The therapeutic siRNAs are generally designed using the knowledge we have gained studying the naturally occurring siRNAs. Typically, siRNAs are 20-25nt in length and are double-stranded. The two strands are defined as the ‘guide stand’ and the ‘passenger strand’. These RNA stands base pair to form a dsRNA flanked by 1 or 2 nucleotide overhangs.
The main prerequisites of siRNA designed as therapeutics are a higher degree of RNA activity, enhanced stability, and its target specificity. In addition, they should have high tissue bioavailability while avoiding inadequate immunological responses. Synthetic siRNAs for therapeutic purposes have the guide strand designed complementary to a region within the mRNA generated from the targeted gene. When exogenous siRNAs enter the cytoplasm, they associate with an Argonaute protein to form an RNA-Induced Silencing Complex (RISC). The guide strand directs the RISC to the target mRNA sequence. However, the RNAse activity of Argonaute cleaves the target mRNA leading to degradation of the target mRNA sequence, abolishing any downstream effects (Figure1).
For siRNAs to silence the intended target, only the guide RNA, which has a sequence complementary to the target mRNA, should be loaded onto the Argonaute protein. Incorrect loading of passenger RNA could form a mature RISC leading to ‘off-target’ effects, silencing unintended genes. The difference in a siRNA’s one strand’s activity relative to the other is termed functional asymmetry. Strand bias in RISC incorporation primarily occurs due to recognizing differences in the termini of the siRNA. The strand whose 5’ end is less stably hybridized to the complementary strand is preferentially loaded into RISC. The therapeutic siRNAs are designed with a chemically modified passenger strand to promote the preferential loading of the guide strand to form a pre-intended mature RISC complex.
The siRNA activity also depends on the nucleotide present at the 5’ termini. The Argonaute preferentially binds with a higher affinity to RNA at 5’ A or U than C or G nucleotides. Rational selection of the 5’-terminal nucleotide in the guide strand ensures high siRNA activity. The mRNA secondary structure precludes siRNA binding through the steric hindrance of RISC binding and cleavage. Based on structural data, it is recommended, given a choice, to target regions of greater accessibility, especially at the 5’ and 3’ ends of the mRNA.
The guide strand’s 2nd-8th bases form the seed region, which complements the target mRNA. The 10th and 11th bases make the mRNA slicing site (Figure 2). These regions require a high degree of complementarity with the target mRNA, and thus there is no tolerance of amino acid substitution in these regions. The 3’overhang (bases 20th and 21st) interact with the PAZ domain of the Argonaute. The bases 17th -19th also interact with the N-terminus of the Argonaute. These bases, which directly interact with the Argonaute, provide an option for substitution by other nucleotides while designing siRNAs as they do not influence base-pair complimentarily with the target mRNA.
About the author:
Dr. Vibhor Mishra is Lead Researcher in the department of pathology at St. Jude Children’s Research Hospital. He is a published author and a science communicator.
Social media links: www.linkedin.com/in/vibhor-mishra-ph-d