Ribonucleic acid interference (RNAi) exploits an ancient part
of the immune system that protects plants and animals against invaders
by the depletion of viral genomic RNA targets in a sequence
manner by making use of small interfering RNAs (siRNAs). The post
gene silencing (PTGS) by RNAi was first discovered in 1998 by Fire et
al. in nematode, Caenorhaditis elegans (Nature 1998,
806-11). The siRNA approach is an indispensable tool today for targeted
gene inhibition in plants, worms and flies for recognizing and
complementary RNAs in the cell in a sequence-specific manner, thereby
the expression of targeted gene in a highly effective way. The siRNA
comprised of a sense strand homologues to the target and an antisense
that binds to the target mRNA. Effective gene silencing by RNAi
(more than 90% inhibition of protein expression) has been shown in
cell culture assays spanning from invertebrates to humans (see below
latest reviews). Although the complete mechanism of the gene silencing
effect induced by RNAi is not clearly understood, a general picture of
this process is slowly emerging.
Naturally RNAi is initiated by an ATP dependant, processive cleavage of the double stranded RNA into 21-23 nucleotide siRNAs by the enzyme called RNase III Dicer, and these siRNAs are incorporated in to various protein factors and form RNA induced silencing complex (RISC). ATP-dependant unwinding of the siRNA duplex generate an active complex, RISC* (the asterisk indicates an active conformation of the complex). Guided by the antisense strand of siRNA, RISC* recognizes and cleaves the complementary mRNA in the cytoplasm with the help of endoribonucleases (siRNAs were found to be ineffective when targeted to introns in the pre-mRNA). It has been shown that the complete unwinding of the antisense strand is not necessary for RNAi. It has also been proposed that the siRNA could get recycled in the process by which it can induce silencing of other copies of mRNA, thereby attributing a catalytic turn-over mechanism for RNAi action. However, it has been demonstrated that single stranded siRNA can also trigger RSIC assisted RNAi. The guidance of RSIC* to the target RNA is sequence specific as one to two nucleotide differences in the target recognition hampers the RNAi function.The generation of siRNAs by Dicer can be bypassed either by the use of synthetic RNA duplexes comprising of 19 nucleotide duplexes (ds-siRNAs) and two overhanging nucleotides at the 3'-ends or by the single stranded siRNAs (ss-siRNA) of length varying from 19-28 nucleotides.
|The proposed mechanism of target mRNA destruction by siRNAs in mammals and flies. The mechanism suggests that the antisense strand of the 21-23 nt siRNA/protein complex (RISC*) can be used with multiple turn-overs destroying multiple copies of the the target mRNA (Ref 1).|
The 21mer synthetic duplex siRNAs, in 20-100 nM concentration (by transfection), have now become a routine tool to validate gene function. Considering the fact that Human Genome consists of ca 30-40,000 protein-coding genes and more than half of their function remains unknown, gene down regulation by siRNA now has become an important method to decipher the functions and interactions of those genes. Since the gene silencing is an effective way to suppress viral genes and human oncogenosis, there also lies a great therapeutic interest in the siRNA induced RNAi. However, the structural requirements of these siRNAs for the prolonged RNAi function are far from being established.
The ds-siRNAs based RNAi offers much more advantage in terms of efficiency for target RNA destruction compared to the use of the ss-siRNAs. The ss-siRNAs unlike ds-siRNAs were found to be less efficient to trigger RNAi. In many cases it took eight times higher concentration of the ss-siRNA to achieve the effective gene silencing potency of the corresponding duplex. Although in vivo expression of siRNAs or its precursors called microRNAs (miRNA) by viral vectors has been shown as an alternative strategy to the use of synthetic siRNAs, this approach is shadowed by the tedious procedure to develop the viral vectors and immunostimulatory side effects associated with the delivery.
Recent data indicate that the in vivo delivery of siRNAs in mice is possible. In two studies, siRNAs were injected under hydrostatic pressure into the mouse tail vein, and silencing of reporter transgenes was observed in different tissues (Nat. Genet. 2002; 32,107-109, Nature 2002, 418, 38-39). The transfection method used in these studies is unlikely to be easy to use in humans because of the detrimental physiological side effects. Considering the high dosage necessary for the RNAi action for the down-regulation of genes, suggests two intrinsic problems: (1) Inherent instability of the siRNAs (as would be expected from any native RNA); (2) lack of effective delivery of siRNA inside the cell. Therefore, biologically stable siRNA mimics and finding appropriate delivery methods (both for the native siRNAs as well as those of the mimics) is perhaps a major barrier for therapeutics in humans. The findings that RNAi approach can effectively snuff out genes against liver disease in mammalian cells has created much excitement. ( Nature Medicine 2003, 9, 347-351). In this report the authors, for the first time, used RNAi effect to halt hepatitis in mice.
Recent papers thus show that RNAi is functional in numerous tissues of therapeutic interest, which makes it challenging for chemists to design and synthesize mimics of the native siRNA, that are stable inside the cell and can be easily delivered.
Prof. Chattopadhyaya is sub-coordinator of SP2: Chemical Tools
in the Integrated
Project RNA Interference
(LSHB-CT-2004-005276), financed by the EU SIXTH FRAMEWORK
(Life sciences, genomics and biotechnology for health). Project's home
Recent Reviews on RNAi
(1) Hutvagner, G., Zamore, P.D.: RNAi: Nature abhors a double
Genet. Dev. 2002, 12, 225-32. (PDF)
(2) Hannon, G.: RNA interference Nature 2002, 418, 244-251. (PDF)
(3) Agami, R.: RNAi and related mechanisms and their potential use in therapy Current Opin. Chem. Biol. 2002, 6, 829-834. (PDF)
(4) McManus, M.T.; Sharp, P.A.: Gene Silencing in Mammals by Small interfering RNAs Nature Rev. Genet. 2002, 3, 737-747. (PDF)
(5) Borchardt, A.; Tuschl, T.: Small Interfering RNAs: A Revolutionary Tool for the Analysis of Gene Function and Gene Therapy Mol. Intervent. 2002, 2, 159-167. (PDF)
(6) Watts, J.K.; Deleavey, G.F.; Damha, M.J.: Chemically modified siRNA: tools and applications Drug Disc. Today 2008, 13, 842-855. (PDF)
(7) Shukla, S.; Sumaria, C.S.; Pradeepkumar, P.I.: Exploring Chemical Modifications for siRNA Therapeutics: A Structural and Functional Outlook ChemMedChem 2010, 5, 328-349. (PDF)
(8) Gaglione, M.; Messere, A.: Recent Progress in Chemically Modified siRNAs Mini-Rev. Med. Chem. 2010, 10, 578-595. (PDF)