Antisense Oligonucleotides and RNA Interference - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 341

Figure 7.4 Basic
steps involved in the
mechanism of RNAi.

dsRNA

Cell membrane

Cleavage of
dsRNA by Dicer

Dicer

siRNAs

siRNAs associated
with RISC complex

RISC

Degradation of substrate
mRNA by RISC and
recycling of RISC complex

RISC

Destruction by
exonucleases

oligonucleotides [97] . Also, the activity of siRNA oligonucleotides has been found
to be affected by the secondary structure of the target mRNA. The RNAi pathway
( Fig. 7.4 ) involves several complicated steps, which we discuss here in brief [96] .

1. The process of RNAi is activated by exposure to dsRNA precursors. In the initiation step,
in the presence of ATP and RNase-III-type endonucleases, the dsRNA precursors are pro-
cessed into smaller nucleotides called siRNAs having length from 21 to 23 nucleotides.
2. The resulting siRNAs are subsequently incorporated into a multiprotein complex known as
the RNA-induced silencing complex (RISC).
3. In the successive step, in the presence of ATP the siRNA molecules undergo unwinding
with the aid of helicases, while getting processed into RISC. This activates the complex,
leaving only the antisense strand of siRNA associated with the RISC, while the sense
strand gets degraded by the exoribonucleases in cytoplasm.
4. The activated RISC then directs the siRNA toward its complementary mRNA sequence.
5. After binding of siRNA to complementary mRNA sequence, the RISC complex cleaves the
target mRNA with the help of Argonaute enzymes associated with the RISC.
6. The cleaved mRNA is then degraded by nucleases in the cytoplasm, thereby inhibiting the
expression of the related protein, while the freed RISC complex recycles to cleave more of
the target mRNA sequences.