The discovery of reagents to inhibit RNA synthesis
and translation has revolutionized the study of biological processes.
These tools include small inhibitory RNA (siRNA), small hairpin
RNA (shRNA) and ribozymes. As RNA interference technology has
grown, so has the list of validated RNA target sequences and
reagents for study. This web site includes a database of validated
siRNA target sequences and links to kits and reagents that may
be used for RNA interference experiments.
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RNA interference refers to the inhibition
of gene expression by small double-stranded RNA molecules. This
phenomenon was initially demonstrated in C. elegans, in
which the injection of dsRNA molecules inhibited complementary
gene expression. Though the use of siRNA has become a widely
used tool for down-regulating gene expression, the existence
of a naturally occurring pathway in eukaryotes has been well
described. The origin of endogenous siRNA (or miRNA) may be transposons,
viruses, repetitive sequences and genes. The process of producing
effective endogenous siRNA is regulated by three enzymes. RNA-dependent
RNA polymerases convert single-stranded RNA into double-stranded
RNA. Alternatively, DNA-dependent RNA polymerases produce dsRNA
by transcribing inverted DNA repeats. The resulting large RNA
molecules are subject to digestion by ribonuclease III (Dicer)
to produce short double-stranded siRNA molecules. Argonaute proteins
are then required to bind siRNA molecules to form a complex known
as RISC (RNA-induced silencing complex). RISCs may then promote
epigentic silencing through RNA-directed DNA methylation or by
target RNA cleavage. Though protein translation may be knocked
down considerably, siRNA does not normally eliminate the expression
of a gene target completely.
siRNA as a Tool
The use of RNA interference for artificially
manipulating gene expression was initially limited by the activation
of cellular antiviral mechanisms. Exposure of cells to sequences
longer than 30 nucleotides induces interferon gene expression
resulting in non-specific RNA degradation and reduced protein
synthesis. However, this problem was circumvented by designing
19 to 22 nucleotide siRNA sequences. Methods for siRNA delivery
into cells include liposome-based addition of purified ribonucleotides
to the media or transfection of plasmid vectors designed to express
siRNA molecules. Plasmid vectors rely on the use of two RNA Polymerase
III promoters (U6 and H1) to drive transcription of the siRNA
molecule. The target sequence (19 to 29 nucleotides) is placed
in a sense and antisense orientation with a small spacer group
in between (short hairpin RNA or shRNA). Once transcribed, a
hairpin structure is formed that can be recognized and cleaved
by Dicer. Alternatively, RNA duplexes may be transcribed without
hairpin structures and directly process by the RISC. Currently,
there are a variety of plasmid and viral vectors that utilize
similar concepts to produce siRNA or shRNA molecules.
Following the discovery of siRNA, several
studies attempted to identify the optimal characteristics required
for siRNA design. Some of the requirements include using sequences
shorter than 30 nucleotides to avoid PKR activation, sequence
stability at the 5' end of the antisense strand relative to the
3' terminus and inserting a TT overhang. Based on studies like
these, a number of algorithms have been developed by academic
and industrial labs to predict the most effective target sequences
for a given gene. Though most of these programs are not perfect,
the likelihood of obtaining a predicted sequence is superior
to designing sequences without consideration of the recommended
features. Synthesis and testing of multiple sequences may be
required. The design of siRNA experiments may contain some potential
pitfalls, thus the design should be done to include appropriate
controls and measurable endpoints. A negative control should
consist of a non-complementary sequence with thermodynamically
similar properties as the effective siRNA sequence. When transfecting
a plasmid vector to introduce siRNA or shRNA, the ratio of lipid
to nucleic acid should be equal and the control vector should
contain a sequence that is transcribed and processed intracellularly.
Validation of the siRNA effect should also be done by measuring
RNA and protein expression.
The concept of down-regulating a single gene
by siRNA can be exploited by high-throughput technologies to
facilitate large scale genomic studies. This can be done in a
microtiter-based format or by designing libraries of siRNA or
shRNA molecules. Libraries may be engineered based on validated
siRNA sequences or by using randomly produced sequences and a
biochemical or cellular assay to identify positive elements.
If multiple iterations are used, the siRNA or shRNA may be rescued,
re-introduced and identified by sequencing. This strategy may
be effectively used to identify drug targets or new components
of signaling pathways.