Numerous experiments
have shown that introduction of long double-stranded RNAs (dsRNA)
into certain organisms (e.g. plants, fungi, metazoans) and cell types
(e.g. mouse embryonic stem cells) can cause degradation of the homologous
mRNA; a phenomenon termed RNA interference or RNAi. In the cell,
the long dsRNAs are cleaved into short 21-25 nt small interfering
RNAs or siRNAs by the ribonuclease DICER. The siRNAs subsequently
assemble with protein components into an RNA-induced silencing complex
(RISC), which tags and destroys the cognate mRNA.
Recent evidence shows that cultured mammalian
cells can also experience specific gene silencing through RNAi.
In early experiments, injection of long dsRNAs into mammalian cells
caused nonspecific mRNA degradation due to activation of an anti-viral
response pathway. More recently, however, experiments have demonstrated
that transfection of mammalian cells with 21-25 nt siRNAs, as opposed
to long dsRNAs, can elicit specific gene silencing. (1) The potential
applications of siRNA-induced RNAi in mammalian cells are exciting.
However, successful use of this tool requires an understanding
of siRNA sequence and structural requirements. In a paper published
in the December 2001 issue of EMBO, Elbashir and colleagues
attempt to address this issue. (2)
By using Drosophila embryo lysates and
the firefly luciferase gene, the authors show that potent siRNAs
appear to have certain common characteristics. The most effective
siRNAs against the luciferase gene turn out to be 21 nt dsRNAs
with 2 nt 3' overhangs. The authors also show that the sequence
specificity of siRNA is very stringent. Single base pair mismatches
between the siRNA and its target mRNA dramatically reduced silencing.
These studies, though limited to a single gene and system, form
a basis for siRNA construction strategies employed by many researchers
attempting to implement siRNA experiments in their labs.
The Approach
Elbashir and colleagues examined siRNA activity
in Drosophila lysates using firefly and sea pansy luciferase
as a reporter system to look at siRNA activity. Assorted panels
of siRNAs against the 5' end of the firefly luciferase gene were
synthesized. The siRNAs within each panel varied with respect to
their length (18-25 nt), length of overhang (0-7 nt 3' overhangs
or 0-4 nt 5' overhangs), sequence of overhang, composition of the
backbone (2'-deoxy- or 2'-O-methyl modified), and sequence specificity
(single or double base substitutions and short inversions).
siRNAs were incubated individually with Drosophila embryo
lysates for 10 minutes. Subsequently, constructs coding for the
firefly and sea pansy luciferases were added to the lysates and
incubated for an additional 60 minutes. siRNA potency was defined
by the extent of knockdown in firefly luciferase activity upon
normalization with sea pansy luciferase levels.
The Findings
Among the siRNAs tested, the most potent ones
had the following common characteristics. They were 21 nt in length
with 2 nt 3' overhangs (i.e. they contained a 19 nt complementary
region between the sense and anti-sense strands). The sequence
of the overhangs did not appear to be critical, as a number of
siRNAs with different overhang sequences were able to function
with the same efficiency (NN/UG and NN/UU). Interestingly, inclusion
of certain 2'-deoxynucleotides in the overhangs did not reduce
the siRNA activity (NN/TdG and NN/TT). However, activity of the
siRNAs was abolished when an entire strand was made with either
2'-deoxy or 2'-O-modified bases.
The authors also showed that target recognition
is highly sequence-specific. One or two base pair mismatches between
the siRNA and the luciferase gene greatly reduced silencing. The
same was true for short 3-4 nt inversions.
The structural and sequence requirements of siRNAs
discovered by Elbashir and colleagues have provided a basis for
construction of active siRNAs. Ambion also recommends that siRNAs
be 21 nt in length, with UU or TT 3'-overhangs. However one should
keep in mind that these structural features are based on findings
that are confined to a limited number of genes and systems. Needless
to say, more studies are needed in order to make generalizations
about structural requirements of active siRNAs.
References
- Elbashir, SM, Harborth, J, Lendeckel, W, Yalcin, A, Weber,
K, and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate
RNA interference in mammalian cell culture. Nature 411:494-498.
- Elbashir, SM, Martinez, J, Patkaniowska, A, Lendeckel, W,
and Tuschl, T. (2001) Functional anatomy of siRNAs for mediating
efficient RNAi in Drosophila melanogaster embryo
lysate. EMBO 20:6877-6888.
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