siRNA Test System
Cat # N0070
Luciferase-based ENGENISTM (Entire
Gene/cDNA or Isolated Sequence) siRNA Test System
is designed to provide a quantitative approach for evaluation of a potential
of any DNA sequence to serve as an efficient target for RNA interference (RNAi).
Current computer-based RNAi target searching algorithms are not perfect, leaving
the probability of selected sequence to be a good target from 30% to 60%. It is
therefore clear that the ability of computer-selected sequence to induce RNA
interference has to be confirmed experimentally. The ENGENISTM
siRNA Test System allows for screening a large number of potential siRNA
target sequences in simple transient transfection experiments, measuring the
reduction in reporter (firefly Luciferase from Photinus pyralis) gene
In most commercially available vectors and kits intended for the
same purpose, the gene silencing potential of a target sequence is tested on the
fused transcript bearing both reporter gene and gene of interest. In such
systems some unpredictable factors like chimeric mRNA folding and target
accessibility may affect the observation of RNAi. Also the distance between the
target site and reporter gene coding region along the mutual messenger RNA plays
a role in the reporter gene silencing: even if mRNA is cleaved at the point of
siRNA target, translation of the reporter gene can still go on till the RNA
degradation by nonspecific nucleases reaches the reporter gene coding region.
Such kind of effects may obscure the real efficiency of particular targets.
Unlike other kits, the ENGENISTM siRNA Test System
offers two possibilities for evaluation of the target sequence efficiency:
on the one hand, using entire gene technology, and on the other hand, using a
technology with isolated target sequences per se. In the latter case,
short target sequences are located at defined positions immediately upstream or
downstream of the Luciferase gene coding region. For entire gene technology, two
variants of transcript can be generated by placing the gene of interest either
in front of Luciferase coding region or behind it.
Independent evaluation of the reporter gene silencing
corresponding to two different positions of isolated target sequence or entire
target gene permits to avoid or reduce the influence of target accessibility
factor and other factors related to RNA conformation, thus making comparison
between various targets more reliable. Another advantage of the ENGENISTM
siRNA Test System is that it makes possible evaluation of the siRNA
target potential even in those cases when the gene of interest is not available
as full-size cDNA clone.
The ENGENISTM siRNA Test System
of four vectors: one effector plasmid (psiRNA) producing double-stranded siRNA,
and three reporter plasmids (psiTEST-target-LUC, psiTEST-LUC-target,
psiTEST-target-IRES-LUC) expressing Luciferase reporter gene fused to siRNA
target sequence or the target gene cDNA.
siRNA testing on isolated targets (Fig. 1) is based on the fact
that the nucleotide sequence of siRNA is identical to the sequence of
corresponding siRNA target. Thus
the same short synthetic DNA fragment has to be cloned into three linearized
plasmids to generate siRNA coding sequence in effector plasmid psiRNA and siRNA
target sequence in two reporter plasmids psiTEST-target-LUC and psiTEST-LUC-target.
Construction of effector and reporter plasmids for siRNA testing on isolated
targets and their mechanism of action
In the frames of entire gene technology, the
siRNA Test System provides two supercoiled vectors psiTEST-LUC-target and
psiTEST-target-IRES-LUC containing multiple cloning sites (MCS) intended for
target gene cloning and subsequent generation of a fused transcript (Fig. 2).
Encephalomyocarditis virus Internal Ribosomal Binding Site (IRES) of the
reporter plasmid psiTEST-target-IRES-LUC enables Luciferase translation from the
second cistron of the fused transcript. MCS structures and vector backbones are
presented in the Section VI.
Fig. 2. Construction of effector and reporter plasmids for
siRNA target testing on fused mRNA and their mechanism of action.
siRNA expression from the effector plasmid psiRNA is driven by
dual promoter expression cassette bearing human U6 and H1 small nuclear RNA
promoters in opposite orientation to each other. Such dual promoter constructs
were shown to efficiently express double-stranded siRNA molecules (1-2) which
could be directly accepted by RNA Interference Searching Complex (RISC), the
multi-enzyme complex with RNase activity specifically digesting messenger RNA at
the siRNA target site (3). This permits to avoid the stage of dicer treatment
which is necessary for hairpin siRNA producing vectors to convert
single-stranded hairpin shRNA to double-stranded siRNA.
Detailed structure of the double-promoter siRNA expressing
cassette is shown in Fig. 3. siRNA coding sequence N1-N19
is placed between the U6 and H1 promoters. RNA synthesis driven by the U6
promoter starts from the nucleotide N1 which has to be G, and
terminates at the stretch of 5 T invading the body of H1 promoter. As a result,
produced RNA has 2 or 3 uridines at its 3' terminus. Similarly, the RNA produced
from the H1 promoter starts with the nucleotide N'19, terminates at 5
T stretch at the beginning of the opposite U6 promoter, and also bears 2 or 3
uridines at its 3' terminus. Two halves of siRNA join together, forming the
functional double-stranded siRNA with protruding 3'-termini.
A mixture of siRNA-producing effector plasmid and one of the
target-bearing reporter plasmids has to be cotransfected transiently into
mammalian cells. Measuring the reporter gene silencing due to the effect of RNA
interference will give a quantitative evaluation of the siRNA target potential.
Fig. 3. siRNA expression from the dual promoter effector