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Argonaute Proteins
The
argonaute proteins represent one of our side projects in creating a
universal
programmable endonuclease.
Abstract:
The proteins
of the argonaute family are the key players in RNA interference, where
they cut
and degrade mRNA. Therefore getting loaded with a siRNA or miRNA that
is the
inverse complement of the target sequence is a necessary step. The
RNA-induced
silencing complex, or RISC as the complex of the siRNA and the
argonaute protein
is often referred to, has been shown to cut the target mRNA exactly
after the
10th base of the guide si-/miRNA. There seem to
be no extensive RNA-specific
interactions, so in theory, the RISC complex should be able to cut
ssDNA as
well. Then, using two inverse complement guide oligonucleotides
combined with a
cycle of heat denaturation to make the DNA duplex accessible to the
RISC should
allow its use as an endonuclease, whose cutting site has been
determined by the
oligos complementary to the target sequence.
The RISC (RNA-induced silencing complex) is one of the most important key players in post transcriptional gene regulation. The DICER complex processes dsRNA or short intramolecular double-stranded regions of pre-miRNA by cutting it down to 20-25 nt fragments. 21 nt long ssRNA fragments, then called siRNA, can be loaded into argonaute proteins, where it serves as a guide oligonucleotide. This complex is then referred to as the RISC. The RISC is then ready to cut any RNA that has complementary sequence to the loaded guide oligonucleotid.
Cleavage
of
the target RNA has been shown to occur exclusively after the 10th
base of the guide oligonucleotide counted from its 5’ end,
ensuring both sequence
as well as cleavage position specificity.
As there is no evidence of extensive interactions with the 2’-OH of the target RNA ribose part of the target, as well as the one of the guide oligonucleotide, it should be possible to cut ssDNA as well. It has indeed been shown, that a ssDNA 21mer (5’-phosphorylated) is even more efficient in acting as the guide than the naturally occurring siRNA. Combination of two complementary 21mer guide oligonucleotides with a heat denaturation step converting the target DNA into the single stranded form should in theory enable the argonaute protein to catalyze a dsDNA cleavage event at any chosen site.
Results and Discussion:
We
were
very thankful to receive two vectors containing argonaute protein
genes, namely
the ones of the thermophilic bacteria Aquifex
aeolicus and Thermus thermophilus,
from Stefan Juranek of the Howard Hughes
Medical Institute Laboratory of RNA
Molecular Biology Rockefeller University. After expression and purification of the
proteins, we first tried to cut ssDNA produced by M13 bacteriophages,
but had
little success - probably due to the extensive secondary structure of
the DNA.
We spared to do some RNA cleavage assays, because we would have had to
establish
all the arrangements for handling RNA first, which would have exceeded
the capacities
we were able to provide for this side project. Next we tried to
generate ssDNA
using a modified thermocycler protocol, where we used just one primer
and a
digestion product as a dsDNA template to ensure the production of ssDNA
of a
determined length. The product was visualized on an agarose gel and
purified prior
to the assay.
In
parallel
we were able to establish a phage display system, where the Aquifex aeolicus argonaute protein was
displayed on M13 phages. Therefore
we created a library of the argonaute protein gene containing multiple
point
mutations introduced by error prone PCR, that were inserted into a
phagmid
vector.
We
hope to
increase the argonaute protein’s affinity towards ssDNA as
well as the ssDNA cleavage
catalysis, by selecting phages bearing argonaute proteins, with that
features out
of the library.
Methods:
Purification
of the protein was achieved by Ni NTA column and size exclusion
chromatography
(see “protein expression and purification”).
ssDNA was produced by
prepearation
of genomic ssDNA of M13 as well as by a modified thermocycler protocol,
similar
to pcr but with just one primer and a digestion product as template to
ensure
constant length of the cycle product.
The
cleavage
test assay was performed analogous to the RNA cutting assays performed
by Wang
et al in “Structure of an argonaute silencing Complex with a
seed-containing
guide DNA and target RNA duplex”, Nature 456,
921-926 (18
December 2008) : “Recombinant T. thermophilus Ago (1 mM final
concentration)
was incubated with a reaction mixture containing 10mM HEPES-KOH, pH7.5,
100mM NaCl,
5mM MnCl2, and 0.5 mM guide strand for 30min at
55 °C in a final
volume of 15 ml…””…RNA
substrate at a final concentration of 0.1 mM was added.
The incubation was continued for 30min at either 55 or 70 °C.
The reaction was
stopped by addition of 185 ml proteinase K solution (1mg/ml proteinase
K, 20mM
HEPES-KOH, pH7.5, 1.5mM EDTA, 100mM NaCl,1.5mM CaCl2 and 1.5% SDS), and
incubated at 55 °C for 10min…” Products
were analyzed on polyacrylamide gel.
The
argonaute protein genes were transformed in a library by error prone
PCR using
Taq-polymerase at high salinity. The product was inserted into a
phagmid vector
that generated a fusionprotein containing the argonaute protein, the
N-terminal
part of the gene-3-proteine as well a signal sequence to export the
protein
into the periplasm.
Panning
is being performed by incubating the argonaute presenting phages
with a 21mer just like in the cleavage assays at the moment. A
complemantary
biotinylated target oligonucleotid is coupled to streptavidin on an
immunotube.
Phages not binding tho ssDNA oligonucleotid will be discarded during
the
washing steps. Those able to bind will be eluded with a buffer
containing the
cation neccasary for the cleavage activity, so argonaute proteins able
to cut ssDNA
should be eluded here. The other proteins, just able to bind, but not
to cut
ssDNA should be eluded by a buffer containing DNaseI (by cutting the
ssDNA link
to the immunotibe).
