Uses of RNA aptamers in molecular biology
David Bunka, Benjamin Mantle, Tamara Belyaeva, Bo Meng, Claire Lane, Adam Nelson,
Simon Phillips, David Rowlands, Sheena Radford and Peter Stockley.
The use of DNA and RNA aptamers is becoming more and more prevalent in modern
molecular biology. These highly adaptable molecules have a wide variety of applications,
including identification of recognition motifs, novel inhibitors of protein function, diagnostic
or therapeutic reagents and even as novel gene regulatory elements. Aptamers are isolated
from complex degenerate libraries of approximately 1015 sequences, in a process referred to
as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) which is
undergoing rapid technical advances (see Figure). We have successfully used our automated
in vitro selection platform to raise aptamers against many targets, a few of which will be
mentioned here.
Partitioning of
target bound
nucleic acids
Nucleic acid pool
is incubated with
target molecules
(Surface Plasmon
Resonance, capillary
electrophoresis)
(Toggle SELEX,
Photo-SELEX)
Nucleic acids are
amplified
(2’-amino, 2’-fluoro,
2’-methyl or 4’-thio
nucleotides,
Tailored-SELEX)
SELEX
(Automated cycles)
Low affinity species
are removed
Bound species
are eluted
In one project, in collaboration with Adam Nelson’s laboratory, we aim to create novel
regulatory elements, “riboswitches”, which can be used in both prokaryotic and eukaryotic
gene expression studies. Our initial target for allosteric regulation of function was the E. coli
methionine repressor, Met J. We have previously reported the isolation of RNA aptamers
which bound to Met J and were able to interfere with binding to promoter DNA in vitro. The
next step of the project was to test these aptamers for in vivo activity. Using a specially
designed tester strain of E. coli, we demonstrated that activity of a reporter gene is increased
1.8-2.5 times in presence of aptamers. Expression of aptamers in the cells was confirmed by
Northern blot. Future work is directed at creating allosterically regulated versions of these
aptamers.
In another project, in collaboration with Simon Phillips’ laboratory, we raised aptamers
against satellite tobacco necrosis virus (STNV) capsids, with the aim of understanding some
of the RNA-protein interactions involved in capsid assembly. Our selections lead to the
isolation of several aptamers with sequence and structural similarities to parts of the viral
genome. Preliminary results suggest that this structure may act as an assembly initiation
signal. In a related project, in collaboration with David Rowlands, we isolated aptamers
against the poliovirus capsid protein pentamer. As with STNV, the aim of this project was to
identify RNA-protein interactions important for viral assembly/RNA packaging and to
determine how the viral capsid specifically packages its genome amongst all the other
cellular RNAs. Our results show that the isolated aptamers specifically interact with capsid
assembly intermediates. Sequence and structure comparisons have identified matches
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between the poliovirus genome and isolated aptamers, again suggesting that we have
identified an assembly initiation signal or recognition element. Studies on the effects of
aptamers on viral assembly in vitro and in vivo are ongoing.
A separate project in collaboration with Sheena Radford had the aim of raising aptamers
which specifically recognise either the fibrillar or monomeric forms of the amyloidogenic
protein, beta-2 microglobulin (β2m). We were successful in isolating aptamers that
preferentially bind to β2m fibrils. Interestingly, these aptamers also recognised β2m fibril
morphologies other than the selection target. This suggests some degree of structural
similarity between these morphologies. Strikingly we also noted that these aptamers were
also able to specifically recognise fibrils formed from a few other amyloidogenic proteins.
This suggests that there are structural similarities between fibrils formed by these different
proteins.
Publications:
Bunka, D. H. J. & Stockley, P. G. (2006). Aptamers come of age - at last! Nature Reviews
Microbiology 4, 588-596.
Funding:
We would like to thank the Wellcome Trust, BBSRC, MRC and EPSRC for funding these
various projects.
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