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FORT BELVOIR, Va. - Better biosensors to detect chemical and
biological threats and enable better post-exposure treatments could
soon save the lives of warfighters and first responders, thanks to
new work on an efficient development of binding-molecules-on-demand
(BMOD). A DTRA CB/JSTO-funded research team at the Foundation for
Applied Molecular Evolution managed by DTRA CB's Dr. Ilya Elashvili
and led by the foundation's Dr. Steven Benner, has just reported a
novel approach to enhance a previously known technology that
develops BMODs for any specific molecular or cellular target with
greater efficiency.
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The artificially expanded genetic information system (AEGIS) used in
this work added two nucleotide “letters” (here, Z and P) to the four
standard nucleotides (G, A, C, and T) by shuffling hydrogen bonding
groups (the blue hydrogen bond donors, the blue prongs in the
cartoons; the red hydrogen bond acceptors, the red dots in the
cartoon). Selection (in vitro) gave a DNA aptamer molecule (sequence
lower right) that, if it contained Z and P (magenta, at positions 23
and 30), bound to breast cancer cells (green curve upper right).
Binding was lost when the Z and/or P was replaced with standard
nucleotides (aqua, blue, purple and yellow curves). (Image by Dr.
Steven Benner, Foundation for Applied Molecular Evolution, February
24, 2014)
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The ability to create high-affinity binding molecules to
a specific target is important for detection, analyses,
prophylaxes, and therapeutic applications. In detection and
analytical schemes, target binding can be monitored directly
or, as is often the practice, by employing observable tags.
In prophylaxes or therapeutics, binding of a molecule to a
specific molecule (such as an enzyme or receptor) or cell
(such as a pathogen or cancer cell) can alter the target's
potency or nature.
This innovation was made possible
by earlier DTRA CB/JSTO-funded work that developed
artificial nucleotides (“NextGen” expanded DNA, also
referred to as Artificially Expanded Genetic Information
Systems or AEGIS) and deepened our basic understanding of
nucleic acids, such as DNA and RNA. We had previously
reported (“Artificial Nucleotides Help Identify Multiple
Real Biothreats,” in June 2013's JSTO in the News) how that
basic research effort enabled the same team of researchers
to improve the ability of the popular Luminex instruments to
detect the targets in multiplex assays with much higher
fidelity than ever before.
This latest work shows how
the use of the artificial nucleotides developed under the
same basic research effort significantly improved the
efficiency of generating binders for a specific target,
which was the subject of a recent Proceedings of the
National Academy of Sciences USA article, “In vitro
selection with artificial expanded genetic information
systems.” In this article, the researchers report using
NextGen-expanded DNA to improve SELEX (Systematic Evolution
of Ligands by Exponential Enrichment) technology for a more
efficient generation of binding molecules. The traditional
SELEX technology uses a library of aptamers to generate
binders in an iterative cycle of selection, amplification,
and mutation process. Since natural DNA aptamers contain
only four nucleotide, G, A, C, T (GACT) building blocks, the
researchers hypothesized that enriching them with the
additional nucleotides from the NextGen DNA would help the
process because of the increased diversity of available
functional groups.
However, the technique involves
Polymerase Chain Reaction (PCR) amplification and deep
sequencing. Therefore, in order for the process to work with
the additional building blocks, not only must the additional
nucleotides exclusively pair with their partners and with
the appropriate matching arrangements of hydrogen bond donor
and acceptor groups, but they must fit Watson–Crick
geometries. This reaction must transpire if the nucleotides
are to be compatible with the polymerases employed. The
scientists worked out these issues earlier in the DTRA
CB/JSTO effort.
Consequently, the research team was
able, in only twelve rounds of selection, to develop high
affinity (dissociation constant [Kd] of 30 nM) DNA aptamer
molecules that bind to a line of breast cancer cells.
Typically, 15–20 rounds of standard GACT SELEX were required
to get similar affinities against cell targets and utilized
the length of the randomized region between 35 and 45
nucleotides. In this study, researchers used only 20
nucleotide-long randomized regions that included two
artificial nucleotides (Z and P) together with the standard
four.
The DNA aptamers offer advantages over
antibodies because they can be readily produced by chemical
synthesis, are easier to store, and have little or no
immunogenicity, which is very useful in therapeutic
applications.
By John Davis
Defense Threat Reduction Agency's Chemical and
Biological Technologies Department
Provided
through DVIDS
Copyright 2014
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