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.
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)
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
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