Scientists, clinician, and physicians alike have long wanted a technology that can routinely deliver molecules that bind to targets important to their research, to diagnostic biomarkers, and to molecules essential to the progression of patient diseases. Currently, macromolecular "binding molecules on demand" (BMODs) are most rapidly available by way of antibody technology. Today, antibodies are gaining niches among therapeutic agents, previously dominated by small molecules generated using the "hard slog" of medicinal chemistry. A quarter century ago, scientists suggested that the replicability and evolvability of DNA and RNA (xNA) might offer an alternative route to macromolecular BMODs. Here, xNA "aptamers" might be selected from libraries of xNA molecule to bind to a target via in vitro selection (SELEX). Aptamers might work under conditions where antibodies do not, especially in environments where proteins unfold. They might eventually displace antibodies or become therapeutic agents, as are many antibodies today. Despite the successes of SELEX, we now understand that the four-nucleotide xNA that it uses has too few functional groups, too little sequence diversity, and too much interference from natural xNA, to meet this vision in its broadest form. Therefore, we propose here to expand SELEX using an artificially expanded genetic information system (AEGIS), a kind of DNA that adds up to eight independently pairing nucleotides to the four found in standard DNA. The proposed work will immediately add two AEGIS nucleotides to SELEX (Z and P, forming a Z:P pair independent of standard C:G and T:A pairs), allowing us to immediately use AEGIS-SELEX to create GACTZP aptamers that bind to lung, breast, and liver cancer cells. Immediate progress is possible because the two collaborating applicant laboratories (Steven Benner at the FfAME and Weihong Tan at UF) have already combined breakthroughs in cell-SELEX, polymerase technology, and AEGIS sequencing, to produce the first AEGIS aptamer. Obtained in just weeks from only 12 rounds of SELEX, this 30 namomolar aptamer offers up the "central hypothesis" for this work: Because AEGIS xNA libraries have richer diversity, they are richer reservoirs of high affinity aptamers than standard xNA libraries. By allowing xNA aptamers to gain up to 60% of the sequence diversity of antibodies, AEGIS-SELEX further offers the opportunity to finally meet the technological goals of SELEX. AEGIS-SELEX should also help expand the science of protein-nucleic acid interactions and molecular recognition in new directions. To achieve this vision, three things must be done, all shown to be feasible by preliminary work: (1) We must improve the fidelity of polymerases that copy AEGIS DNA. (2) We must improve sequencing technology for AEGIS DNA. (3) We must add more AEGIS nucleotides to the Z and P that have already been proven;and (4) we must compare AEGIS-SELEX to standard SELEX. Following a "two for the price of one" strategy, we will do this benchmarking by creating useful aptamers that target circulating liver, breast, and lung cancer cells.

Public Health Relevance

Scientists, clinician, and physicians alike have long wanted a technology that can routinely deliver molecules that bind to targets important to molecules that their research programs study, their diagnostic biomarkers, and involved in the progression of disease in their patients. This project will deliver this technology, exploiting an innovative combination of synthetic biology and molecular evolution, a combination based on recent breakthroughs in the two collaborating laboratories. In addition to providing a platform to advance the sciences of protein-DNA interactions and molecular recognition, the proposed work will immediately deliver molecules that bind to breast, liver and lung cancer cells, including those that may be circulating in patient blood.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Drug Discovery and Molecular Pharmacology Study Section (DMP)
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Fabian, Miles
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Foundation for Applied Molecular Evolutn
United States
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