Technology to Create Spiegel ERAbodies on Demand: Biostable Universal Antibody Replacements Foundation for Applied Molecular Evolution Elisa Biondi ABSTRACT Researchers in biomedical, diagnostic, and clinical areas want to create (or buy), on demand, reagents that bind to proteins and other targets that may be involved in a biological process that they are studying. Antibod- ies have long served this role. However, as biologics, antibodies are at the center of an "irreproducibility crisis" in biomedical research, and, even when suitable, take months and thousands of dollars to make. This has driven efforts to create antibody replacements, both protein (e.g. Darpins) and RNA (e.g. aptamers). The first are difficult to manipulate, while the second have low stability and disappointing affinity. We hypothesize that an unnatural platform with an "Expanded RNA Alphabet" (ERA) and extra functional groups with extra binding potential will meet this long-standing unmet need. While expanded DNA alphabets are now advanced, a first innovation is that ERAs have not yet been the target of any preliminary data. We hypothesize that nanomolar binding will be routinely achieved because ERAbodies will have access to (a) higher information density that will lead to (b) better defined folds, both by using an RNA scaffold and by having functionality that supports folding, (c) greater structural diversity that gives ERAbodies more modes for tight binding, and (d) more folding motifs that allow ERAbodies to have more compact structures. They are also hypothesized to have all of the advantages of classical aptamers, including value as the starting points for subsequent rounds of evolution, modifiability using signaling entities, low cost, fast turnaround, and direct chemical synthesis. This R21 project will prove the value of this new technology platform, which will allow researchers to order or directly create in weeks, binders for targets that they themselves select. We hypothesize a further innovation by merging yet unexplored ERA technology with the classical concept of mirror symmetry. To create ERAbodies that are stable in biological systems, we will make these in mirror image ("Spiegel") form. Aim 1. A single R21 demonstration project will show that ERAbodies can be made with building blocks created by palladium chemistry. Although this technology is agnostic with respect to applications (it can create binders for any target), this demonstration will have impact by targeting a protein with outsized medical significance, PD-L1 (Programmed Death Ligand 1). We will test the hypothesis that L-ERA reagents, much like standard L-RNA, are stable against RNases in living systems. This workflow step will consume 18 months. We will expand methods to sequence ERA and benchmark the fidelity of enzymatic synthesis. In the workflow, this will be completed in the first 6 months, as patterns of transliteration by various reverse transcriptases will be used to define a sequencing procedure applicable to various 6- and 8-letter ERA systems. The target metrics are binding affinity (nanomolar), binding specificity (100:1 discrimination), and in vitro sera stability (<0.1% RNase degradation over 2 hours).

Technology to Create Spiegel ERAbodies on Demand: Biostable Universal Antibody Replacements Foundation for Applied Molecular Evolution Elisa Biondi NARRATIVE Laboratory and clinical biomolecular research is the foundation for advances in diagnostics, therapy, and patient management, as it provides detailed understanding of the relation between organic molecules and behaviors of the living state, natural and diseased. Key to its scientific method is the testing of hypotheses that relate specific molecules, often peptides and proteins, to specific behaviors. This often requires the search for a commercial antibody or, failing that, a month to year effort to create one, which in turn often leads to the discovery of another example of an antibody that exemplifies the "irreproducibility crisis" caused by such biologics. In this proposal, we will meet this unmet need by creating an unnatural platform with an "Expanded RNA Alphabet" (ERA) that routinely creates, on demand, nanomolar binders that will serve as research tools. Special attention will be made to create ERAbodies in forms that are stable in biological mixtures. If successful, this new capability should transform biomolecular research.