Minimal Antibody Analogs on Nucleic Acid Scaffolding

The antibody-antigen lock-and-key recognition scheme, at the onset of the immune response, is a fundamental aspect of vertebrate life whose mechanism continues to be studied, and has been continuously under the spotlight ever since the beginning of the COVID-19 pandemic. Though simple at its core, the concept of the lock-and-key hides a fairly complex biochemical process involving the maturation of very specific antibodies from a nave set where partial binding to a new antigen occurs. There is evidence suggesting that the immune system does not engage the full combinatorial arsenal of binding elements to trigger an effective response but rather, that specific amino acids found at precise locations on the paratope (the antigen recognition region of the antibody) are able to direct an efficient antigen binding. Engineering the molecular lock promises to deliver highly effective approaches to identify and target foreign keys, but current techniques suffer from high costs, low- throughput and/or low diversity. Monoclonal antibodies can achieve strong epitope binding affinity but are expensive to produce and very labor-intensive. Nucleic acids on the other hand are much easier to handle and prepare than proteins and they can be designed to display protein-like properties (aptamers), but those are usually limited in chemical complexity to what polymerases are able to identify and process as substrates. We propose to explore an entirely new concept in which the recognition element of conventional antibodies is synthesized within a nucleic acid molecule that is folded in such a way that it presents a paratope-like structure. This nucleic acid-based scaffold would only serve to create a system of loops decorated with amino acid side-chains that are commonly found in high affinity antibodies: tyrosine (Tyr) and serine (Ser). We expect to be able to measure strong binding interactions with minimal amino acid use not only because Tyr and Ser usually dominate in paratope groups, but also because we would prepare very complex combinatorial libraries of antibody analogs by employing high- throughput, parallel nucleic acid synthesis. To do so, microarray photolithography would allow us to obtain several hundreds of thousands of unique synthetic antibodies using automated nucleic acid assembly and phosphoramidite chemistry and, importantly, to assay each and every individual member of this family independently in a single binding event. In addition, a major advantage of this method is the ability to scan the binding landscape, revealing the peaks and troughs of the binding strength across the entire paratope without discontinuity. Candidates for Minimal synthetic Antibody Analogs on Nucleic Acid Scaffolding (MAANAS) are amenable to further refinement by prospecting around a core of critical tyrosine and/or serine side-chain locations. MAANAS are simple to synthesize owing to the extremely robust phosphoramidite coupling reaction, and can be derivatized with alternative functional groups in a straightforward manner. MAANAS have the potential to transform the bioengineering of antibodies by providing a platform to explore nucleic/amino acid chimeras without relying on enzymatic reactions and in a completely cell- free environment. Because MAANAS can be produced on demand, rapidly and have very long shelf- lives, they will become a powerful tool for biochemists and synthetic biologists to study, manipulate and redesign the mechanism of paratope-epitope interaction.

New nucleobases, going beyond the ACGT alphabet, have been introduced into DNA. These modified bases contain functional arms that carry the side chain of serine and tyrosine amino acids, which is expected to impart novel function into DNA. We were able to grow DNA chips containing these new bases and found that these modifications can affect the stability and the binding affinity to given protein targets. We can therefore now perform high-throughput synthesis of chemically modified DNA analogs as a way to massively increase the speed at which we can screen and select for improved DNA variants.