Using antibodies to control DNA-templated chemical reactions

In the complex cellular environment, precise control of chemical reactivity is crucial to avoid undesired side reactions. Nature achieves this through mechanisms that co-localize biomolecules, increasing their effective local concentrations and triggering reactions otherwise improbable at low cellular concentrations. Inspired by this, artificial compartmentalization of reactants has been explored using various approaches, including molecular containers and templates. DNA-templated synthesis (DTS) is a versatile templating method utilizing the predictable Watson-Crick interactions of DNA to co-localize reactive groups conjugated to oligonucleotide sequences, enabling reactions under highly dilute conditions. DTS offers advantages in terms of predictability, cost, and ease of conjugation, finding applications in sensing, drug release, and small-molecule drug discovery. However, a limitation is its reliance solely on nucleic acids as templating agents. This research aims to overcome this limitation by demonstrating a strategy for controlling DNA-templated chemical reactions using specific IgG antibodies as co-templating agents. This leverages both the bivalent binding of IgG antibodies and the versatility of nucleic acids as scaffolds to conjugate reactive groups and recognition molecules, enabling synthetic antibody-directed chemical reactions with potential applications in clinical diagnosis and drug delivery.

The paper extensively reviews the existing literature on compartmentalization strategies for controlling chemical reactivity, highlighting the use of molecular containers like capsules, boxes, zeolites, covalent organic frameworks, and metal-organic frameworks. It emphasizes the advantages of template-based approaches, particularly DNA-templated synthesis (DTS), and its applications in various fields, including nucleic acid detection, drug release, and small-molecule drug discovery. The authors point out the limitations of DTS, which is reliant on nucleic acids for templating. They highlight the potential benefits of using other biomolecules, including clinically relevant biomarkers, to enhance the versatility and applicability of DTS.

The researchers designed a strategy for antibody-directed DNA-templated synthesis. This involved conjugating a reactive group and an antigen to each of a pair of complementary DNA oligonucleotides, designed such that duplex formation is only favored upon antibody binding. The bivalent binding of the antibody co-localizes the oligonucleotides, bringing the reactive groups into close proximity and increasing the reaction rate. To optimize this process, they systematically investigated different lengths of complementary domains in the oligonucleotides, assessing the impact on duplex stability and reaction efficiency. This involved using FRET (Fluorescence Resonance Energy Transfer) pairs to monitor duplex formation, both with an antibody-mimic DNA strand and with specific IgG antibodies. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and phosphoramidate ligation were employed as model reactions. The reaction yields were determined using denaturing PAGE (polyacrylamide gel electrophoresis) and ESI-MS (Electrospray Ionization-Mass Spectrometry). Kinetic modelling was used to further analyze the reaction dynamics and relate the observed yields to the dissociation constants of the oligonucleotide duplexes. The study also performed control experiments using Fab fragments, competitive inhibitors, and non-specific antibodies to confirm the role of bivalent antibody binding in driving the reaction. Furthermore, the applicability of the method to a clinically-relevant system was demonstrated by the antibody-directed synthesis of a thrombin-inhibiting aptamer, whose functionality was confirmed using fibrinogen clotting assay.

The authors successfully demonstrated antibody-directed DNA-templated synthesis. They found that the optimal length of the complementary domain in the oligonucleotide pairs was crucial for achieving efficient antibody-mediated reaction enhancement. Using an antibody-mimic DNA strand, they showed a clear correlation between the duplex stability and the reaction yield, with optimal results observed for intermediate duplex lengths. The CuAAC reaction was successfully triggered by specific IgG antibodies, with no product formation observed in the absence of the antibody or when using non-specific antibodies or Fab fragments. The reaction yield was concentration-dependent, reaching a maximum at a specific antibody concentration. This approach was shown to be versatile and adaptable to different types of antibodies and reactions. Importantly, they demonstrated the successful antibody-directed synthesis of a functional thrombin-inhibiting aptamer, highlighting the potential therapeutic applications of the method. The orthogonal nature of the system was proven by successful execution of two different reactions in the same solution, each triggered by a different specific antibody. Kinetic modelling provided a framework to understand and predict the reaction yields as a function of relevant parameters. The results obtained with both the antibody-mimic and real antibodies were fitted well by the model.

The study successfully demonstrates the use of IgG antibodies to control DNA-templated chemical reactions, overcoming a critical limitation of traditional DTS by incorporating protein biomarkers into the templating process. The findings highlight the versatility and orthogonality of the method, showcasing its potential for synthesizing clinically relevant molecules. The successful synthesis of a functional thrombin-inhibiting aptamer reinforces the practical implications of this technology. The ability to control multiple reactions simultaneously with different antibodies in a single solution opens exciting possibilities for complex biochemical manipulations. The work lays the foundation for the development of in vivo targeted therapies and diagnostics, where drug synthesis is precisely controlled by the presence of specific biomarkers. The modular nature of the approach allows for easy adaptation to different antibodies and reactions, broadening its potential application scope. Future research could focus on expanding the range of reactions and antibodies, optimizing reaction conditions for in vivo applications, and exploring additional applications in various fields of medicine and biotechnology.

This research introduces a novel strategy for controlling DNA-templated chemical reactions using specific IgG antibodies as co-templating agents. The method is versatile, orthogonal, and adaptable to various antibodies and reactions. Successful synthesis of a functional thrombin-inhibiting aptamer demonstrated the potential for therapeutic applications. Future work could focus on in vivo applications and exploring the potential for more complex molecular synthesis using this technique.

The study primarily focused on in vitro experiments. Further research is needed to adapt and optimize the method for in vivo applications, considering factors like antibody stability, delivery mechanisms, and potential interactions with the biological environment. The current system uses relatively small antigens; investigations into larger and more complex antigens might be needed to assess the generalizability of the method. Although the kinetic model provides a good fit for the observed data, more complex models might be needed to account for additional factors influencing reaction kinetics and specificity in a more diverse set of experimental conditions.