Programmable and Conditional Proteolysis Targeting Chimeras Enabled by DNA Nanotechnology - SUMMARY/ABSTRACT Approximately 85% of the proteome is considered undruggable by traditional, occupancy-driven pharmacology employing small molecular inhibitors. Proteolysis-targeting chimeras (PROTACs) and associated molecules that induce targeted protein degradation via the ubiquitin–proteasome system have emerged as a revolutionary strategy for addressing “undruggable” targets. However, despite the groundbreaking advances and entry of some PROTACs into clinical trials, substantial challenges and considerable room for further development remain. Limitations of current PROTACs include poor cell permeability, limited availability of E3 ligands, and poor selectivity, highlighting a critical need for ongoing innovations in Targeted Protein Degradation (TPD) technology. To address these issues, the possibility of developing an efficacious drug delivery system, coupled with conditional activation of target protein degradation, stands out as a pivotal enhancement. In this context, nucleic acid-based modalities offer considerable promise for advancing TPD, building on the successful biomedical applications of nucleic acid-based drugs like antisense oligonucleotides (ASOs). The programmable and addressable nature of DNA or RNA nanostructures has been successfully harnessed, allowing for functional integration with small molecules, proteins, and peptides to realize applications in drug delivery, vaccines, and biosensors, among others. Based on these prior successes, we hypothesize that by leveraging the inherent programmability and addressability of DNA, precise and effective delivery of protein degradation modalities into cells and conditional activation of degradation can be achieved. For proof-of-concept validation, we have established an efficient cytoplasmic delivery platform utilizing a multifunctional DNA nanodevice and developed a DNA-based target protein degradation system (DTAC) capable of degrading CDK4/6 proteins at nanomolar concentrations. Building on these accomplishments, we intend to integrate our delivery platform with the DNA- based protein degradation system, aiming to pioneer the next generation of TPD technologies. Our approach aims to 1) optimize the programmability of our DNA duplex-based protein degradation system by transitioning from DNA duplex structures to multitargeting DNA nanostructures; 2) assess protein degradation variations caused by spatial distances and evaluate the efficiency of multi-target degradation; 3) integrate and optimize the cytoplasmic DNA nanostructure delivery system with the DNA-based protein degradation system; and 4) validate and optimize the conditional activation of protein degradation through two distinct design approaches: i) toehold mediated conditional activity of DTAC and ii) allosteric mediated conditional activity of DTAC.
