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Chemical cross-linking has been widely used to modify the physical and chemical properties of materials. Molecularly imprinted polymers (MIPs) are a subclass of cross-linked polymers with designable binding sites that make them highly useful in a broad range of chemical and biological applications. Computational efforts to model, characterize, and design cross-linked polymers are limited in part due to challenges in obtaining their matrix-like and probabilistic structures experimentally. Computational prediction of polymer cross-linking is resource-intensive and underexplored. Here, we propose LNKD (Linking Nodes in KD-trees), a resource-efficient algorithm for predicting pairs of reactive atoms in pre-cross-linked 3D structures of monomers that applies not only to the modeling of MIPs, but also chemical cross-linking in other materials. LNKD performs a spatial query around all reactive atoms in a structure and uses a cross-linking probability function to predict pairs of atoms most likely to form cross-links. Additionally, we introduce a protocol for modeling molecularly imprinted nanoparticles (MINPs), a type of MIP, that combines molecular dynamics simulations with LNKD. We validate the method by its accurate modeling of MINPs and their binding properties in comparison to experimental results. For the MINPs tested, LNKD found cross-linking pairs for 88–95% of the 780 total reactive atoms in approximately three seconds on a laptop and docking results reproduce experimental trends in ligand binding. 

We have developed a non-cationic transfection vector in the form of bottlebrush polymer-antisense oligonucleotide (ASO) conjugates. Termed pacDNA (polymer-assisted compaction of DNA), these agents show improved biopharmaceutical characteristics and antisense potency in vivo while suppressing non-antisense side effects. Nonetheless, there still is a lack of the mechanistic understanding of the cellular uptake, subcellular trafficking, and gene knockdown with pacDNA. Here, we show that the pacDNA enters human non-small cell lung cancer cells (NCI-H358) predominantly by scavenger receptor-mediated endocytosis and macropinocytosis and trafficks via the endolysosomal pathway within the cell. The pacDNA significantly reduces a target gene expression (KRAS) in the protein level but not in the mRNA level, despite that the transfection of certain free ASOs causes ribonuclease H1 (RNase H)-dependent degradation of KRAS mRNA. In addition, the antisense activity of pacDNA is independent of ASO chemical modification, suggesting that the pacDNA always functions as a steric blocker. 

The mutant form of the guanosine triphosphatase (GTPase) KRAS is a key driver in human tumors but remains a challenging therapeutic target, making KRAS MUT cancers a highly unmet clinical need. Here, we report a class of bottlebrush polyethylene glycol (PEG)–conjugated antisense oligonucleotides (ASOs) for potent in vivo KRAS depletion. Owing to their highly branched architecture, these molecular nanoconstructs suppress nearly all side effects associated with DNA–protein interactions and substantially enhance the pharmacological properties of the ASO, such as plasma pharmacokinetics and tumor uptake. Systemic delivery to mice bearing human non–small-cell lung carcinoma xenografts results in a significant reduction in both KRAS levels and tumor growth, and the antitumor performance well exceeds that of current popular ASO paradigms, such as chemically modified oligonucleotides and PEGylation using linear or slightly branched PEG. Importantly, these conjugates relax the requirement on the ASO chemistry, allowing unmodified, natural phosphodiester ASOs to achieve efficacy comparable to that of chemically modified ones. Both the bottlebrush polymer and its ASO conjugates appear to be safe and well tolerated in mice. Together, these data indicate that the molecular brush–ASO conjugate is a promising therapeutic platform for the treatment of KRAS -driven human cancers and warrant further preclinical and clinical development.