Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation.

RNA interactome studies have revealed that hundreds of zinc-finger proteins (ZFPs) are candidate RNA-binding proteins (RBPs), yet their RNA substrates and functional significance remain largely uncharacterized. Here, we present a systematic multi-omics analysis of the DNA- and RNA-binding targets and regulatory roles of more than 100 ZFPs representing 37 zinc-finger families. We show that multiple ZFPs are previously unknown regulators of RNA splicing, alternative polyadenylation, stability, or translation.

Discovery and Evaluation of TLR-Targeted Immune Agonists.

Toll-like receptor (TLR) activation converts immunologically inactive tumors into immunologically active tumors by activating tumor residing antigen-presenting cells and recruitment of cytotoxic T lymphocytes. Targeted immune agonists (TIAs) are antibody drug conjugates with small-molecule TLR agonist payloads. The mechanism of action of TIAs involves tumor antigen recognition, Fcγ-receptor-dependent phagocytosis, and TLR-mediated activation to drive tumor killing by myeloid cells.

Design and Optimization of Selectivity-Tunable Toll-like Receptor 7/8 Agonists as Novel Antibody-Drug Conjugate Payloads.

Toll-like receptors 7 and 8 are involved in modulating the adaptive and innate immune responses, and their activation has shown promise as a therapeutic strategy in the field of immuno-oncology. While systemic exposure to TLR7/8 agonists can result in poor tolerance, combination therapies and targeted delivery through antibody-drug conjugates (ADCs) can help mitigate adverse effects. Described herein is the identification of a novel and potent series of pyrazolopyrimidine-based TLR7/8 agonists with tunable receptor selectivity.

High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.