Biology subjects

Hoy, A.

Publications and source records attributed to Hoy, A..

3 recordsLinked to original sources

bioRxiv2025

Transcriptome-wide mapping reveals an RNA-dependent mechanism of platinum cancer drugs.

Off-target interactions frequently compromise the clinical utility of anticancer agents by driving dose-limiting toxicity and therapeutic resistance. Although RNA has been predicted to be an off-target for numerous FDA-approved drugs, the extent and functional significance of RNA off-targeting among anticancer small molecules remain poorly understood. Using a systematic drug-binding screen, we identified cisplatin, a frontline chemotherapeutic that acts canonically through DNA adduct formation, as a prominent RNA binder. We employed cisplatin as a model compound to characterize the mechanistic basis and functional impact of RNA-small molecule off-targeting. To map transcriptome-wide cisplatin-RNA interactions, we developed PlatRNA-seq, a click-chemistry-enabled RNA-binding profiling platform. Genomic and functional analyses reveal that cisplatin preferentially accumulates at RNA G-quadruplex (rG4) structures near 5' transcript ends, inducing R-loop formation. Critically, we demonstrate that cisplatin cytotoxicity is partially mediated through RNA binding, revealing a noncanonical mechanism of action. Collectively, these findings illustrate the functional consequences of RNA-small-molecule off-targeting and provide a generalizable framework for investigating small-molecule-RNA interactions, opening new avenues for therapeutic innovation.

molecular biology↗

bioRxiv2024

Allosteric modulation of protein kinase A in individuals affected by NLPD-PKA , a neurodegenerative disease in which the RIβ-L50R variant is expressed

Protein kinase A (PKA) is a crucial signaling enzyme in neurons, with its dysregulation being implicated in neurodegenerative diseases. Assembly of the PKA holoenzyme, comprising a dimer of heterodimers of regulatory (R) and catalytic (C) subunits, ensures allosteric regulation and functional specificity. Recently, we defined the RI{beta}-L50R variant as a causative mutation that triggers protein aggregation in a rare neurodegenerative disease. However, the mechanism underlying uncontrolled PKA allosteric regulation and its connection to the functional outcomes leading to clinical symptoms remain elusive. In this study, we established an in vitro model using patient-derived cells for a personalized approach and employed direct measurements of purified proteins to investigate disease mechanisms in a controlled environment. Structural analysis and circular dichroism spectroscopy revealed that cellular proteins aggregation resulted from misfolded RI{beta}-subunits, preventing holoenzyme assembly and anchoring through A Kinase Anchoring Proteins (AKAPs). While maintaining high affinity to the C subunit, the resulting RI{beta}-L50R:C heterodimer exhibits reduced cooperativity, requiring lower cAMP concentrations for dissociation. Consequently, there was an increased translocation of C-subunit into the nucleus, impacting gene expression. We successfully controlled C subunit translocation by introducing a mutation that decreased RI{beta}:C dissociation in response to elevated cAMP levels. This research thus sets the stage for developing therapeutic strategies that modulate PKA assembly and allostery, thus exerting control over the unique molecular signatures identified in the disease-associated transcriptome profile.

cell biology↗

bioRxiv2022

Bio-orthogonal chemistry-based conjugation strategy facilitates investigation of impacts of s2U, s4U, m1A and m6A guide RNA modifications on CRISPR activity.

The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential towards treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on incorporation of modifications into the single guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as m1A, m6A, s2U and s4U. The impacts of these RNA modifications on overall CRISPR activity was investigated in vitro and in Cas9-expressing HEK293T cells.

biochemistry↗