Optimizing the Chemiluminescence of a Light-Producing Deoxyribozyme.

Supernova is a chemiluminescent deoxyribozyme recently discovered in our group. It transfers the phosphate group from the 1,2-dioxetane substrate CDP-Star to its 5' hydroxyl group, which triggers a decomposition reaction and the production of light. Here we investigated the effects of reaction conditions on the ability of Supernova to generate a chemiluminescent signal (using a plate reader assay) and to phosphorylate itself (using a ligation assay).

Overlapping but distinct: a new model for G-quadruplex biochemical specificity.

G-quadruplexes are noncanonical nucleic acid structures formed by stacked guanine tetrads. They are capable of a range of functions and thought to play widespread biological roles. This diversity raises an important question: what determines the biochemical specificity of G-quadruplex structures? The answer is particularly important from the perspective of biological regulation because genomes can contain hundreds of thousands of G-quadruplexes with a range of functions.

General and Modular Strategy for Designing Potent, Selective, and Pharmacologically Compliant Inhibitors of Rhomboid Proteases.

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude.

Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases.

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme.

Altered biochemical specificity of G-quadruplexes with mutated tetrads.

A fundamental motif in canonical nucleic acid structure is the base pair. Mutations that disrupt base pairs are typically destabilizing, but stability can often be restored by a second mutation that replaces the original base pair with an isosteric variant. Such concerted changes are a way to identify helical regions in secondary structures and to identify new functional motifs in sequenced genomes.