The Varkud Satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free energy relationship measurements with molecular dynamics, molecular solvation theory, and
ab initio
quantum mechanical/molecular mechanical free energy simulations to gain insight into catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfers in the transition state. Further, we provide evidence for a critical Mg
2+
ion in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg
2+
adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the VS, hairpin, and hammerhead ribozyme classes.
We describe a customizable approach to cancer therapy in which a gold nanoparticle (Au-NP) delivers a drug that is selectively activated within the cancer cell by the presence of an mRNA unique to the cancer cell. Fundamental to this approach is the observation that the amount of drug released from the Au-NP is proportional to both the presence and abundance of the cancer cell specific mRNA in a cell. As proof-of-principle, we demonstrate both the efficient delivery and selective release of the multi-kinase inhibitor dasatinib from Au-NPs in leukemia cells with resulting efficacy in vitro and in vivo. Furthermore, these Au-NPs reduce toxicity against hematopoietic stem cells and T-cells. This approach has the potential to improve the therapeutic efficacy of a drug and minimize toxicity while being highly customizable with respect to both the cancer cell specific mRNAs targeted and drugs activated.
Endonucleolytic ribozymes constitute a class of non-coding RNAs that catalyze single strand RNA scission. With crystal structures available for all of the known ribozymes, a major challenge involves relating functional data to the physically observed RNA architecture. In the case of the HDV ribozyme, there are three high-resolution crystal structures, the product state of the reaction and two precursor variants, with distinct mechanistic implications. Here, we develop new strategies to probe the structure and catalytic mechanism of a ribozyme. First, we use double mutant cycles to distinguish differences in functional group proximity implicated by the crystal structures. Second, we use a corrected form of the Brønsted equation to assess the functional significance of general acid catalysis in the system. Our results delineate the functional relevance of atomic interactions inferred from structure, and suggest that the HDV ribozyme transition state resembles the cleavage product in the degree of proton transfer to the leaving group.
Two synthetic approaches to linear dasatinib-DNA conjugates via click chemistry are described. One approach involves the reaction of excess azido dasatinib derivative with 5'-(5-hexynyl) tagged DNAs and the other involves the reaction of excess alkynyl linked dasatinib with 5'-azido tagged DNA. The second approach using alkynyl derived dasatinib and 5'-azido tagged DNA yielded the corresponding dasatinib-DNA conjugates in higher yield (47% versus 10-33% for the first approach). Studies have shown these linear dasatinib-DNA conjugates derived gold nanoparticles exhibit efficacy against leukemia cancer cells with reduced toxicity toward normal cells compared to free dasatinib.
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