Publications and Presentations

Sullenger, Karen S.; Turner, R. Steven

doi:10.1007/978-94-6300-022-2_16

Cited by 3 publications

(6 citation statements)

References 15 publications

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“…RNA strand cleavage by 2′-O-transphosphorylation is universal in biology and has farreaching implications for medicine [10][11] . The goal of gaining a predictive understanding of the catalytic mechanisms of RNA cleavage reactions in the context of well-studied biological systems is important from a fundamental scientific perspective, as well as from a technology engineering viewpoint [12][13] .…”

Section: Rna Cleavage Reactionsmentioning

confidence: 99%

“…Of key interest to the present discussion is how RNA cleavage is catalyzed by small catalytic RNA molecules known as nucleolytic ribozymes [5][6] . Nucleolytic ribozymes serve as platforms for the design of new biomedical technology 13 and therapeutics [10][11] , and as models for our understanding of RNA catalysis and its implications for theories of evolution 18 . The central challenge is to gain a predictive understanding of precisely how these small RNA molecules, with their limited repertoire of chemical functional groups, are able to function as "high-speed" ribozymes 16 .…”

Section: Nucleolytic Ribozymes (Small Self-cleaving Rnas)mentioning

confidence: 99%

“…The central challenge is to gain a predictive understanding of precisely how these small RNA molecules, with their limited repertoire of chemical functional groups, are able to function as "high-speed" ribozymes 16 . Such an understanding would enable molecular engineering and guide the design of new catalytic RNA-based technology 13 and medicine [10][11] .…”

Section: Nucleolytic Ribozymes (Small Self-cleaving Rnas)mentioning

confidence: 99%

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An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes

et al. 2019

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A predictive understanding of the mechanisms of RNA cleavage is important for the design of emerging technology built from biological and synthetic molecules that have promise for new biochemical and medicinal applications. Over the past 15 years, RNA cleavage reactions involving 2'-O-transphosphorylation have been discussed using a simplified framework introduced by Breaker that consists of four fundamental catalytic strategies (designated α, β, γ, and δ) that contribute to rate enhancement. As more detailed mechanistic data emerge, there is need for the framework to evolve and keep pace. We develop an ontology for discussion of strategies of enzymes that catalyze RNA cleavage via 2'-O-transphosphorylation that stratifies Breaker's framework into primary (1°), secondary (2°) and tertiary (3°) contributions to enable more precise interpretation of mechanism in the context of structure and bonding. Further, we point out instances where atomic-level changes give rise to changes in more than one catalytic contribution, a phenomenon we refer to as 'functional blurring'. We hope that this ontology will help clarify our conversations and pave the path forward toward a consensus view of these fundamental and fascinating mechanisms. The insight gained will deepen our understanding of RNA cleavage reactions catalyzed by natural protein and RNA enzymes, as well as aid in the design of new engineered DNA and synthetic enzymes.

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Section: Rna Cleavage Reactionsmentioning

confidence: 99%

Section: Nucleolytic Ribozymes (Small Self-cleaving Rnas)mentioning

confidence: 99%

Section: Nucleolytic Ribozymes (Small Self-cleaving Rnas)mentioning

confidence: 99%

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An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes

et al. 2019

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“…Much of what is known about these mechanisms have been gleaned from detailed experimental and computational studies of small nucleolytic ribozymes that catalyze site-specific cleavage (and ligation) of RNA. [3][4][5] These ribozymes are widespread in both bacterial and human genomes [6][7][8][9] where they likely play complex roles in RNA processing and regulation of gene expression, and have impact in biotechnology, 10,11 medicine, [12][13][14] and theories into the origins of life itself. [15][16][17] In the last 5 years, the number of known naturally occurring nucleolytic ribozyme classes has roughly doubled, sparking a surge of experimental effort aimed toward their structural and functional characterization.…”

Section: Introductionmentioning

confidence: 99%

“…Twister ribozymes catalyze RNA transphosphorylation that leads to site-specific cleavage of the RNA phosphodiester backbone. This is a fundamentally important reaction in biology that is catalyzed by naturally occurring nucleolytic ribozymes (hammerhead, [20][21][22] hairpin, 23,24 HDV, 25,26 VS, 27,28 glmS, 29,30 twister, 8,31 pistol, 9,32 TS, 9,33 and hatchet 9 ribozymes), and protein enzymes (e.g., RNase A 34 ), as well as artificially engineered DNA enzymes (e.g., [8][9][10][11][12][13][14][15][16][17]. In this reaction, the 2′O makes a nucleophilic attack on the phosphorus atom of the adjacent scissile phosphate to form a pentavalent transition state (or metastable intermediate), followed by departure of the O5′ leaving group to produce 2′,3′cyclic phosphate and 5′OH cleavage products.…”

Section: Introductionmentioning

confidence: 99%

Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology

2019

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The catalytic properties of RNA have been a subject of fascination and intense research since their discovery over 30 years ago. Very recently, several classes of nucleolytic ribozymes have emerged and been characterized structurally. Among these, the twister ribozyme has been center-stage, and a topic of debate about its architecture and mechanism owing to conflicting interpretations of different crystal structures, and in some cases conflicting interpretations of the same functional data. In the present work, we attempt to clean up the mechanistic "debris" generated by twister ribozymes using a comprehensive computational RNA enzymology approach aimed to provide a unified interpretation of existing structural and functional data. Simulations in the crystalline environment and in solution provide insight into the origins of observed differences in crystal structures, and coalesce on a common active site architecture, and dynamical ensemble in solution. We use GPU-accelerated free energy methods with enhanced sampling to ascertain microscopic nucleobase pK a values of the implicated general acid and base, from which predicted activity-pH profiles can be compared directly with experiments. Next, ab initio quantum mechanical/molecular mechanical (QM/MM) simulations with full dynamic solvation under periodic boundary conditions are used to determine mechanistic pathways through multi-dimensional free energy landscapes for the reaction. We then characterize the rate-controlling transition state, and make predictions about kinetic isotope effects and linear free energy relations. Computational mutagenesis is performed to explain the origin of rate effects caused by chemical modifications and make experimentally testable predictions. Finally, we provide evidence that helps to resolve conflicting issues related to the role of metal ions in catalysis. Throughout each stage, we highlight how a conserved L-platform structural motif, to-gether with a key L-anchor residue, forms the characteristic active site scaffold enabling each of the catalytic strategies to come together not only for the twister ribozyme, but the majority of the known small nucleolytic ribozyme classes.

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Endosomolytic and Tumor-Penetrating Mesoporous Silica Nanoparticles for siRNA/miRNA Combination Cancer Therapy

Wang

Xie

Kilchrist

et al. 2020

ACS Appl. Mater. Interfaces

133

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Combination therapies consisting of multiple short therapeutic RNAs, such as small interfering RNA (siRNA) and microRNA (miRNA), have enormous potential in cancer treatment as they can precisely silence specific set of oncogenes and target multiple related disease pathways. However, clinical use of siRNA/miRNA combinations is limited by the availability of safe and efficient systemic delivery systems with sufficient tumor penetrating and endosomal escaping capabilities. This study reports on the development of multifunctional tumor-penetrating mesoporous silica nanoparticles (iMSNs) for simultaneous delivery of siRNA (siPlk1) and miRNA (miR-200c), using encapsulation of a photosensitizer indocyanine green (ICG) to facilitate endosomal escape and surface conjugation of iRGD peptide to enable deep tumor penetration. Increased cell uptake of the nanoparticles was observed in both 3D tumor spheroids in vitro and in orthotopic MDA-MB-231 breast tumors in vivo. Using a galectin-8 recruitment assay, we showed that reactive oxygen species generated by ICG upon light irradiation functioned as an endosomolytic stimulus that caused release of the siRNA/miRNA combination from endosomes. Co-delivery of the therapeutic RNAs displayed combined cell killing activity in cancer cells. Systemic intravenous treatment of metastatic breast cancer with the iMSNs loaded with siPlk1 and miR-200c resulted in a significant suppression of the primary tumor growth and in marked reduction of metastasis upon short light irradiation of the primary tumor. This work demonstrates that siRNA-miRNA combination assisted by photodynamic effect and tumor penetrating delivery system may provide a promising approach for metastatic cancer treatment.

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Publications and Presentations

Cited by 3 publications

References 15 publications

An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes

An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes

Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology

Endosomolytic and Tumor-Penetrating Mesoporous Silica Nanoparticles for siRNA/miRNA Combination Cancer Therapy

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