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25 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS	Alu RNA pseudoknot alterations influence SRP9/SRP14 association Daniel Gussakovsky , Mira J. F. Brown , Higor Sette Pereira , Markus Meier , Gay P. Padilla-Meier , Nicole A. Black , Evan P. Booy , Jörg Stetefeld , Trushar R. Patel , Sean A. Mckenna Rna DOI: 10.1261/rna.080359.124 Diamond Proposal Number(s): [26855] Show Abstract ▼ Abstract: There are over 1 million Alu elements in the human genome which can be transcribed into discrete, RNA polymerase III transcribed non-coding Alu RNAs. These Alu RNAs often interact with and are regulated by the protein heterodimer SRP9/SRP14. This interaction is dependent on a 5’ pseudoknot domain in the Alu RNA that is thought to be held together by a canonical nucleotide triad within a U-turn motif. Herein, we discover a significant reduction in BC200 expression after mutation of a critical guanosine in the U-turn motif within its pseudoknot domain. We studied a recently discovered short human Alu RNA, EB120 that lacked the canonical Alu RNA U-turn nucleotide triad. We tested the expression of EB120 in 18 different human cell lines and tissues. EB120 was found to lack association with SRP9/SRP14 in a cellular context. Small angle X-ray scattering followed by atomistic computation structure prediction suggests the BC200 Alu domain and its U-turn mutant both possess a canonical Alu RNA fold, while EB120 lacks one. Our results highlight the structural diversity of Alu RNA, and the impact mutations may have on Alu RNA function.	May 2025
B21-High Throughput SAXS	Suppressing Tymovirus replication in plants using a variant of ubiquitin Anuradha De Silva , Kihun Kim , John Weiland , Jihyun Hwang , Jacky Chung , Higor S. Pereira , Trushar R. Patel , Joan Teyra , Ankoor Patel , Mohammed M. Mira , Mazdak Khajehpour , Melvin Bolton , Claudio Stasolla , Sachdev S. Sidhu , Brian L. Mark Plos Pathogens 21 DOI: 10.1371/journal.ppat.1012899 Diamond Proposal Number(s): [26855] Open Access Show Abstract ▼ Abstract: RNA viruses have evolved numerous strategies to overcome host resistance and immunity, including the use of multifunctional proteases that not only cleave viral polyproteins during virus replication but also deubiquitinate cellular proteins to suppress ubiquitin (Ub)-mediated antiviral mechanisms. Here, we report an approach to attenuate the infection of Arabidopsis thaliana by Turnip Yellow Mosaic Virus (TYMV) by suppressing the polyprotein cleavage and deubiquitination activities of the TYMV protease (PRO). Performing selections using a library of phage-displayed Ub variants (UbVs) for binding to recombinant PRO yielded several UbVs that bound the viral protease with nanomolar affinities and blocked its function. The strongest binding UbV (UbV3) candidate had a EC50 of 0.3 nM and inhibited both polyprotein cleavage and DUB activity of PRO in vitro. X-ray crystal structures of UbV3 alone and in complex with PRO reveal that the inhibitor exists as a dimer that binds two copies of PRO. Consistent with our biochemical and structural findings, transgenic expression of UbV3 in the cytosol of A. thaliana suppressed TYMV replication in planta, with the reduction in viral load being correlated to UbV3 expression level. Our results demonstrate the potential of using UbVs to protect plants from tymovirus infection, a family of viruses that contain numerous members of significant agricultural concern, as well as other plant viruses that express functionally related proteases with deubiquitinating activity.	Jan 2025
B21-High Throughput SAXS	Development of a single‐domain antibody to target a G‐quadruplex located on the hepatitis B virus covalently closed circular DNA genome Gerardo B. Figueroa , Simmone D'Souza , Higor S. Pereira , Gunjan Vasudeva , Sara B. Figueroa , Zachary E. Robinson , Maulik D. Badmalia , Vanessa Meier‐stephenson , Jennifer A. Corcoran , Guido Van Marle , Yi Ni , Stephan Urban , Carla S. Coffin , Trushar R. Patel Journal Of Medical Virology 96 DOI: 10.1002/jmv.29692 Diamond Proposal Number(s): [26855] Open Access Show Abstract ▼ Abstract: To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.	Jun 2024
B21-High Throughput SAXS	Factor-dependent internal ribosome entry site and -1 programmed frameshifting signal in the bemisia-associated dicistrovirus 2 Yihang Chen , Subash Chapagain , Jodi Chien , Higor Sette Pereira , Trushar R. Patel , Alice K. Inoue-Nagata , Eric Jan Viruses 16 DOI: 10.3390/v16050695 Open Access Show Abstract ▼ Abstract: The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.	Apr 2024
B21-High Throughput SAXS	tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut Natalie Krahn , Jingji Zhang , Sergey V. Melnikov , Jeffery Tharp , Alessandra Villa , Armaan Patel , Rebecca J. Howard , Haben Gabir , Trushar R. Patel , Jörg Stetefeld , Joseph Puglisi , Dieter Söll Nucleic Acids Research DOI: 10.1093/nar/gkad1188 Diamond Proposal Number(s): [22113] Open Access Show Abstract ▼ Abstract: Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G–U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G–C base pairs (G3:C70 and G5:C68).	Nov 2023
B21-High Throughput SAXS	Mapping and characterization of G‐quadruplexes in monkeypox genomes Higor Sette Pereira , Darren L. Gemmill , M. Quadir Siddiqui , Gunjan Vasudeva , Trushar R. Patel Journal Of Medical Virology 95 DOI: 10.1002/jmv.28783 Diamond Proposal Number(s): [26855] Open Access Show Abstract ▼ Abstract: Monkeypox virus (MPXV) is a double-stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non-endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost-effective antivirals could ease severe health outcomes. G-quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic-scale mapping of different MPXV isolates highlighted two conserved putative quadruplex-forming sequences MPXV-exclusive in 590 isolates. Subsequently, we assessed the G-quadruplex formation using circular dichroism spectroscopy and solution small-angle X-ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4-binding partners—Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small-molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G-quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G-quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.	May 2023
B21-High Throughput SAXS	Investigating RNA–RNA interactions through computational and biophysical analysis Tyler Mrozowich , Sean M. Park , Maria Waldl , Amy Henrickson , Scott Tersteeg , Corey r. Nelson , Anneke De Klerk , Borries Demeler , Ivo l. Hofacker , Michael t. Wolfinger , Trushar R. Patel Nucleic Acids Research 24 DOI: 10.1093/nar/gkad223 Diamond Proposal Number(s): [26855] Open Access Show Abstract ▼ Abstract: Numerous viruses utilize essential long-range RNA–RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA–RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA–RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA–RNA interaction using size-exclusion chromatography coupled with multi-angle light scattering and analytical ultracentrifugation. Next, we demonstrate that the 5′ and 3′ terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA–RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA–RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.	Mar 2023
B21-High Throughput SAXS	Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription Scott Tersteeg , Tyler Mrozowich , Amy Henrickson , Borries Demeler , Trushar R. Patel Biochemistry And Cell Biology 7 DOI: 10.1139/bcb-2022-0118 Diamond Proposal Number(s): [26855] Open Access Show Abstract ▼ Abstract: Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.	Aug 2022
B21-High Throughput SAXS	Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats Michael H D'Souza , Tyler Mrozowich , Maulik D. Badmalia , Mitchell Geeraert , Angela Frederickson , Amy Henrickson , Borries Demeler , Michael t. Wolfinger , Trushar R. Patel Nucleic Acids Research 50, 5881 - 5898 DOI: 10.1093/nar/gkac414 Diamond Proposal Number(s): [26855] Open Access Show Abstract ▼ Abstract: Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.	Jun 2022
B21-High Throughput SAXS	Targeting Xist with compounds that disrupt RNA structure and X inactivation Rodrigo Aguilar , Kerrie B. Spencer , Barry Kesner , Noreen F. Rizvi , Maulik D. Badmalia , Tyler Mrozowich , Jonathan D. Mortison , Carlos Rivera , Graham F. Smith , Julja Burchard , Peter J. Dandliker , Trushar R. Patel , Elliott B. Nickbarg , Jeannie T. Lee Nature DOI: 10.1038/s41586-022-04537-z Diamond Proposal Number(s): [26855, 22113] Show Abstract ▼ Abstract: Although more than 98% of the human genome is non-coding, nearly all of the drugs on the market target one of about 700 disease-related proteins. The historical reluctance to invest in non-coding RNA stems partly from requirements for drug targets to adopt a single stable conformation. Most RNAs can adopt several conformations of similar stabilities. RNA structures also remain challenging to determine. Nonetheless, an increasing number of diseases are now being attributed to non-coding RNA and the ability to target them would vastly expand the chemical space for drug development. Here we devise a screening strategy and identify small molecules that bind the non-coding RNA prototype Xist. The X1 compound has drug-like properties and binds specifically the RepA motif6 of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that RepA can adopt multiple conformations but favours one structure in solution. X1 binding reduces the conformational space of RepA, displaces cognate interacting protein factors (PRC2 and SPEN), suppresses histone H3K27 trimethylation, and blocks initiation of X-chromosome inactivation. X1 inhibits cell differentiation and growth in a female-specific manner. Thus, RNA can be systematically targeted by drug-like compounds that disrupt RNA structure and epigenetic function.	Mar 2022

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