RNA Secondary Structure As a First Step for Rational Design of the Oligonucleotides Towards Inhibition of Influenza a Virus Replication
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Replication and Kinetic Trapping of Nucleic Acids in Alternative Environments
REPLICATION AND KINETIC TRAPPING OF NUCLEIC ACIDS IN ALTERNATIVE ENVIRONMENTS A Dissertation Presented to The Academic Faculty by Adriana Lozoya Colinas In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Chemistry and Biochemistry Georgia Institute of Technology December 2020 COPYRIGHT © 2020 BY ADRIANA LOZOYA COLINAS REPLICATION AND KINETIC TRAPPING OF NUCLEIC ACIDS IN ALTERNATIVE ENVIRONMENTS Approved by: Dr. Nicholas V. Hud, Advisor Dr. Amanda Stockton School of Chemistry and Biochemistry School of Chemistry and Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Martha A. Grover Dr. Adegboyega (Yomi) Oyelere School of Chemical & Biomolecular School of Chemistry and Biochemistry Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Loren Williams School of Chemistry and Biochemistry Georgia Institute of Technology Date Approved: October 16, 2020 ACKNOWLEDGEMENTS I would like to thank my mom and dad for all their support and setting up an example for me to follow. I really appreciate everything you have done to encourage me to succeed and follow my dreams. I want to thank Mario for always supporting me. I know it hasn’t always been easy being far away, thank you for being patient and supportive with me. Thank you for the time and adventures we lived together. To all my Latino family at Georgia Tech, thank you for making me feel closer to home. We spent a lot of time together, learned a lot from each other and shared our culture, all of which have made my PhD experience more enjoyable. I would also like to acknowledge my advisor, Nick Hud, for being supportive and sharing his passion for science with me. -
Guide for Morpholino Users: Toward Therapeutics
Open Access Journal of Drug Discovery, Development and Delivery Special Article - Antisense Drug Research and Development Guide for Morpholino Users: Toward Therapeutics Moulton JD* Gene Tools, LLC, USA Abstract *Corresponding author: Moulton JD, Gene Tools, Morpholino oligos are uncharged molecules for blocking sites on RNA. They LLC, 1001 Summerton Way, Philomath, Oregon 97370, are specific, soluble, non-toxic, stable, and effective antisense reagents suitable USA for development as therapeutics and currently in clinical trials. They are very versatile, targeting a wide range of RNA targets for outcomes such as blocking Received: January 28, 2016; Accepted: April 29, 2016; translation, modifying splicing of pre-mRNA, inhibiting miRNA maturation and Published: May 03, 2016 activity, as well as less common biological targets and diagnostic applications. Solutions have been developed for delivery into a range of cultured cells, embryos and adult animals; with development of a non-toxic and effective system for systemic delivery, Morpholinos have potential for broad therapeutic development targeting pathogens and genetic disorders. Keywords: Splicing; Duchenne muscular dystrophy; Phosphorodiamidate morpholino oligos; Internal ribosome entry site; Nonsense-mediated decay Morpholinos: Research Applications, the transcript from miRNA regulation; Therapeutic Promise • Block regulatory proteins from binding to RNA, shifting Morpholino oligos bind to complementary sequences of RNA alternative splicing; and get in the way of processes. Morpholino oligos are commonly • Block association of RNAs with cytoskeletal motor protein used to prevent a particular protein from being made in an organism complexes, preventing RNA translocation; or cell culture. Morpholinos are not the only tool used for this: a protein’s synthesis can be inhibited by altering DNA to make a null • Inhibit poly-A tailing of pre-mRNA; mutant (called a gene knockout) or by interrupting processes on RNA • Trigger frame shifts at slippery sequences; (called a gene knockdown). -
Long Non-Coding Rnas Are Largely Dispensable for Zebrafish Embryogenesis, Viability and Fertility
bioRxiv preprint doi: https://doi.org/10.1101/374702; this version posted July 23, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Long non-coding RNAs are largely dispensable for zebrafish embryogenesis, viability and fertility Mehdi Goudarzi1, Kathryn Berg1, Lindsey M. Pieper1, and Alexander F. Schier1,2,3,4,5 1Department oF Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. 2Center For Brain Science, Harvard University, Cambridge, MA 02138, USA., 3FAS Center For Systems Biology, Harvard University, Cambridge, MA 02138, USA., 4Allen Discovery Center For Cell Lineage Tracing, University oF Washington, Seattle, 5 WA 98195, USA., Biozentrum, University oF Basel, Switzerland. Correspondence should be addressed to M.G. ([email protected]) or A.F.S. ([email protected]). Hundreds oF long non-coding RNAs (lncRNAs) have been identiFied as potential regulators oF gene expression, but their Functions remain largely unknown. To study the role oF lncRNAs during vertebrate development, we selected 25 zebraFish lncRNAs based on their conservation, expression proFile or proximity to developmental regulators, and used CRISPR-Cas9 to generate 32 deletion alleles. We observed altered transcription oF neighboring genes in some mutants, but none oF the lncRNAs were required For embryogenesis, viability or Fertility. Even RNAs with previously proposed non-coding Functions (cyrano and squint) and other conserved lncRNAs (gas5 and lnc-setd1ba) were dispensable. In one case (lnc-phox2bb), absence oF putative DNA regulatory-elements, but not of the lncRNA transcript itselF, resulted in abnormal development. -
Targeting Protein Translation, RNA Splicing, and Degradation by Morpholino-Based Conjugates in Plasmodium Falciparum
Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum Aprajita Garga, Donna Wesolowskib, Dulce Alonsob,1, Kirk W. Deitschc, Choukri Ben Mamouna, and Sidney Altmanb,2 aDepartment of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; bDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; and cDepartment of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065 Contributed by Sidney Altman, August 11, 2015 (sent for review May 27, 2015; reviewed by Ron Dzikowski and Rima Mcleod) Identification and genetic validation of new targets from available same targets, MO conjugates have also been used to inhibit RNA genome sequences are critical steps toward the development of splicing and initiation of protein translation (9, 17, 18). new potent and selective antimalarials. However, no methods are To enhance cellular uptake of morpholino oligomers and other currently available for large-scale functional analysis of the Plasmo- drug-like molecules, arginine-rich peptides and polyguanidino dium falciparum genome. Here we present evidence for successful dendrimers have been used (19–23). For morpholino-based anti- use of morpholino oligomers (MO) to mediate degradation of target microbial activity, two types of conjugates have been developed, mRNAs or to inhibit RNA splicing or translation of several genes of PPMOs and vivo morpholino oligomers (VMOs, octa-guanidinium P. falciparum involved in chloroquine transport, apicoplast biogen- dendrimer-conjugated MOs; Materials and Methods). PPMOs are esis, and phospholipid biosynthesis. Consistent with their role in the produced following conjugation of a specific MO to a cell-pen- parasite life cycle, down-regulation of these essential genes resulted etrating, arginine-rich peptide, whereas VMOs are synthesized in inhibition of parasite development. -
Advances in Oligonucleotide Drug Delivery
REVIEWS Advances in oligonucleotide drug delivery Thomas C. Roberts 1,2 ✉ , Robert Langer 3 and Matthew J. A. Wood 1,2 ✉ Abstract | Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge. Oligonucleotides are nucleic acid polymers with the In addition to their ability to recognize specific tar- potential to treat or manage a wide range of diseases. get sequences via complementary base pairing, nucleic Although the majority of oligonucleotide therapeutics acids can also interact with proteins through the for- have focused on gene silencing, other strategies are being mation of three-dimensional secondary structures — a pursued, including splice modulation and gene activa- property that is also being exploited therapeutically. For tion, expanding the range of possible targets beyond example, nucleic acid aptamers are structured -
Guide for Morpholino Users: Toward Therapeutics
Open Access Journal of Drug Discovery, Development and Delivery Special Article - Antisense Drug Research and Development Guide for Morpholino Users: Toward Therapeutics Moulton JD* Gene Tools, LLC, USA Abstract *Corresponding author: Moulton JD, Gene Tools, Morpholino oligos are uncharged molecules for blocking sites on RNA. They LLC, 1001 Summerton Way, Philomath, Oregon 97370, are specific, soluble, non-toxic, stable, and effective antisense reagents suitable USA for development as therapeutics and currently in clinical trials. They are very versatile, targeting a wide range of RNA targets for outcomes such as blocking Received: January 28, 2016; Accepted: April 29, 2016; translation, modifying splicing of pre-mRNA, inhibiting miRNA maturation and Published: May 03, 2016 activity, as well as less common biological targets and diagnostic applications. Solutions have been developed for delivery into a range of cultured cells, embryos and adult animals; with development of a non-toxic and effective system for systemic delivery, Morpholinos have potential for broad therapeutic development targeting pathogens and genetic disorders. Keywords: Splicing; Duchenne muscular dystrophy; Phosphorodiamidate morpholino oligos; Internal ribosome entry site; Nonsense-mediated decay Morpholinos: Research Applications, the transcript from miRNA regulation; Therapeutic Promise • Block regulatory proteins from binding to RNA, shifting Morpholino oligos bind to complementary sequences of RNA alternative splicing; and get in the way of processes. Morpholino oligos are commonly • Block association of RNAs with cytoskeletal motor protein used to prevent a particular protein from being made in an organism complexes, preventing RNA translocation; or cell culture. Morpholinos are not the only tool used for this: a protein’s synthesis can be inhibited by altering DNA to make a null • Inhibit poly-A tailing of pre-mRNA; mutant (called a gene knockout) or by interrupting processes on RNA • Trigger frame shifts at slippery sequences; (called a gene knockdown). -
Strategies for Genetic Inactivation of Long Noncoding Rnas in Zebrafish
Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Strategies for Genetic Inactivation of Long Noncoding RNAs in Zebrafish Perrine Lavalou1, Helene Eckert1, Louise Damy1, Florian Constanty1, Sara Majello1, Angelo Bitetti1, Antoine Graindorge1, Alena Shkumatava1,* 1Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, France *Corresponding author E-mail: [email protected] (AS) ABSTRACT The number of annotated long noncoding RNAs (lncRNAs) continues to groW, hoWever their functional characterization in model organisms has been hampered by the lack of reliable genetic inactivation strategies. While partial or full deletions of lncRNA loci disrupt lncRNA expression, they do not permit the formal association of a phenotype With the encoded transcript. Here, we examined several alternative strategies for generating lncRNA null alleles in zebrafish and found that they often resulted in unpredicted changes to lncRNA expression. Removal of the transcriptional start sites (TSSs) of lncRNA genes resulted in hypomorphic mutants due to the usage of either constitutive or tissue-specific alternative TSSs. Deletions of short, deeply conserved lncRNA regions can also lead to overexpression of truncated transcripts. By contrast, a knock-in of a polyadenylation signal enabled complete inactivation of malat1, the most abundant vertebrate lncRNA. In summary, lncRNA null alleles require extensive in vivo validation and We propose insertion of transcription termination sequences as the most reliable approach to generate lncRNA- deficient zebrafish. 1 Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press INTRODUCTION Thousands of lncRNAs have been identified in multiple vertebrate species (Hezroni et al., 2015; Necsulea et al., 2014), but their biological functions remain mostly unknoWn. -
RNA Structural Motif Recognition Based on Least-Squares Distance
Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press BIOINFORMATICS RNA structural motif recognition based on least-squares distance YING SHEN,1 HAU-SAN WONG,2,4 SHAOHONG ZHANG,3 and LIN ZHANG1 1School of Software Engineering, Tongji University, Shanghai 200092, China 2Department of Computer Science, City University of Hong Kong, Kowloon, Hong Kong 3Department of Computer Science, Guangzhou University, Guangzhou 510006, China ABSTRACT RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods. Keywords: RNA structural motif; RNA motif recognition INTRODUCTION RNA molecule (i.e., the search space). There are two prevalent ways to search for RNA structural motifs. The first class of LongnoncodingRNA(ncRNA),whichconsistsof>200ntand methods is to find motifs based on their geometric features. has little or no protein-coding capability, is a remarkable class of RNAs. They are important in various biological processes NASSAM (Harrison et al. 2003) represents RNA motifs and (Dinger et al. -
Identification of Inhibitors of Ribozyme Self-Cleavage in Mammalian Cells Via High-Throughput Screening of Chemical Libraries
JOBNAME: RNA 12#5 2006 PAGE: 1OUTPUT: Saturday April 114:41:58 2006 csh/RNA/111792/RNA23004 Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Identification of inhibitors of ribozymeself-cleavage in mammalian cells via high-throughput screening of chemical libraries LAISING YEN,1 MAXIMEMAGNIER,1 RALPH WEISSLEDER,2 BRENT R. STOCKWELL,3 andRICHARD C. MULLIGAN 1 1 Department of Genetics, Harvard Medical School, and Division of Molecular Medicine, Children’sHospital, Boston, Massachusetts 02115, USA 2 Center for Molecular Imaging Research, Massachusetts GeneralHospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA 3 Department of BiologicalSciences and DepartmentofChemistry,Fairchild Center,Columbia University,New York, New York 10027, USA ABSTRACT We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established acell-based assay in which expression of a luciferase ( luc)reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. -
Cross-Talk Between Overlap Interactions in Biomolecules: a Case Study of the Β-Turn Motif
molecules Article Cross-Talk between Overlap Interactions in Biomolecules: A Case Study of the b-Turn Motif Jayashree Nagesh Solid State and Structural Chemistry Unit, Indian Institute of Science Bangalore, Bengaluru 560012, Karnataka, India; [email protected] Abstract: Noncovalent interactions play a pivotal role in regulating protein conformation, stability and dynamics. Among the quantum mechanical (QM) overlap-based noncovalent interactions, n ! p∗ is the best understood with studies ranging from small molecules to b-turns of model proteins such as GB1. However, these investigations do not explore the interplay between multiple overlap interactions in contributing to local structure and stability. In this work, we identify and characterize all noncovalent overlap interactions in the b-turn, an important secondary structural element that facilitates the folding of a polypeptide chain. Invoking a QM framework of natural bond orbitals, we demonstrate the role of several additional interactions such as n ! s∗ and p ! p∗ that are energetically comparable to or larger than n ! p∗. We find that these interactions are sensitive to changes in the side chain of the residues in the b-turn of GB1, suggesting that the n ! p∗ may not be the only component in dictating b-turn conformation and stability. Furthermore, a database search of n ! s∗ and p ! p∗ in the PDB reveals that they are prevalent in most proteins and have significant interaction energies (∼1 kcal/mol). This indicates that all overlap interactions must be taken into account to obtain a comprehensive picture of their contributions to protein structure and energetics. Lastly, based on the extent of QM overlaps and interaction energies, we propose geometric criteria using which these additional interactions can be efficiently tracked in broad database searches. -
Microrna-24A Is Required to Repress Apoptosis in the Developing Neural Retina
Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION mary miRNAs (pri-miRNAs). These transcripts are then microRNA-24a is required to processed by the enzymes Drosha and Dicer to generate repress apoptosis in the the mature single-stranded miRNA of ;22 nucleotides, which is then incorporated into the RNA-induced silenc- developing neural retina ing complex (RISC), characterized by the presence of the 1 Argonaute family of proteins (Pasquinelli et al. 2005). James C. Walker and Richard M. Harland This complex is responsible for the regulatory function of the miRNAs, leading to translational repression or Department of Molecular and Cell Biology and Center for degradation of target mRNAs. Integrative Genomics, University of California at Berkeley, Several miRNAs have been implicated in the regula- Berkeley, California 94720, USA tion of apoptosis in Drosophila (Xu et al. 2004). In various Programmed cell death is important for the proper de- forms of cancer, miR-21 has been shown to be an anti- apoptotic factor (Chan et al. 2005; Cheng et al. 2005) and velopment of the retina, and microRNAs (miRNAs) may miR-34 has been shown to be a downstream target of p53 be critical for its regulation. Here, we report that miR- and an inducer of cell death (He et al. 2007). However, 24a is expressed in the neural retina and is required for knowledge is still lacking about the in vivo roles of most correct eye morphogenesis in Xenopus. Inhibition of miRNAs during vertebrate development. Recent work in miR-24a during development causes a reduction in eye mice has shown that Dicer inactivation, specifically in size due to a significant increase in apoptosis in the ret- the retina, results in neuronal degeneration (Damiani ina, whereas overexpression of miR-24a is sufficient to et al. -
Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry-A New Method Amenable to Automated Synthesizer
Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry-A New Method Amenable to Automated Synthesizer Ujjwal Ghosh†a, Jayanta Kundu†a, Atanu Ghosh†, Arnab Das†, Dhriti Nagar¶, Aurnab Ghose¶ and Surajit Sinha†* †School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of Science, 2A and 2B Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India ¶Indian Institute of Science Education and Research, Pune, Maharashtra 411008, India aAuthors have equal contribution *Corresponding author: [email protected] KEYWORDS: Morpholino oligonucleotides, Trityl and Fmoc chemistry, DNA Synthesizer, Activators ETT and Iodine, Antisense reagents. Abstract: Phosphorodiamidatemorpholino oligo- nucleotides (PMO) are routinely used for gene silencing and the recently developed PMO-based drug “Exondys51” has highlighted the importance of PMO as excellent antisense reagents. Howev- er, the synthesis of PMO has remained challeng- ing. Here a method for the synthesis of PMO us- ing either trityl or Fmoc-protected active morpho- lino monomers using chlorophosphoramidate chemistry in the presence of a suitable coupling agent on a solid support has been reported. After screening several coupling agents (tetrazole, 1,2,4-triazole, ETT, iodine, LiBr and dicyanoimidazole), ETT and iodine were found to be suitable for efficient coupling. Fmoc chemistry was not known for PMO synthesis because the preparation of Fmoc-protected chloro- phosphoramidate monomers was not trivial. Synthesis of Fmoc-protected activated monomers and their use in PMO synthesis is reported for the first time. 25-mer PMO has been synthesized using both the methods and vali- dated in vivo in the zebrafish model by targeting the no tail gene.