DOCSLIB.ORG

Mini-Review: Current Strategies to Knockdown Long Non-Coding Rnas Kim a Lennox and Mark a Behlke* Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mini-Review: Current Strategies to Knockdown Long Non-Coding Rnas Kim a Lennox and Mark a Behlke* Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA Lennox KA, Behlke MA. J Rare Dis Res Treat. (2016) 1(3): 66-70 Journal of www.rarediseasesjournal.com Rare Diseases Research & Treatment Mini Review Open Access Mini-review: Current strategies to knockdown long non-coding RNAs Kim A Lennox and Mark A Behlke* Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA Article Info Text Article Notes The discovery of functional long non-coding RNAs (lncRNAs) Received: September 12, 2016 has challenged the paradigm that most RNAs encode proteins. Accepted: November 25, 2016 LncRNAs are an abundant class of RNAs >200 nt in length that were *Correspondence: previously assumed to be transcriptional noise. It is now widely Dr. Mark A Behlke, Integrated DNA Technologies, Inc., accepted that some of these lncRNAs have diverse regulatory roles Coralville, IA, 52241, USA, Tel: 319-626-8432; Fax: 319- in gene expression, can serve as structural components for shaping 626-8466, Email: [email protected] nuclear organization and can even act as decoys for other RNAs © 2016 Mark A Behlke. This article is distributed under the or proteins1-3. While tens of thousands of lncRNA transcripts have terms of the Creative Commons Attribution 4.0 International been cataloged, the biological function of the majority of lncRNAs License. still remains a mystery. Determining the individual functionality Keywords of members of this recently discovered class of RNAs is important Long non-coding RNAs lncRNA for better understanding of developmental biology, evolution and Noncoding RNA genetic disease. Antisense RNAi 4 siRNA distributions that are critical for their function . Some lncRNAs CRISPR are Similarenriched to inproteins, the nucleus individual and are lncRNAs involved have in specific regulating subcellular nuclear Knockdown processes such as transcription and RNA processing. Other lncRNAs are enriched in the cytoplasm where they can impact protein localization or modulate mRNA stability and translation. Some lncRNAs are more equally distributed between the nucleus and the cytoplasm. The location of the primary reservoir of a lncRNA can impact the ability to knock down expression levels of that species. Three strategies are commonly used to knockdown or knockout lncRNAs, including: degradation of the RNA by RNA interference (RNAi), degradation of the RNA by RNase H activate antisense oligonucleotides (ASOs), or gross deletion/alteration at the DNA level using CRISPR/Cas9 genome editing methods (Figure 1). Each of these tools have their own advantages and disadvantages, and and the transcriptional landscape within which it resides. RNAi is a commonlysuccess can employed be influenced knockdown by the subcellular technique localization that utilizes of the the lncRNA multi- protein RNAi-induced silencing complex (RISC) to suppress mRNAs5. The human RISC loading complex (RLC), is comprised of three proteins (Dicer, TRBP and Ago2) responsible for processing longer dsRNAs into the mature siRNAs and loading these siRNAs into Ago2. It has previously been demonstrated that RNAi-mediated mRNA degradation occurs in the cytoplasm6. In a series of fractionation and co-immunoprecipitation experiments, Stalder et al. more binding with its targeted mRNA and Ago2-mediated cleavage of the mRNAspecifically occurs demonstrate primarily at that the roughcanonical endoplasmic RISC loading, reticulum, subsequent where mRNAs are translated into proteins7. RNase H-mediated antisense RNA knockdown capitalizes on the endogenous RNase H1 enzyme, Page 66 of 70 Lennox KA, Behlke MA. J Rare Dis Res Treat. 2016 1(3): 66-70 Journal of Rare Diseases Research & Treatment Figure 1: Schematic of the three common methods used to knockdown or knockout lncRNAs. RNA interference (RNAi) utilizes the multi- protein RNAi-induced silencing complex (RISC) containing a siRNA to specifically degrade the targeted RNA. Antisense oligonucleotides (ASOs) bind to their targeted RNA and use endogenous RNase H1, an enzyme that cleaves the RNA in an RNA/DNA heteroduplex. CRISPR-Cas9 genome editing makes alterations at the genomic level by using a target specific crRNA hybridized to the tracrRNA, which is complexed to the Cas9 protein. which is most abundant in the nucleus where it is thought RNase-H mediated antisense knockdown, since RNase to function in DNA replication and repair8-11. Alternatively, H is predominantly found in the nucleus. Conversely, we steric blocking ASOs can be used to block splice junctions thought RNAi, a mostly cytoplasmic process, might be to reduce accumulation of mature lncRNA transcripts or more effective when targeting cytoplasmic populations of block access to key functional domains without triggering lncRNAs. To test this possibility, a systematic study was degradation of the target RNA12,13. Steric blocking ASOs performed in cell culture comparing the effectiveness of ASOs (20mer DNA-PS, 20mer 2’OMe-PS gapmers or 16mer LNA-PS gapmers (Exiqon), where PS = phosphorothioate morpholinoare made of backbones chemically14. modifiedBoth RNAi residues and RNase that H-activedo not linkages, LNA = locked nucleic acid and 2’OMe = 2’-O-Methyl ASOssupport rely RNase upon H1 naturally cleavage, present such as effector2’-modified molecules ribose orto RNA) or RNAi (DsiRNAs, siRNA, or Silencer® Select siRNAs degrade the lncRNA. In contrast, CRISPR/Cas9 genome editing methods rely upon a bacterial endonuclease variable localization patterns17. Nuclear lncRNAs (MALAT1 enzyme that can be targeted to desired sites in the genome (Thermoand NEAT1), Fisher cytoplasmic Scientific)) lncRNAs against (DANCR seven andlncRNAs OIP5-AS1) with or dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were stranded DNA breaks at or around the target site15,16. targeted by either ASOs (18 sites/target) or RNAi (28 sites/ byThe a cellularsite-specific repair guide machinery RNA where heals itthe generates double-stranded double- breaks, leaving small “scars” in the genome, or can even be depending on parameters such as GC content and target used to delete large blocks of DNA and thereby eliminate accessibility,target). As there sites is were a wide selected range by of using efficacy optimized between design sites the lncRNA at the genomic level. CRISPR/Cas9 methods can also be used to introduce new sequences at the target house design criteria (ASOs) to maximize the probability loci, such as transcriptional terminators that will prevent ofalgorithms comparing (RNAi) highly or activeusing siteswell-defined between stringentto the twoin- production of full-length lncRNAs. Understanding the knockdown strategies. The results were consistent with nature of the lncRNA of interest can better assist with our hypothesis: nuclear lncRNAs were more easily knocked selecting the optimal knockdown technique. down when using ASOs, while cytoplasmic targets were With an extensive background of mRNA knockdown more easily knocked down when using RNAi (Figure 2). experience, we expected that applying the same repertoire When comparing the potency of the various ASO chemical of reagents to suppress lncRNAs would be easily accomplished. However, we found that some lncRNAs and 2’OMe-PS ASOs had very similar knockdown patterns betweenmodification the strategies,different sites, it was the found 2’OMe-PS that while gapmers the DNA-PS were seemed to be a correlation that RNAi-based methods consistently more potent. This was expected, as the 2’OMe (smallwere difficult interfering to suppress, RNAs, siRNAs;and eventually or Dicer noticed substrate what RNAs, DsiRNAs) were less effective against nuclear- The 2’OMe-PS gapmer and LNA-PS gapmer ASOs typically localized lncRNA targets. We hypothesized that these showedbases increase similar the potency binding at affinity the 10 ofnM the dose; ASO however,to the target. the nuclear lncRNAs would be more easily suppressed using LNA-PS gapmers were often more potent at lower doses. Page 67 of 70 Lennox KA, Behlke MA. J Rare Dis Res Treat. 2016 1(3): 66-70 Journal of Rare Diseases Research & Treatment Nuclear lncRNAs 120 A 100 N R g 80 n i n i a 60 m e R 40 % 20 0 MALAT1 NEAT1 MALAT1 NEAT1 Cytoplasmic lncRNAs 120 A 100 N R g 80 n i ASOs n i 60 a m RNAi e R 40 % 20 0 DANCR OIP5-AS1 DANCR OIP5-AS1 Dual-localized lncRNAs 120 A 100 N R g 80 n i n i 60 a m e R 40 % 20 0 TUG1 CasC7 HOTAIR TUG1 CasC7 HOTAIR Figure 2: Overview of lncRNA knockdown results comparing method efficacy with lncRNA subcellular localization (17). Antisense oligonucleotides (LNA-PS gapmers, 2’OMe-PS gapmers or DNA-PS) or RNAi reagents (siRNAs, DsiRNAs or Silencer® Select siRNAs) were transfected into HeLa cells with Lipofectamine® 2000 for 24 h at 10 nM dose. ASOs = antisense oligonucleotides; PS = phosphorothioate linkages. There was no overall difference in activity between any of So far, we have commented on the “relative ease” of the various RNAi reagents tested. Duration of knockdown either knockdown technique to suppress their targets was not addressed in this study; suppression lasting 3-7 based on whether the lncRNA was predominantly nuclear days is typically seen in cell culture (where dilution from or cytoplasmic. A closer look at the data shows that ASOs cell division can be limiting by reducing concentration are also effective at degrading cytoplasmic RNAs (Figure of the ASOs or siRNAs), whereas knockdown for several 2). ASOs not only have the innate ability to target nuclear- weeks or even a month can be seen in non-dividing retained RNAs, but they can also degrade nascent RNAs cells in vivo18,19. While this study only examined lncRNA prior to cytoplasmic exportation. Also, a recent study knockdown in cell culture, similar relative activities examining the rate-limiting step of mRNA degradation between the different reagent classes would be expected by ASOs found that RNase H1 was also present in low in vivo if suitable delivery tools were employed. For in vivo levels in the cytoplasm23. While ASO-mediated mRNA use, a clear performance advantage would be predicted degradation occurred more rapidly in the nucleus, it also for the short LNA-PS gapmers if administered by naked IV occurred at a slower rate in the cytoplasm.
Load more

Recommended publications
  • Engineering Nucleic Acid Chemistry for Precise and Controllable CRISPR
    Science Bulletin 64 (2019) 1841–1849 Contents lists available at ScienceDirect Science Bulletin journal homepage: www.elsevier.com/locate/scib Review Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing ⇑ Weiqi Cai a,b, Ming Wang a,b, a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecule Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b University of Chinese Academy of Sciences, Beijing 100049, China article info abstract Article history: The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/ Received 21 June 2019 Cas9) genome editing technology is revolutionizing our approach and capability to precisely manipulate Received in revised form 16 July 2019 the genetic flow of mammalians. The facile programmability of Cas9 protein and guide RNA (gRNA) Accepted 23 July 2019 sequence has recently expanded biomedical application of CRISPR/Cas9 technology from editing mam- Available online 30 July 2019 malian genome to various genetic manipulations. The therapeutic and clinical translation potential of CRISPR/Cas9 genome editing, however, are challenged by its off-target effect and low genome editing effi- Keywords: ciency. In this regard, developing new Cas9 variants and conditional control of Cas9/gRNA activity are of CRISPR/Cas9 genome editing great potential for improving genome editing accuracy and on-target efficiency. In this review, we sum- RNA engineering Nucleic acid chemistry marize chemical strategies that have been developed recently to engineer the nucleic acid chemistry of Gene therapy gRNA to enhance CRISPR/Cas9 genome editing efficacy, specificity and controllability.
    [Show full text]
  • Patenting Interfering RNA
    Patenting Interfering RNA J. Douglas Schultz SPE Art Unit 1635 (571) 272-0763 [email protected] Oligonucleotide Inhibitors: Mechanisms of Action RNAi - Mechanism of Action • dsRNA induces sequence-specific degradation of homologous gene transcripts • RISC metabolizes dsRNA to small 21-23- nucleotide siRNAs – RISC contains dsRNase (“Dicer”), ssRNase (Argonaut 2 or Ago2) • RISC utilizes antisense strand as “guide” to find cleavable target siRNA Mechanism of Action miRNA Mechanism of Action Interfering RNA Glossary of Terms • RNAi – RNA interference • dsRNA – double stranded RNA • siRNA – small interfering RNA, double stranded, 21-23 nucleotides • shRNA – short hairpin RNA (doubled stranded by virtue of a ssRNA folding back on itself) • miRNA – micro RNA • RISC – RNA-induced silencing complex – Dicer – RNase endonuclease siRNA miRNA • Exogenously delivered • Endogenously produced • 21-23mer dsRNA • 21-23mer dsRNA • Acts through RISC • Acts through RISC • Induces homologous target • Induces homologous target cleavage cleavage • Perfect sequence match • Imperfect sequence match – Results in target degradation – Results in translation arrest RNAi Patentability issues Sample Claims: • A siRNA that inhibits expression of a nucleic acid encoding protein X. OR • A siRNA comprising a 2’-modification, wherein said modification comprises 2’-fluoro, 2’-O-methyl, or 2’- deoxy. (Note: no target recited) OR • A method of reducing tumor cell growth comprising administering siRNA targeting protein X. RNAi Patentability Issues 35 U.S.C. 101 – Utility • Credible/Specific/Substantial/Well Established. • Used to attempt modulation of gene expression in human diseases • Routinely investigate gene function in a high throughput fashion or to (see Rana RT, Nat. Rev. Mol. Cell Biol. 2007, Vol. 8:23-36).
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub
    US 20040O86860A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub. Date: May 6, 2004 (54) METHODS OF PRODUCING RNAS OF Publication Classification DEFINED LENGTH AND SEQUENCE (51) Int. Cl." .............................. C12Q 1/68; C12P 19/34 (76) Inventor: Muhammad Sohail, Marston (GB) (52) U.S. Cl. ............................................... 435/6; 435/91.2 Correspondence Address: MINTZ, LEVIN, COHN, FERRIS, GLOWSKY (57) ABSTRACT AND POPEO, PC. ONE FINANCIAL CENTER Methods of making RNA duplexes and single-stranded BOSTON, MA 02111 (US) RNAS of a desired length and Sequence based on cleavage of RNA molecules at a defined position, most preferably (21) Appl. No.: 10/264,748 with the use of deoxyribozymes. Novel deoxyribozymes capable of cleaving RNAS including a leader Sequence at a (22) Filed: Oct. 4, 2002 Site 3' to the leader Sequence are also described. Patent Application Publication May 6, 2004 Sheet 1 of 2 US 2004/0086860 A1 DNA Oligonucleotides T7 Promoter -TN-- OR 2N-2-N-to y Transcription Products GGGCGAAT-N-UU GGGCGAAT-N-UU w N Deoxyribozyme Cleavage - Q GGGCGAAT -------' Racction GGGCGAAT N-- UU N-UU ssRNA products N-UU Anneal ssRNA UU S-2N- UU siRNA product FIGURE 1: Flowchart summarising the procedure for siRNA synthesis. Patent Application Publication May 6, 2004 Sheet 2 of 2 US 2004/0086860 A1 Full-length transcript 3'-digestion product 5'-digestion product (5'GGGCGAATA) A: Production of single-stranded RNA templates by in vitro transcription and digestion With a deoxyribozyme V 2- 2 V 22inv 22 * 2 &3 S/AS - 88.8x, *...* or as IGFR -- is as 4.
    [Show full text]
  • Evaluation of Locked Nucleic Acid–Modified Small Interfering RNA in Vitro and in Vivo
    833 Evaluation of locked nucleic acid–modified small interfering RNA in vitro and in vivo Olaf R. Mook, Frank Baas, Marit B. de Wissel, model. Therefore, LNA-modified siRNA should be pre- and Kees Fluiter ferred over unmodified siRNA. [Mol Cancer Ther 2007; 6(3):833–43] Department of Neurogenetics, Academic Medical Center, Amsterdam, the Netherlands Introduction RNA interference (RNAi) is a natural process that affects Abstract gene silencing in eukaryotic systems at transcriptional, RNA interference has become widely used as an experi- posttranscriptional, and/or translational levels (1). Small mental tool to study gene function. In addition, small interfering RNA (siRNA) molecules are the key intermedi- interfering RNA (siRNA) may have great potential for the ates in this process, which can potentially inhibit the treatment of diseases. Recently, it was shown that siRNA expression of any given target gene. siRNA molecules hold can be used to mediate gene silencing in mouse models. great promise as biological tools and as potential thera- Locally administered siRNAs entered the first clinical trials, peutic agents for targeted inhibition of disease-causing but strategies for successful systemic delivery of siRNA genes. are still under development. Challenges still exist about the To optimize the use of siRNA as a therapeutic therapy, stability, delivery, and therapeutic efficacy of siRNA. In several modes of delivery have been tested in vivo. the present study, we compare the efficacy of two Improved delivery in animals has been achieved by methods of systemic siRNA delivery and the effects of complexation with cationic liposomes (2), polyethyleni- siRNA modifications using locked nucleic acids (LNA) in a mine (3), and Arg-Gly-Asp–polyethylene glycol–polyethy- xenograft cancer model.
    [Show full text]
  • Locked Nucleic Acid
    lysi ana s & io B B io f m o e l d a Journal of Mishra and Mukhopadhyay, J Bioanal Biomed 2013, 5:2 i n c r i n u e DOI: 10.4172/1948-593X.1000e114 o J ISSN: 1948-593X Bioanalysis & Biomedicine Editorial OpenOpen Access Access Locked Nucleic Acid (LNA)-based Nucleic Acid Sensors Sourav Mishra and Rupa Mukhopadhyay* Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700 032, India In recent times, much developments in the field of ‘nucleic acid may prevent interactions with the solid substrates, [15] and it can have sensors’, especially using alternative nucleic acid probes like peptide multiple water bridges that provide it with extra stability compared to nucleic acid (PNA) and locked nucleic acid (LNA), have taken place. DNA or RNA. It has been shown that both the highest Tm increase Although most of these are concerned with ‘solution phase’ studies, per LNA modification and the best mismatch discrimination are some reports have been made on ‘on-surface’ detection. The latter type achieved for short LNA sequences [16]. In addition to that, LNA of detection is particularly important to nurture, considering clinical phosphoramidites and their oligomers are commercially available, diagnostic applications using microarrays. In this article, we’ll briefly and LNA nucleotides can be mixed with those of the natural nucleic present the primary developments reported in the past two decades, acids for generating heterogeneous probe molecules. These properties along with possibilities for future developments, in case of the LNA- of LNA hold the promise that it can be a potentially better alternative based sensors.
    [Show full text]
  • Tissue-Specific Delivery of CRISPR Therapeutics
    International Journal of Molecular Sciences Review Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors Karim Shalaby 1 , Mustapha Aouida 1,* and Omar El-Agnaf 1,2,* 1 Division of Biological and Biomedical Sciences (BBS), College of Health & Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), Doha 34110, Qatar; [email protected] 2 Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Doha 34110, Qatar * Correspondence: [email protected] (M.A.); [email protected] (O.E.-A.) Received: 12 September 2020; Accepted: 27 September 2020; Published: 5 October 2020 Abstract: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis.
    [Show full text]
  • Ribozyme-Mediated, Multiplex CRISPR Gene Editing and Crispri in Plasmodium Yoelii
    bioRxiv preprint doi: https://doi.org/10.1101/481416; this version posted November 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Ribozyme-Mediated, Multiplex CRISPR Gene Editing and CRISPRi in Plasmodium yoelii Michael P. Walker1 and Scott E. Lindner1 * 1Department of Biochemistry and Molecular Biology, the Huck Center for Malaria Research, Pennsylvania State University, University Park, PA. *Correspondence: Scott E. Lindner, [email protected] Running Title: CRISPR-RGR in Plasmodium yoelii Keywords: Plasmodium, CRISPR, CRISPRi, Ribozyme, HDR, ALBA bioRxiv preprint doi: https://doi.org/10.1101/481416; this version posted November 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Abstract 2 Functional characterization of genes in Plasmodium parasites often relies on genetic 3 manipulations to disrupt or modify a gene-of-interest. However, these approaches are limited by 4 the time required to generate transgenic parasites for P. falciparum and the availability of a 5 single drug selectable marker for P. yoelii. In both cases, there remains a risk of disrupting native 6 gene regulatory elements with the introduction of exogenous sequences. To address these 7 limitations, we have developed CRISPR-RGR, a SpCas9-based gene editing system for 8 Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) sgRNA expression strategy. 9 Using this system with P. yoelii, we demonstrate that both gene disruptions and coding sequence 10 insertions are efficiently generated, producing marker-free and scar-free parasites with homology 11 arms as short as 80-100bp.
    [Show full text]
  • Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas Reinhardtii
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations and Theses in Biological Sciences Biological Sciences, School of Spring 2013 Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii Xinrong Ma University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/bioscidiss Part of the Biology Commons, Cellular and Molecular Physiology Commons, Microbiology Commons, and the Molecular Genetics Commons Ma, Xinrong, "Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii" (2013). Dissertations and Theses in Biological Sciences. 51. https://digitalcommons.unl.edu/bioscidiss/51 This Article is brought to you for free and open access by the Biological Sciences, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations and Theses in Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII by Xinrong Ma A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biological Sciences Under the Supervision of Professor Heriberto Cerutti Lincoln, Nebraska May, 2013 SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII Xinrong Ma, Ph.D. University of Nebraska, 2013 Advisor: Heriberto Cerutti RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes, with regulatory roles in a variety of biological processes, including cell cycle, cell differentiation, physiological and metabolic pathways, and stress responses.
    [Show full text]
  • 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.
    [Show full text]
  • CRISPR/Cas9 with Single Guide RNA Expression Driven by Small Trna Promoters Showed Reduced Editing Efficiency Compared to a U6 Promoter
    Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press LETTER TO THE EDITOR CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter YUDA WEI,1,2 YAN QIU,1,2 YANHAO CHEN,1,2 GAIGAI LIU,1,2 YONGXIAN ZHANG,1,2 LUWEI XU,3 and QIURONG DING1,2 1CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 2University of Chinese Academy of Sciences, Shanghai 200031, China 3Taizhou Hospital of Traditional Chinese Medicine, Taizhou 225300, China ABSTRACT Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd and coworkers indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression. Though the underlying mechanisms are still under investigation, our study suggests that the CRISPR/Cas9 system with tRNA promoter- driven sgRNA expression needs to be reevaluated before it can be used for therapeutic genome editing. Keywords: CRISPR/Cas9; genome editing; tRNA promoter The use of clustered regularly interspaced short palindromic have a DNA packaging limit of ∼5 kb, and the standard repeats (CRISPR)/CRISPR-associated (Cas) systems for in AAV–SaCRISPR system almost fills the cargo limitation, it vivo genome editing has emerged as a potential treatment would be helpful to further reduce the total length of the for human diseases (Cox et al.
    [Show full text]
  • Alpha-L-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce
    International Journal of Molecular Sciences Article Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro Prithi Raguraman 1,2, Tao Wang 1,2, Lixia Ma 3, Per Trolle Jørgensen 4 , Jesper Wengel 4 and Rakesh N. Veedu 1,2,4,* 1 Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150 Australia; [email protected] (P.R.); [email protected] (T.W.) 2 Perron Institute for Neurological and translational Science, Perth 6005, Australia 3 School of Statistics, Henan University of Economics and Law, Zhengzhou 450001, China; [email protected] 4 Nucleic Acid Center, Department of Physics and Chemistry and Pharmacy, University of Southern Denmark, M 5230 Odense, Denmark; [email protected] (P.T.J.); [email protected] (J.W.) * Correspondence: [email protected] Received: 19 February 2020; Accepted: 29 March 2020; Published: 31 March 2020 Abstract: Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 20-O-methyl phosphorothioate (20-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 20-OMePS control AOs.
    [Show full text]
  • 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).
    [Show full text]