DOCSLIB.ORG

Alpha-L-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 20-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules. Keywords: α-l-LNA; locked nucleic acids; antisense oligonucleotides; DMD 1. Introduction Alpha-l-locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA) with the inverted stereochemistry at C20, C30 and C40 positions (Figure1). Like LNAs, α-l-LNA also exhibits high binding affinity to complementary RNA and DNA oligonucleotides when incorporated into oligonucleotides [1,2]. Apart from this, α-l-LNA nucleotides also displayed a high degree of resistance to nucleases in addition to showing decreased cytotoxicity [3]. The favourable biophysical properties of α-l-LNA have led to several studies towards evaluating their potential, including for therapeutic applications [4–8]. Int. J. Mol. Sci. 2020, 21, 2434; doi:10.3390/ijms21072434 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2434 2 of 12 Int. J. Mol. Sci. 2020, 21, 2434 2 of 12 FigureFigure 1. Structural 1. Structural representation representation of theof the phosphorothioate phosphorothioa nucleicte nucleic acid acid analogues analogues used used in this in this study. study. Duchenne muscular dystrophy (DMD) is a serious muscle-wasting disorder, caused by lack of dystrophinDuchenne protein, muscular which dystrophy is essential (DMD) for the normalis a serious functioning muscle- ofwasting the muscle disorder, cells, caused including by musclelack of contractiondystrophin protein and movement., which is Dystrophinessential for actsthe normal as an anchor, functioning connecting of the themuscle cytoskeleton cells, including of the muscle fibrecontraction and the and extracellular movement. matrix. Dystrophin Lack of acts dystrophin as an anchor, protein connecting usually occurs the cytoskeleton due to mutations of the in muscle one or morefibre and exons the or extracellular deletion of exonsmatrix. of Lack the dystrophin of dystrophin gene protein [9]. Antisense usually oligonucleotideoccurs due to mutations (AO)-mediated in one spliceor more modulation exons or deletion has been of well exons explored of the todystrophin reinstate thegene reading [9]. Antisense frame in oligonucleotide order to produce (AO the)- partiallymediated functional splice modulation dystrophin has proteinbeen well [10 explored–12]. One to suchreinstate drug, the Exondys reading 51frame (Sarepta in order Therapeutics) to produce composedthe partially of phosphorodiamidate functional dystrophin morpholino protein oligomer [10–12] (PMO). One chemistry,such drug was, conditionallyExondys 51 approved(Sarepta inTherapeutics) 2016 by the composed US Food and of Drugphosphorodiamidate Administration (FDA)morpholino for clinical oligomer use [13(PMO)]. Although chemistry the, PMOwas drugconditionally is approved approved for DMD, in 2016 the productionby the US Food cost and of PMOs Drug isAdministration extremely high, (FDA) and importantly, for clinical use it is [13] not. compatibleAlthough the with PMO standard drug is phosphoramidite approved for DMD, chemistry. the production This problem cost could of PMO be addresseds is extremely by developing high, and shorterimportantly, phosphorothioate it is not compatible AOs, which with couldstandard help phosphoramidite reduce the cost of chemistry. the AOs. ItThis is worth problem adding could here be thataddressed a parallel by trialdeveloping conducted shorter for another phosphorothi AO candidateoate AOs Drisapersen, which could composed help ofreduc 20-OMePSe the cost chemistry of the (FigureAOs. It1 is) (BioMarinworth adding Pharmaceutical) here that a para wasllel rejected trial byconducted the FDA for [ 14 another]. Herein, AO we candidate report the Drisapersen systematic α l evaluationcomposed of of 2′--OMePS-LNA modifiedchemistry 2 (Figure0-OMePS 1) AOs(BioMarin and their Pharmaceutical) efficiency to inducewas rejected exon-skipping by the FDA in [14]mdx. mouse myotubes in vitro. Herein, we report the systematic evaluation of α-L-LNA modified 2′-OMePS AOs and their efficiency to induce exon-skipping in mdx mouse myotubes in vitro. 2. Results 2. ResultsFirst, we synthesised 20mer α-l-LNA-modified 20-OMePS AO (NAC 9078) containing three α-l-LNA nucleotides at positions 15, 18 and 20 and their corresponding fully-modified 2 -OMePS First, we synthesised 20mer α-L-LNA-modified 2′-OMePS AO (NAC 9078) containing0 three α- control AO (20mer, 3642). The AOs were then systematically truncated by removing one and two L-LNA nucleotides at positions 15, 18 and 20 and their corresponding fully-modified 2′-OMePS nucleotides respectively from the 5 and 3 ends to produce 18mer AOs (NAC 9079 with three α-l-LNA control AO (20mer, 3642). The AOs0 were0 then systematically truncated by removing one and two modification at positions 13, 15 and 18, and NAC 9080 with five α-l-LNA nucleotides at positions 2, nucleotides respectively from the 5′ and 3′ ends to produce 18mer AOs (NAC 9079 with three α-L- 7, 10, 14 and 17) and a 16mer AO (NAC 9081 with three modifications at positions 12, 14 and 16) as LNA modification at positions 13, 15 and 18, and NAC 9080 with five α-L-LNA nucleotides at well as their 2 -OMePS controls (18mer, 4036; 16mer, 4039). The binding affinity of the AOs against positions 2, 7, 010, 14 and 17) and a 16mer AO (NAC 9081 with three modifications at positions 12, 14 the complementary RNA target was examined by performing a thermal stability analysis (Table1). and 16) as well as their 2′-OMePS controls (18mer, 4036; 16mer, 4039). The binding affinity of the AOs The Dmd exon-23 skipping efficiency of the AOs was then analysed in vitro using the H-2Kb-tsA58 against the complementary RNA target was examined by performing a thermal stability analysis (H2K) mdx mouse myotubes. Briefly, the cells were plated on a 24-well plate and allowed to differentiate (Table 1). The Dmd exon-23 skipping efficiency of the AOs was then analysed in vitro using the H- into myotubes over 24 h. These cells were then transfected with the AOs using Lipofectin, a cationic 2Kb-tsA58 (H2K) mdx mouse myotubes. Briefly, the cells were plated on a 24-well plate and allowed lipid-based delivery agent, at 2.5, 5, 12.5, 25, 50 and 100 nM concentrations. The treated cells were to differentiate into myotubes over 24 h. These cells were then transfected with the AOs using collected after 24 h of incubation, and subsequently the RNA was isolated followed by RT-PCR Lipofectin, a cationic lipid-based delivery agent, at 2.5, 5, 12.5, 25, 50 and 100 nM concentrations. The analysis. The PCR products were later analysed by 2% agarose gel electrophoresis. The gel images treated cells were collected after 24 h of incubation, and subsequently the RNA was isolated followed were quantified by performing a densitometry analysis using the ImageJ software. by RT-PCR analysis. The PCR products were later analysed by 2% agarose gel electrophoresis. The 2.1.gel images Evaluation were of quantifiedα-l-LNA AOs by per to Induceforming Exon-23 a densitometry Skipping analysis using the ImageJ software. The exon-23 skipping efficiency of α-l-LNA modified 20-OMePS AOs was evaluated in parallel to the corresponding 20-OMePS control unmodified AOs of similar lengths. The 20mer NAC 9078 Int. J. Mol. Sci. 2020, 21, 2434 3 of 12 Int. J. Mol. Sci. 2020, 21, 2434 Table 1. Sequences used in this study. 3 of 12 AO Name Sequence (5′-3′ direction) Tm (°C) NAC 9078 (20mer) GGC CAA ACC UCG GCαT UAαC CαT 65.2 containing three α-l-LNA nucleotides with a melting temperature (Tm) of 65.2 ◦C (Table1) showed efficient exon-23 skippingNAC 9079 (Figure (18mer)2 ). TheGCC exon AAA skipping CCU CGG e ffiαCciencyUαT AC wasαC similar64.2 or even better when NAC 9080 (18mer) GαCC AAA αCCU αCGG CαTU AαCC 68.8 directly compared with the corresponding 20-OMePS AOs (Figure2A).
Load more

Recommended publications
  • 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]
  • Locked Nucleic Acid: Modality, Diversity, and Drug Discovery
    Downloaded from orbit.dtu.dk on: Sep 23, 2021 Locked nucleic acid: modality, diversity, and drug discovery Hagedorn, Peter H.; Persson, Robert; Funder, Erik D.; Albæk, Nanna; Diemer, Sanna L.; Hansen, Dennis J.; Møller, Marianne R; Papargyri, Natalia; Christiansen, Helle; Hansen, Bo R. Total number of authors: 13 Published in: Drug Discovery Today Link to article, DOI: 10.1016/j.drudis.2017.09.018 Publication date: 2018 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Hagedorn, P. H., Persson, R., Funder, E. D., Albæk, N., Diemer, S. L., Hansen, D. J., Møller, M. R., Papargyri, N., Christiansen, H., Hansen, B. R., Hansen, H. F., Jensen, M. A., & Koch, T. (2018). Locked nucleic acid: modality, diversity, and drug discovery. Drug Discovery Today, 23(1), 101-114. https://doi.org/10.1016/j.drudis.2017.09.018 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
    [Show full text]
  • A Short Adaptive Path from DNA to RNA Polymerases
    A short adaptive path from DNA to RNA polymerases Christopher Cozensa, Vitor B. Pinheiroa, Alexandra Vaismanb, Roger Woodgateb, and Philipp Holligera,1 aMedical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; and bSection on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Edited by John Kuriyan, University of California, Berkeley, CA, and approved April 10, 2012 (received for review December 21, 2011) DNA polymerase substrate specificity is fundamental to genome long (14) and more commonly stall at +6–7 nt (14–16, 18) even integrity and to polymerase applications in biotechnology. In the after prolonged incubation. At the same time, there is compel- current paradigm, active site geometry is the main site of specificity ling structural and phylogenetic evidence for an adaptive path control. Here, we describe the discovery of a distinct specificity linking DNA to RNA polymerase activity in the evolution of the checkpoint located over 25 Å from the active site in the polymerase single-subunit RNA polymerases (ssRNAPs) of mitochondria thumb subdomain. In Tgo, the replicative DNA polymerase from and T-odd bacteriophages (e.g., T7 RNA polymerase), which are Thermococcus gorgonarius, we identify a single mutation (E664K) thought to derive from an ancestral polA-family DNA poly- within this region that enables translesion synthesis across a tem- merase (19–22). Thus, we (17) and others (6, 10, 16) have argued plate abasic site or a cyclobutane thymidine dimer. In conjunction that there must be a determinant of polymerase substrate spec- with a classic “steric-gate” mutation (Y409G) in the active site, ificity that has remained unidentified and that precludes syn- E664K transforms Tgo DNA polymerase into an RNA polymerase thesis of longer RNAs in the steric-gate mutants.
    [Show full text]
  • Locked Nucleic Acid Technologytm: a Brief Overview
    Locked Nucleic Acid TechnologyTM: A brief overview Jesper Wengel Nucleic Acid Center University of Southern Denmark 5230 Odense M Denmark 2 LNATM Structure EXIQON Nucleic acid duplexes fall into two major conformational types, the A-type and the B-type, dictated by the puckering of the single nucleotides, a C3´-endo (N-type) conformation in the A-type and a C2´- | Locked Nucleic Acid Technology™ Nucleic | Locked endo (S-type) conformation in the B-type (Figure 1) (Saenger, 1984). The A-type is adopted by RNA when dsRNA duplex regions are found, whereas the dsDNA in the genome adopt a B-type. Targeting DNA and RNA with high binding affinity can be effectuated by a conformational restriction in an N-type conformation. In LNA, the O2´ and the C4´ atoms are linked by a methylene group hereby introducing a conformational lock of the molecule into a near perfect N-type conformation. We now define LNA as oligonucleotides containing one or more of the 2´-O,4´-C-methylene-β-D-ribofuranosyl nucleosides called LNA monomers (Figure 1). Figure 1 OH OH O O Base Base Base Base O O O O HO O OH O O O ˉO P O S-type N-type ˉO P O Figure 1. Nucleoside conformations and the structure and locked conformation of LNA monomers. A major structural characteristic of LNA is its close resemblance to the natural nucleic acids. This leads to easy handling as LNA sequences have similar physical properties, including water solubility. Furthermore, LNA sequences are synthesised by the conventional phosphoramidite chemistry allowing automated synthesis of fully modified LNA-sequences as well as chimeras with DNA, RNA, modified monomers or labels.
    [Show full text]
  • Opportunities and Challenges in the Delivery of Mrna-Based Vaccines
    pharmaceutics Review Opportunities and Challenges in the Delivery of mRNA-Based Vaccines Abishek Wadhwa , Anas Aljabbari , Abhijeet Lokras , Camilla Foged and Aneesh Thakur * Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark; [email protected] (A.W.); [email protected] (A.A.); [email protected] (A.L.); [email protected] (C.F.) * Correspondence: [email protected]; Tel.: + 45-3533-3938; Fax: +45-3533-6001 Received: 28 December 2019; Accepted: 26 January 2020; Published: 28 January 2020 Abstract: In the past few years, there has been increasing focus on the use of messenger RNA (mRNA) as a new therapeutic modality. Current clinical efforts encompassing mRNA-based drugs are directed toward infectious disease vaccines, cancer immunotherapies, therapeutic protein replacement therapies, and treatment of genetic diseases. However, challenges that impede the successful translation of these molecules into drugs are that (i) mRNA is a very large molecule, (ii) it is intrinsically unstable and prone to degradation by nucleases, and (iii) it activates the immune system. Although some of these challenges have been partially solved by means of chemical modification of the mRNA, intracellular delivery of mRNA still represents a major hurdle. The clinical translation of mRNA-based therapeutics requires delivery technologies that can ensure stabilization of mRNA under physiological conditions. Here, we (i) review opportunities and challenges in the delivery of mRNA-based therapeutics with a focus on non-viral delivery systems, (ii) present the clinical status of mRNA vaccines, and (iii) highlight perspectives on the future of this promising new type of medicine.
    [Show full text]
  • Michip) Based on Locked Nucleic Acids (LNA
    JOBNAME: RNA 12#5 2006 PAGE: 1OUTPUT: Saturday April 115:14:46 2006 cshl/RNA/111792/RNA23324 Downloaded from rnajournal.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Asensitive array formicroRNA expression profiling (miChip) based on lockednucleic acids (LNA) MIRCOCASTOLDI, 1,3 SABINE SCHMIDT, 2 VLADIMIR BENES, 2 MIKKEL NOERHOLM, 5 ANDREAS E. KULOZIK,1,4 MATTHIAS W. HENTZE, 3,4 and MARTINA U. MUCKENTHALER1,4 1 Department of Pediatric Oncology,Hematology and Immunology, University of Heidelberg, Germany 2 Genomics Core Facility, 3 Gene Expression Unit, and 4 Molecular Medicine Partnership Unit, EMBL, Heidelberg, Germany 5 Research and Development, Exiqon, Vedbaek, Denmark ABSTRACT MicroRNAs represent aclass of short ( ; 22 nt), noncoding regulatory RNAs involved in development, differentiation, and metabolism. We describe anovel microarray platform for genome-wide profiling of mature miRNAs (miChip) using locked nucleic acid (LNA)-modified capture probes. The biophysical properties of LNA were exploited to design probe sets for uniform, high-affinity hybridizations yielding highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. MiChip will greatly simplify miRNA expression profiling of biological and clinical samples. Keywords: microRNAs; microarrays; LNA; RNA translation; mouse INTRODUCTION agivencell or tissue (Doench and Sharp 2004;deMoor et al. 2005). The accurate profiling of miRNA expressionthus MicroRNAs (miRNAs) constitute aclass of recently dis- represents an important tooltoinvestigate physiological covered short regulatory RNAs that control gene expression and pathophysiological states. post-transcriptionallyin, e.g., development, differentiation, Different methodologies havebeenused to profile and metabolism (Krichevsky et al.2003; Abbott et al.
    [Show full text]
  • The Ability of Locked Nucleic Acid Oligonucleotides to Pre-Structure The
    University of Southern Denmark The ability of locked nucleic acid oligonucleotides to pre-structure the double helix A molecular simulation and binding study Xu, You; Gissberg, Olof; Pabon-Martinez, Y Vladimir; Wengel, Jesper; Lundin, Karin E; Smith, C I Edvard; Zain, Rula; Nilsson, Lennart; Villa, Alessandra Published in: PLOS ONE DOI: 10.1371/journal.pone.0211651 Publication date: 2019 Document version: Final published version Document license: CC BY Citation for pulished version (APA): Xu, Y., Gissberg, O., Pabon-Martinez, Y. V., Wengel, J., Lundin, K. E., Smith, C. I. E., Zain, R., Nilsson, L., & Villa, A. (2019). The ability of locked nucleic acid oligonucleotides to pre-structure the double helix: A molecular simulation and binding study. PLOS ONE, 14(2), [e0211651]. https://doi.org/10.1371/journal.pone.0211651 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to [email protected] Download date: 07. Oct. 2021 RESEARCH ARTICLE The ability of locked nucleic acid oligonucleotides to pre-structure the double helix: A molecular simulation and binding study You Xu1¤a, Olof Gissberg2, Y.
    [Show full text]
  • Cooperative Enhancement of Deoxyribozyme Activity by Chemical Modification and Added Cationic Copolymer
    論文 / 著書情報 Article / Book Information Title Cooperative enhancement of deoxyribozymeactivity by chemical modification and addedcationic copolymer Authors Ken Saito, Naohiko Shimada, ATSUSHI MARUYAMA Citation Science and Technology of Advanced Materials, Vol. 17, , p. 437-442 Pub. date 2016, 6 DOI http://dx.doi.org/10.1080/14686996.2016.1208627 Creative Commons See the 2nd page. Powered by T2R2 (Tokyo Institute Research Repository) Science and Technology of Advanced Materials ISSN: 1468-6996 (Print) 1878-5514 (Online) Journal homepage: http://www.tandfonline.com/loi/tsta20 Cooperative enhancement of deoxyribozyme activity by chemical modification and added cationic copolymer Ken Saito, Naohiko Shimada & Atushi Maruyama To cite this article: Ken Saito, Naohiko Shimada & Atushi Maruyama (2016) Cooperative enhancement of deoxyribozyme activity by chemical modification and added cationic copolymer, Science and Technology of Advanced Materials, 17:1, 437-442, DOI: 10.1080/14686996.2016.1208627 To link to this article: http://dx.doi.org/10.1080/14686996.2016.1208627 © 2016 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis Accepted author version posted online: 04 Jul 2016. Published online: 29 Jul 2016. Submit your article to this journal Article views: 46 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tsta20 Download by: [Tokyo Institute of Technology] Date: 29 August 2016,
    [Show full text]
  • (SIRC) Shuttles Mirnas, Sirnas, and Oligonucleotides to the Nucleus
    A stress-induced response complex (SIRC) shuttles miRNAs, siRNAs, and oligonucleotides to the nucleus Daniela Castanottoa,1, Xiaowei Zhanga, Jessica Alluinb, Xizhe Zhangc, Jacqueline Rügera, Brian Armstrongd, John Rossib, Arthur Riggsc, and C. A. Steina,b,1 aDepartment of Medical Oncology, City of Hope, Duarte, CA 91010; bDepartment of Molecular and Cellular Biology, City of Hope, Duarte, CA 91010; cDepartment of Diabetes & Metabolic Diseases, City of Hope, Duarte, CA 91010; and dDepartment of Neuroscience, City of Hope, Duarte, CA 91010 Edited by Marvin H. Caruthers, University of Colorado at Boulder, Boulder, CO, and approved May 7, 2018 (received for review December 11, 2017) Although some information is available for specific subsets of concentrations of arsenite [arsenic trioxide (As III)] results in miRNAs and several factors have been shown to bind oligonucle- greatly increased ON, siRNA, or miRNA targeting and sup- otides (ONs), no general transport mechanism for these molecules pression of nuclear RNAs, demonstrating the validity of our has been identified to date. In this work, we demonstrate that the findings. The fine-tuning of this process may be of extreme im- nuclear transport of ONs, siRNAs, and miRNAs responds to cellular portance for the manipulation of miRNAs, siRNAs, and ONs in stress. Furthermore, we have identified a stress-induced response a therapeutic setting. complex (SIRC), which includes Ago-1 and Ago-2 in addition to the transcription and splicing regulators YB1, CTCF, FUS, Smad1, Smad3, Results and Smad4. The SIRC transports endogenous miRNAs, siRNAs, and ONs Bind Shuttling Proteins That Are Involved in the Cell Stress ONs to the nucleus.
    [Show full text]
  • Development of Oligonucleotide Based Artificial Ribonucleases 2ʼ-O-Meoban’S and Pnazymes
    From DEPARTMENT OF BIOSCIENCES AND NUTRITION Karolinska Institutet, Stockholm, Sweden Development of Oligonucleotide Based Artificial Ribonucleases 2ʼ-O-MeOBAN’s and PNAzymes Merita Murtola Stockholm 2009 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Larserics Digital Print. © Merita Murtola, 2009 ISBN 978-91-7409-725-2 to my parents ABSTRACT The present thesis is based on three parts. The first and second part describe the development of two different classes of metal-ion dependent artificial ribonucleases, 2'- O-methyl-ribonucleic acid based artificial ribonucleases (2'-O-MeOBANs) and peptide nucleic acid based artificial ribonucleases (PNAzymes). These studies may be regarded as a development of traditional antisense methodology. Antisense mediated inhibition of gene expression can be achieved by antisense oligonucleotide hybridization with the target mRNA causing a steric blocking or generating a substrate for the endogenous RNase H. In the case of artificial ribonucleases the catalytic transestrification unit is covalently attached to the oligonucleotide scaffold and this enables the catalytic cleavage of target mRNA without the assistance of cellular enzymes and allows for a larger variety of modifications to the oligonucleotide. The developed 2'-OMeOBANs and PNAzymes are individually tailored artificial enzymes capable of sequence selective cleavage of target RNA. The general basis for the systems is that these artificial enzymes selectively hybridize with the target RNA, which brings the catalytic group to the vicinity of the scissile phosphodiester linkage thus inducing cleavage of the RNA. Several 2'-O-MeOBANs and PNAzymes have been developed and evaluated with respect to cleavage of a model of the leukemia related M-BCR/ABL mRNA.
    [Show full text]
  • Locked and Unlocked Nucleosides in Functional Nucleic Acids
    Molecules 2011, 16, 4511-4526; doi:10.3390/molecules16064511 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Review Locked and Unlocked Nucleosides in Functional Nucleic Acids Holger Doessing and Birte Vester * Nucleic Acid Center, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark; E-Mail: [email protected] (H.D.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +45-6550-2406; Fax: +45-6550-2467. Received: 3 May 2011; in revised form: 19 May 2011 / Accepted: 25 May 2011 / Published: 27 May 2011 Abstract: Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes. Keywords: locked nucleic acid (LNA); unlocked nucleic acid (UNA); aptamer; ribozyme; deoxyribozymes (DNAzyme) 1. Introduction Ribozymes, DNAzymes, and aptamers, collectively referred to as ‘functional nucleic acids’, are RNA or DNA structures with sequence-specific folds. These functional nucleic acids achieve their tertiary folds and activity through a combination of different molecular interactions and motifs: Hydrogen bonds, hydrophobic interactions, van der Waals forces, canonical and non-canonical base pairs, base stacking, coaxial stacking, tetraloops, G-quadruplexes, and metal ion coordination [1,2]. However, the use of nucleic acids in therapeutics and bio- and nanotechnologies is troubled by denaturation and/or biodegradation of the nucleic compounds.
    [Show full text]