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Bis-Locked Nucleic Acids: a New Tool for Double Helix Invasion From Department of Laboratory Medicine Karolinska Institutet, Stockholm, Sweden BIS-LOCKED NUCLEIC ACIDS: A NEW TOOL FOR DOUBLE HELIX INVASION Sylvain Geny Stockholm 2015 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by E-Print AB 2015 © Sylvain Geny, 2015 ISBN 978-91-7676-014-7 Bis-Locked Nucleic Acids: a new tool for double helix invasion THESIS FOR DOCTORAL DEGREE (Ph.D.) By Sylvain Geny Principal Supervisor: Opponent: Prof C. I. Edvard Smith Prof Keith Fox Karolinska Institutet University of Southampton Department of Laboratory Medicine Centre for Biological Sciences Clinical Research Center Examination Board: Co-supervisor(s): Prof Joakim Lundeberg Dr Karin Lundin Kungliga Tekniska Högskolan Karolinska Institutet School of Biotechnology Department of Laboratory Medicine Division of Gene Technology Clinical Research Center Prof Matti Sällberg Dr Samir EL Andaloussi Karolinska Institutet Karolinska Institutet Department of Laboratory Medicine Department of Laboratory Medicine Division of Clinical Microbiology Clinical Research Center Assoc Prof Björn Högberg Karolinska Institutet Department of Medical Biochemistry and Biophysics To my family and my friends. ABSTRACT Deoxyribonucleic acid (DNA) is a macromolecule that contains the information for all living organisms to achieve important cellular processes such as growth and replication. DNA consists of two strands coiled around each other to form a double helix. Several interactions including Watson-Crick pairing and stacking between the bases stabilize the double helix. To ensure the integrity of genetic information, the bases are on the inside the double helix whereas the phosphate and sugar groups are on the outside. Thus, most DNA information is not accessible until DNA is unwound prior to replication or transcription. In cells, enzymes called helicases achieve DNA strands separation. The anti-gene strategy aims to introduce a short single-stranded oligonucleotide (ON) to bind genomic DNA at a specific site to block the mRNA transcription. As a result, mRNA and protein expression for a specific gene are expected to be reduced, which could be beneficial in certain clinical contexts. However, this therapeutic strategy is hampered by the low stability of the DNA binding, the difficulty to reach the genomic target and to block the RNA polymerase, suggesting the needs for new DNA binding ONs. In this thesis, we developed clamp-ONs, bis-locked nucleic (bisLNAs), binding via Hoogsteen and Watson-Crick interactions. In paper I, we assessed their binding characteristics and demonstrated their ability to invade supercoiled DNA at intranuclear pH and salt conditions. In paper II, we investigated the step-by-step mechanism for bisLNA invasion. Based on this mechanism, we designed a new generation of bisLNAs using non- natural modifications such as a stacking linker and 2´-glycylamino LNAs. In paper III, we investigated how the bisLNAs invade in a more complex environment using rolling circle amplification for detection. 1 LIST OF SCIENTIFIC PAPERS This thesis is based on the following articles I. Moreno, P.M.D.*, Geny, S.*, Pabon Y.V., Bergquist H., Zaghloul E.M., Rocha C.S., Oprea II, Bestas B., Andaloussi S.E., Jorgensen P.T, Lundin K.E, Pedersen E.B, Wengel J., Smith C.I.E. Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA, Nucleic Acids Res., 2013, 41, 5, 3257–3273 II. Geny S., Moreno P.M.D.*, Krzywkowski T.*, Andersen N.K., Isse A.J., El- Madani A.M., Lou C., Pabon Y.V., Gissberg O., Anderson B.A., Zaghloul A.M., Zain R., Hrdlicka P., Jorgensen P.T., Nilsson M., Lundin K.E., Pedersen E.B., Wengel J., Smith C.I.E. Next generation bis-locked nucleic acids with stacking linker and 2´-glycylamino-LNA show enhanced invasion into duplex DNA (manuscript submitted) III. Geny S.*, Krzywkowski T.*, Gissberg O., Lundin K.E., Pedersen E.B., Wengel J., Nilsson M., Smith C.I.E. Rolling circle amplification assisted by bis-locked nucleic acids invasion into supercoiled DNA (manuscript) *Equal contribution 2 CONTENTS 1 INTRODUCTION ........................................................................................................... 1 1.1 Gene therapy ......................................................................................................... 1 1.2 Anti-gene therapy .................................................................................................. 5 1.3 DNA structure ....................................................................................................... 8 1.4 DNA mode of binding ......................................................................................... 12 1.4.1 Triple helix formation ............................................................................ 12 1.4.2 Double helix invasion ............................................................................. 15 1.5 Oligonucleotide synthesis ................................................................................... 17 1.6 Nucleotide analogues .......................................................................................... 19 1.6.1 TFO analogues ....................................................................................... 19 1.6.2 Peptide nucleic acids .............................................................................. 20 1.6.3 Locked nucleic acids .............................................................................. 22 1.7 Intercalators ......................................................................................................... 24 2 AIMS ............................................................................................................................. 26 3 METHODOLOGY ........................................................................................................ 27 3.1 Fluorescence binding assay ................................................................................. 27 3.2 Electrophoresis mobility shift assay ................................................................... 27 3.3 S1 nuclease assay ................................................................................................ 28 3.4 Melting assay ....................................................................................................... 29 3.5 Footprinting ......................................................................................................... 30 3.6 Rolling circle amplification ................................................................................ 31 4 RESULTS, DISCUSSION AND PERSPECTIVES .................................................... 33 4.1 Paper I .................................................................................................................. 33 4.2 Paper II ................................................................................................................ 36 4.3 Paper III ............................................................................................................... 39 5 CONCLUDING REMARKS ........................................................................................ 41 6 ACKNOWLEDGMENTS ............................................................................................ 42 7 REFERENCES .............................................................................................................. 45 3 LIST OF ABBREVIATIONS A adenine ag anti-gene AGO2 argonaute 2 AR androgen receptor C cytosine ChIP chromatin immunoprecipitation CMV cytomegalovirus CRISPR clustered regularly interspaced short palindromic repeat CPG controlled pore glass Da Dalton DNA deoxyribonucleic acid DMD Duchenne muscular dystrophy DSB double-stranded break dsDNA double-stranded DNA EMSA electrophoretic mobility shift assay glyLNA 2´-glycylamino-LNA G guanine HG Hoogsteen HPLC high pressure liquid chromatography HIV human immunodeficiency virus HDR homology directed repair LNA locked nucleic acid MALDI matrix assisted laser desorption ionisation mRNA messenger RNA miRNA micro RNA MS mass spectrometry 4 NMR nuclear magnetic resonance nt nucleotide ON oligonucleotide pc pseudo complementary PEI polyethyleneimine PLP padlock probe PNA peptide nucleic acid PR progesterone RCA rolling circle amplification RCP rolling circle product RNA ribonucleic acid RNAi RNA interference RISC RNA-induced silencing complex SCID severe combined immunodeficiency siRNA silencing RNA ssDNA single-stranded DNA SSO splice-switching oligonucleotide SNA spherical nucleic acid T thymine TALEN transcription activator-like effector nuclease TFO triplex forming oligonucleotide Tm melting temperature TINA twisted intercalating nucleic acid TISH triplex in situ hybridization WC Watson-Crick ZFN zinc finger nuclease 5 1 INTRODUCTION 1.1 GENE THERAPY 1.1.1 Introduction Gene therapy can be defined as the intentional modulation of gene expression in specific cells for therapeutic purposes. The concept of gene therapy arises from between 1960s and 1970s. In this decade, it became clear that genetic diseases as well as degenerative and infectious diseases could be corrected at a genetic level [1, 2]. In 1990, the first authorized clinical trials were carried out using retroviruses to transfect ex vivo T-cells [3] or cancer cells [4] to treat severe combined immunodeficiency (SCID), caused by adenine deaminase deficiency (ADA- SCID) or melanoma without success. In 1999, an 18 years old undergoing a gene therapy trial for
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