Cyclohexenyl nucleic acids: conformationally flexible oligonucleotides

Cyclohexenyl nucleic acid (CeNA) is a nucleic acid mimic, where the (deoxy)ribose sugar has been replaced by cyclohexenyl moieties. In order to study the conformation of cyclohexenyl nucleosides by NMR, the HexRot program was developed to calculate conformations from scalar coupling constants of cyclohexenyl compounds, analogous to the methods applied for (deoxy)ribose nucleosides. The conformational equilibria and the values of the thermodynamic parameters are very similar between a cyclohexenyl nucleoside [energy difference between (2)H(3) (N-type) and (2)H(3) (S-type) is 1.8 kJ/mol and equilibrium occurs via the eastern hemisphere with a barrier of 10.9 kJ/mol] and a natural ribose nucleoside (energy difference between N-type and S-type is 2 kJ/mol and equilibrium occurs via the eastern hemisphere with a barrier of 4–20 kJ/mol). The flexibility of the cyclohexenyl nucleoside was demonstrated by the fast equilibrium between two conformational states that was observed in a CeNA-U monomer, combined with the (2)H(3) conformation of the cyclohexene moiety when incorporated into a Dickerson dodecamer and the (2)H(3) conformation when incorporated in a d(5′-GCGT*GCG-3′)/d(5′-CGCACGC-3′) duplex, as determined by the NMR spectroscopy. This represents the first example of a synthetic nucleoside that adopts different conformations when incorporated in different double-stranded DNA sequences

The naturally occurring N6-threonyl adenine in anticodon loop of Schizosaccharomyces pombe tRNA(i) causes formation of a unique U-turn motif

Modified nucleosides play an important role in structure and function of tRNA. We have determined the solution structure of the anticodon stem–loop (ASL) of initiator tRNA of Schizosaccharomyces pombe. The incorporation of N6-threonylcarbamoyladenosine at the position 3′ to the anticodon triplet (t(6)A37) results in the formation of a U-turn motif and enhances stacking interactions within the loop and stem regions (i.e. between A35 and t(6)A37) by bulging out U36. This conformation was not observed in a crystal structure of tRNAi including the same modification in its anticodon loop, nor in the solution structure of the unmodified ASL. A t(6)A modification also occurs in the well studied anti-stem–loop of lys-tRNA(UUU). A comparison of this stem–loop with our structure demonstrates different effects of the modification depending on the loop sequence

Structural investigations of alpha-homo DNA/RNA, CeNA/RNA and CeNA/DNA.

Het onderzoek naar gemodificeerde nucleïnezuren is een breed onderzoeksg ebied waarin we verschillende deelgebieden kunnen onderscheiden. Eerst i s er het onderzoek naar de modificaties die voorkomen in de natuur. Maar daarnaast werd er ook reeds een overweldigende hoeveelheid gemodificeer de nucleïnezuren gesynthethiseerd. De studie van deze gemodificeerde str ucturen is grotendeels toegespits op moleculen (1) die RNA en DNA naboot sen maar daarnaast enkele verbeterde eigenschappen vertonen zoals sterke re hybrydisatie en een hogere stabiliteit ten opzichte van afbraak door nucleasen, (2) waaraan extra functionaliteit is toegevoegd en (3) molecu len die ons informatie kunnen geven over de etiologie van nucleïnezuren. Structurele studies van gemodificeerde nucleïnezuren hebben substantiëel bijgedragen tot het beter begrijpen van de structuur/activiteit relatie van deze moleculen. In het labo voor medicinale chemie te Leuven werden verschillende nucleïnezuren die een modificatie bevatten, aangemaakt do or chemische synthese en de structurele eigenschappen ervan werden onder zocht met behulp van NMR spectroscopie. Een eerste molecule waarvan de eigenschappen werden onderzocht is a-homo -DNA. In dit molecule is de nucleobase op het anomere koolstof atoom van een pyranose ring geplaatst, terwijl de hydroxyl groep en de hydroxymet hyl groep respectievelijk op positie 4 en 5 geplaatst zijn. De structuur bepaling van een a-homo-DNA/RNA duplex bracht aan het licht dat a-homo-D NA en RNA hybridizeren tot een nieuw soor helix, verschillend van de sta ndaard A- of B-helix. De nieuwe helix wordt gekenmerkt door helix parame ters die sterk verschillen van nauurlijke duplexen. Een tweede interessant gemodificeerd molecule waarin de natturlijke ribo se ring vervangen werd door een cyclohexeen ring, is CeNA (Cyclohexene n ucleic acid). NMR studies konden aantonen dat CeNA nucleotiden flexibele moleculen zijn, net zoals DNA en RNA nucleotiden. De structuurophelderi ng van verschillende DNA duplexen met een geïncorporeerd CeNA nucleotide met behulp van NMR toonde aan dat het nucleotide verschillende conforma ties kan aannemen wanneer het zich bevindt in verschillende DNA sequenti es. Een volledige CeNA/RNA duplex vertoonde een helix structuur die zich bevindt tussen de A- en B-helix maar meer aansluit bij de A-helix. Een laatste gemodificeerd nucleïnezuur dat bestudeerd werd is een Dicker son dodecameer waarin T8 vervangen werd door een 3 O-b-D-ribofuranosyl-2 -deoxy-thimidine. Deze modificatie zorgt voor een extra ribose in de ru ggengraat van het dodecameer. Na structuuropheldering met behulp van NMR werd duidelijk dat de extra ribose tussen T8 en C9 slechts kleine veran deringen teweeg brengt in de structuur van het dodecameer en dat de nieu we ruggengraat in staat is om de normale fosfodiester brug goed na te bo otsen.DANKWOORD iii PUBLICATION LIST v ABBREVIATIONS vii Chapter 1: INTRODUCTION 1 1.1 RNA and DNA, the code of life 1 1.1.1 From DNA to proteins 1 1.1.2 Non coding RNA 2 1.2 DNA and RNA, structural characteristics 4 1.2.1 Chemical structure 4 1.2.2 The double helix 7 1.2.3 Other secondary and tertiary structures of RNA 12 1.3 Modified oligonucleotides 13 1.3.1 Research interests 13 1.3.2 Structure of modified oligonucleotides 20 Chapter 2: NMR OF NUCLEIC ACIDS 31 2.1 Introduction 31 2.2 NMR experiments 32 2.2.1 The second dimension 32 2.2.2 Homonuclear COrrelation SpectroscopY (COSY) 33 2.2.3 HETeronuclear CORrelation spectroscopy (HETCOR) 35 2.2.4 Nuclear Overhauser and Exchange SpectroscopY (NOESY) 36 2.2.5 TOtal COrrelation SpectroscopY (TOCSY) 38 2.3 NMR of Nucleic Acids 39 2.3.1 Assignment strategies 39 2.3.2 Generating restraints 44 Chapter 3: AIMS OF THIS STUDY 47 Chapter 4: CYCLOHEXENE NUCLEIC ACIDS (CENA) 49 4.1 Introduction 49 4.2 Structural characterisation of cyclohexenyl nucleotides 50 4.2.1 Puckering parameters of a cyclohexene ring 50 4.2.2 Calculation of potential energy surfaces of cyclohexenyl nucleosides: 52 4.2.3 Determination of five conformational parameters of cyclohexenyl nucleosides 56 4.2.4 Development of the HexRot program: 56 4.2.5 Determination of thermodynamic parameters of cyclohexenyl nucleosides by NMR spectroscopy. 58 4.3 Structure determination of a DNA duplex with an incorporated cyclohexenyl nucleotide 60 4.3.1 Assignment of the Resonance signals 61 4.3.2 Generation of experimental restraints 63 4.3.3 Structure determination of the duplex 65 4.4 Structure determination of an RNA/CeNA duplex 68 4.4.1 Assignment of the Resonance signals 68 4.4.2 Generation of experimental restraints 69 4.4.3 Structure determination of the duplex 71 4.5 Discussion 73 4.6 Materials and Methods 77 Chapter 5: -HOMO-DNA 81 5.1 Introduction 81 5.2 Structure determination of an RNA/-homo-DNA duplex 83 5.2.1 Assignment of the Resonance signals 84 5.2.2 Generation of experimental restraints 86 5.2.3 Structure determination of the duplex 88 5.3 Discussion 90 5.4 Materials and Methods 92 Chapter 6: DISACCHARIDE NUCLEOSIDES 95 6.1 Introduction 95 6.1.1 Free disaccharide nucleosides 95 6.1.2 Disaccharides incorporated in oligonucleotides 101 6.2 Structural characterization of an oligonucleotide containing 3'-O--D-ribofuranosyl-2'-deoxyribonucleosides 103 6.2.1 Structural characterization of the monomer 106 6.2.2 Assignment of the resonance signals 109 6.2.3 Generation of experimental restraints 112 6.2.4 Structure determination of the duplex 113 6.3 Materials and Methods 116 Chapter 7: GENERAL CONCLUSIONS 119 SUMMARY 121 SAMENVATTING 123 APPENDIX 125 REFERENCE LIST 137nrpages: 148status: publishe

Structure of the alpha-homo-DNA:RNA duplex and the Function of Twist and Slide To Catalogue Nucleic Acid Duplexes

High-resolution NMR studies of an alpha-homo-DNA:RNA duplex reveal the formation of a right-handed parallel-oriented helix. It differs significantly from a standard A- or B-type helix by a small twist value (26.2 degrees ), which leads to a helical pitch of 13.7 base pairs per helical turn, a negative inclination (-1.78 A) and a large x displacement (5.90 A). The rise (3.4 A) is similar to that found in B-DNA. The solution of this new helix structure has stimulated us to develop a mathematical and geometrical model based on slide and twist parameters to describe nucleic acid duplexes. All existing duplexes can be positioned within this landscape, which can be used to understand the helicalization process.status: publishe

Pre-microRNA binding aminoglycosides and antitumor drugs as inhibitors of Dicer catalyzed microRNA processing

Over-expressions of miRNAs are being increasingly linked with many diseases including different types of cancer. In this study, the role of some known small molecular therapeutics has been investigated for their ability to bind with the pre-miRNA target (hsa-mir-155) and thereby to interfere with the Dicer catalyzed miRNA processing. Potential binding and inhibition effects have been demonstrated by some of these analogs. They can be used as leads for further development of potent small molecular miRNA-antagonists.status: publishe

Structural and Binding Study of Modified siRNAs with the Argonaute 2 PAZ Domain by NMR Spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17 % compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.status: publishe

The naturally occurring N6-threonyl adenine in anticodon loop of Schizosaccharomyces pombe tRNAi causes formation of a unique U-turn motif

Modified nucleosides play an important role in structure and function of tRNA. We have determined the solution structure of the anticodon stem-loop (ASL) of initiator tRNA of Schizosaccharomyces pombe. The incorporation of N6-threonylcarbamoyladenosine at the position 3' to the anticodon triplet (t6A37) results in the formation of a U-turn motif and enhances stacking interactions within the loop and stem regions (i.e. between A35 and t6A37) by bulging out U36. This conformation was not observed in a crystal structure of tRNAi including the same modification in its anticodon loop, nor in the solution structure of the unmodified ASL. A t6A modification also occurs in the well studied anti-stem-loop of lys-tRNA(UUU). A comparison of this stem-loop with our structure demonstrates different effects of the modification depending on the loop sequence.status: publishe

Disaccharide nucleosides, an important group of natural compounds

status: publishe