Inhibitory properties of double helix forming circular oligonucleotides
Inhibitory properties of double helix forming circular oligonucleotidesElena Azhayeva, Alex Azhayev*, Seppo Auriola, Unni Tengvall, Arto Urtti1 and Harri Lönnberg2
Department of Pharmaceutical Chemistry and 1Department of Pharmaceutics, University of Kuopio, FIN-70211 Kuopio, Finland and 2Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
Received August 27, 1997;Revised and Accepted October 30, 1997
ABSTRACT
Several circular oligonucleotides were synthesized and characterized by electrospray ionization mass spectrometry. Experiments on termination of primer extension catalysed by DNA polymerases, Klenow fragment and Tth have demonstrated that a double helix forming circular 2'-deoxyribooligomer containing a 25mer sequence complementary to the target single-stranded DNA along with a 34mer random mismatching stretch appears to be a potent inhibitor of replication in vitro. Studies on inhibition of luciferase gene expression in a cell-free transcription-translation system have shown that a duplex forming circular 2'-deoxyribooligonucleotide containing a 25mer sequence complementary to the target mRNA and a 14mer random mismatching stretch can serve as an effective antisense compound as a standard linear complementary oligomer. Features of double helix forming circular oligonucleotides composed of 2'-deoxyribonucleosides seem to be useful for the design of new antigene and antisense agents.
Antigene (1 ) and antisense (2 -4 ) oligonucleotides have been found to display properties useful for drug design. These compounds are often chemically modified to improve their pharmacokinetics and stability. Extensive research has been focused on modification of the sugar-phosphate backbone in an attempt to prepare potential therapeutics based on phosphorothioates, methylphosphonates and 2'-O-alkylribooligomers (5 -10 ). Some reports show that excessive use of some of these modifications changes the affinity of an oligonucleotide for a target nucleic acid and, more interestingly, leads to formation of complexes of a conformation that appears to alter interaction with enzymes involved in the biochemical process of interest (10 -12 ). It is well known, for example, that although introduction of certain chemical modifications leads to an enhanced affinity of antisense compounds for RNA, their complexes with nucleic acids appear to be poor RNase H substrates. Our recent investigations on binding of triplex forming circular oligonucleotides and a hybrid complex forming looped oligonucleotides with single-stranded DNA and their influence on replication in vitro (13 ) led us to the conclusion that inhibitory effects result from the spatial structure of oligonucleotide-target complexes rather than from mere differences in their thermodynamic stability. This conclusion led to the idea of utilization of duplex forming circular oligonucleotides as mediators of certain biochemical reactions.
We report here results on the preparation and characterization of several disulphide cross-linked circular oligonucleotides. We also include some experiments employing these compounds and describing termination of primer extension catalysed by two different DNA polymerases and inhibition of luciferase reporter gene expression in a cell-free system.
The oligomers were prepared on solid support 1 bearing an ester linkage (14 ), on support 2 bearing a disulphide bond (15 ) or on commercial nucleoside-bound solid supports (Glen Research). Monomer 3, bearing an ester function, was prepared as described earlier (16 ) and monomer 4, having a disulphide bond, was a commercial product (Glen Research). 2'-Deoxyribonucleoside 3'-phosphoramidites and 2'-O-methylribonucleoside 3'-phosphoramidites were from Glen Research.
Oligomers reported here were assembled on a PE Applied Biosystems 392 DNA synthesizer on a 1 µM scale. Introduction of the cystamine residue into assembled oligonucleotides bearing ester functions was achieved with 1 M cystamine (free base) in water overnight. Free thiol groups were generated from the disulphide functions by treating with dithiothreitol in the presence of triethylamine (17 ). After isolation of oligomers bearing thiol functions on both the 5'- and the 3'-termini cross-linking was achieved as reported earlier (13 ,17 ).
Gel electrophoresis and HPLC. PAGE was carried out as described (10 ,14 ). Anion exchange HPLC to purify and characterize oligonucleotides was performed on a PolyWax LP (300 Å, 5 µm, 4.6 × 100 mm) column (PolyLC). RP HPLC was run on a Nucleosil C18 (300 Å, 5 µm, 4 × 250 mm) column (Macherey-Nagel).
Electrospray ionization mass spectrometry. Electrospray ionization mass spectrawere recorded on an LCQ quadrupole ion trap mass spectrometer equipped with an electrospray ionization source (Finnigan MAT). The spray needle was set to -2.5 kV. The spray was stabilized using a nitrogen sheath flow with the value set to 68. The stainless steel capillary was heated to 200°C. The capillary voltage was -39 V and the tube lens offset was -10 V. The full-scan mass spectra were measured using 200 ms for collection of ions in the trap; 5-12 microscans were summed. Typically 3-10 scans were averaged during measurement of each injection. The oligomers were dissolved in 80% acetonitrile containing 25 mM triethylamine. The flow was set to 3 µl/min and 2-5 µl samples containing 1-20 pmol oligonucleotide were injected into the system. The molecular weights of the oligomers were calculated using the computer program Gretas Garbos (created by W.Hines and B.W.Gibson, UCSF).
Replication termination experiments were conducted on synthetic 75mer single-stranded DNA template M1 or M2. These experiments employed Escherichia coli Klenow fragment (Pharmacia) or Tth polymerase (Hytest) and were performed as reported earlier (13 ). A mixture of 1 pmol template and 10-100 pmol inhibitory oligomer was used in this study.
Expression of luciferase was studied using a coupled transcription-translation system (TNT T7/SP6 Coupled Wheat Germ Extract System; Promega). RSV-Luc luciferase expression plasmid (0.2 µg) and different concentrations of oligonucleotides were employed. The plasmid was used in its circular form utilizing cDNA upstream of the T7 site. Reactions were performed at 30°C for 90 min, according to the kit protocol. After completion of transcription-translation the amount of luciferase expressed was verified by measuring light emission on a luminometer (Bio-Orbit) using standard luciferase assay reagent [20 mM tricine, 1.07 mM (MgCO3)4 Mg(OH)2·5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, pH 7.8, 2 µM coenzyme A, 470 µM luciferin and 530 µM ATP].
The present work demonstrates that double helix forming circular 2'-deoxyribooligonucleotides containing sequences complementary to the target nucleic acids and possessing random mismatching sequences may be employed to influence certain key biochemical processes. By tuning the structure of these compounds one can form nucleic acids complexes which would bring about either inhibition or preservation of the enzymatic reactions of interest. Additionally, these compounds may display new properties (e.g. increased stability). Although knowledge of the spatial structures of complexes of modified circular oligonucleotides with nucleic acids has yet to be gained and although certain efforts concerning proper tuning of these compounds lie ahead, we believe that interesting features of the cross-linked oligonucleotides reported here may be useful for the design of new antigene and antisense compounds.
2 Crooke,S.T. (1995) In Wolff,M.E. (ed.), Oligonucleotide Therapeutics. Burger's Medicinal Chemistry and Drug Discovery. Wiley, New York, NY, Vol. 1, pp. 863-900.
5 Uhlman,E. and Peyman,A. (1990) Chem. Rev., 90, 543-584.
6 Bischofberger,N. and Shea,R.G. (1992) In Propst,C.L. and Thomas,J. (eds), Nucleic Acid Targeted Drug Design. Marcel Dekker Inc., New York, NY, pp. 579-613.
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