ABSTRACT
Thrombin-binding aptamer (TBA) is a 15-nt DNA oligomer that efficiently inhibits thrombin. It has been shown that TBA folds into an anti-parallel unimolecular G-quadruplex. Its three-dimensional chair-like structure consists of two G-tetrads connected by TT and TGT loops. TBA undergoes fast degradation by nucleases in vivo. To improve the nuclease resistance of TBA, a number of modified analogs have been proposed. Here, we describe anomeric modifications of TBA. Non-natural α anomers were used to replace selected nucleotides in the loops and core. Significant stabilization of the quadruplex was observed for the anomeric modification of TT loops at T4 and T13. Replacement of the core guanines either prevents quadruplex assembly or induces rearrangement in G-tetrads. It was found that the anticoagulant properties of chimeric aptamers could be retained only with intact TT loops. On the contrary, modification of the TGT loop was shown to substantially increase nuclease resistance of the chimeric aptamer without a notable disturbance of its anticoagulant activity.
Subject(s)
Aptamers, Nucleotide/chemistry , G-Quadruplexes , Anticoagulants/chemistry , Anticoagulants/pharmacology , Aptamers, Nucleotide/pharmacology , Base Sequence , Circular Dichroism , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Thermodynamics , Thrombin TimeABSTRACT
A series of DNA aptamers bearing triazole internucleotide linkages that bind to thrombin was synthesized. The novel aptamers are structurally analogous to the well-known thrombin-inhibiting G-quadruplexes TBA15 and TBA31. The secondary structure stability, binding affinity for thrombin and anticoagulant effects of the triazole-modified aptamers were measured. A modification in the central loop of the aptamer quadruplex resulted in increased nuclease resistance and an inhibition efficiency similar to that of TBA15. The likely aptamer-thrombin binding mode was determined by molecular dynamics simulations. Due to their relatively high activity and the increased resistance to nuclease digestion imparted by the triazole internucleotide linkages, the novel aptamers are a promising alternative to known DNA-based anticoagulant agents.
Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/pharmacology , Nucleotides/chemistry , Serine Proteinase Inhibitors/pharmacology , Thrombin/antagonists & inhibitors , Thrombin/metabolism , Triazoles/chemistry , Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/metabolism , Binding Sites , Dose-Response Relationship, Drug , G-Quadruplexes , Molecular Conformation , Molecular Dynamics Simulation , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/metabolism , Structure-Activity Relationship , Thrombin/chemistry , Triazoles/pharmacologyABSTRACT
New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was shown to protect the oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested as PCR primers. Modifications remote from the 3'-terminus were tolerated by polymerases. Our results suggest that these new oligonucleotide analogues are among the most promising triazole DNA mimics characterized to date.
Subject(s)
DNA/chemistry , Molecular Probes/chemical synthesis , Oligonucleotides/chemistry , Triazoles/chemistry , Deoxyribonucleases/chemistry , Hydrolysis , Molecular Mimicry , Nucleic Acid Denaturation , Nucleic Acid Hybridization , Polymerase Chain ReactionABSTRACT
A simple and stereoselective synthesis of a protected 4-(aminomethyl)-1-(2-deoxy-ß-D-ribofuranosyl)-1,2,3-triazole cyanoethyl phosphoramidite was developed for the modification of synthetic oligonucleotides. The configuration of the 1,2,3-triazolyl moiety with respect to the deoxyribose was unambiguously determined in ROESY experiments. The aminomethyl group of the triazolyl nucleotide was fully functional in labelling reactions. Furthermore, the hybridization behavior of 5' triazole-terminated oligonucleotide was similar to that of 5' aminohexyl-terminated oligomer with the same sequence. Internal modifications of the oligonucleotide strands resulted in significant decrease of duplex stability.
Subject(s)
Click Chemistry/methods , Nucleosides/chemistry , Organophosphorus Compounds/chemistry , Triazoles/chemistry , Click Chemistry/economics , Cyclization , Nucleosides/chemical synthesis , Organophosphorus Compounds/chemical synthesis , Stereoisomerism , Triazoles/chemical synthesisABSTRACT
Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5'-GT-2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3' (R(2AP)CW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5'-CTCAGTCTACGC GAA GCGTAGACTGAG-3' (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R(2AP)CW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19 degrees C to 33 degrees C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R(2AP)CW triplex and up to six YOYO molecules to CW hairpin.
Subject(s)
Benzoxazoles/chemistry , DNA/chemistry , Fluorescent Dyes/chemistry , Intercalating Agents/chemistry , Quinolinium Compounds/chemistry , Base Sequence , Molecular Structure , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oligonucleotides/chemistry , ThermodynamicsABSTRACT
DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.
