Synthesis of a carboxamide linked T * T dimer and its incorporation in oligonucleotides (original) (raw)
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Nucleic Acids Research, 1999
We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (U P). The intramolecular triplex A 6-L-T 6-L-(U P) 5 T (L = two octanediol residues) shows a single UV-melting transition which is >20_ higher than that of the parent triplex A 6-L-T 6-L-T 6 at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by U P , in a pH dependent fashion. Triplex stability increases by ∼12 K for each T to U P substitution. Quantitative footprinting studies have examined the interaction of three U P-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5′-AGGAAG-AGAAAAAAGAA. At pH 5.0, the U P-containing oligonucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (U P) 6 CU P T binds ∼1000-fold more tightly than the unmodified oligonucleotide T 6 CTT. Oligonucleotides containing fewer U P residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.
Tetrahedron, 1996
5′-O-(4,4′-dimethoxytrityl)-5-methyl-N4-(1-pyrenylmethyl)cytidine(5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(1,2,4-triazolyl)thymidine derivative which was synthesized from 5-O-DMT protected thymidine by acetylation with acetic anhydride and subsequent reaction with triethylamine, 1,2,4-triazole and POCl3. Stabilities are reported for DNA duplexes, three-way junctions and triplexes when 5 is inserted. Most interestingly, the three-way junctions are stabilized when 5 is used for insertion into the junction region. This break-through for recognizing the foot of a stem could have far-reaching importance because new targets for antisense oligos is now rendered possible on intact secondary structures.Graphic
Synthesis and Characterization of a [3-15N]-Labeled Cis-Syn Thymine Dimer-Containing DNA Duplex
The Journal of Organic Chemistry, 2006
Cis-syn thymine dimers are the major photoproducts of DNA and are the principal cause of mutations induced by sunlight. To better understand the nature of base pairing with cis-syn thymine dimers we have synthesized a decamer oligodeoxynucleotide (ODN) containing a cis-syn thymine dimer labeled at the N3 of both T's with 15 N by two efficient routes from [3-15 N]-thymidine phosphoramidite. In the post-synthetic irradiation route, an ODN containing an adjacent pair of [3-15 N]-labeled T's was irradiated and the cis-syn dimer-containing ODN isolated by HPLC. In the mixed building block route, a mixture of cis-syn and trans-syn dimer-containing ODNs was synthesized from a mixture of [3-15 N]-labeled thymine dimer phosphoramidites after which the cis-syn dimer-containing ODN was isolated by HPLC. The N3-nitrogen and imino proton signals of an 15 N-labeled thymine dimercontaining decamer duplex were assigned by 2D 1 H-15 N heterocorrelated HSQC NMR spectroscopy and the 15 N-1 H coupling constant was found to be 1.8 Hz greater for the 5′-T than for the 3′-T. The larger coupling constant is indicative of weaker H-bonding that is consistent with the more distorted nature of the 5′-base pair found in solution state NMR and crystallographic structures.
Bioorganic & Medicinal Chemistry Letters, 2013
A double-headed nucleoside wherein an additional thymine is attached to the 2 0 -O-position of uridine via a methylene linker is prepared and incorporated into oligonucleotides. With single incorporations of the modified nucleotide monomer, these oligonucleotides form duplexes with the complementary DNA sequences which are thermally less stable as compared to the unmodified duplexes. However, stabilization of bulged duplexes or three way junctions is observed. A cross-strand interaction between two additional thymines is also seen in a DNA-duplex, when specifically introduced in a so-called (+1)-zipper motif, however, much weaker than obtained with the corresponding analogue with the methylene linker directly attached to the 2 0 -C-position. This demonstrates that the ability to act as a compressed dinucleotide is unique for the latter and due to its perfect preorganization of the additional base in the duplex core. The ability of DNA to self assemble into a double helical structure using specific Watson-Crick base pairing between nucleobases from complementary strands has led to the construction of various functional nanostructures. 1,2 The unique structure provides an excellent scaffold to organize various functional moieties in a predicable manner. 1,2 Double-headed nucleotides (defined as nucleotides with additional nucleobases) present an opportunity to explore the hydrogen-bonding as well as stacking interactions from the additional nuclobases with an idea of finding novel nucleic acid structures. We have shown that nucleotide monomer W ( with an additional thymine attached to the 2 0 -position of the 2 0 -deoxyuridine through an ethylene linker, when incorporated in the branching point of a three way junction (TWJ) induces a thermal stabilization of the secondary nucleic acid structure. 3 Recently we introduced monomer X with a shorter methylene linker between an additional thymine and the 2 0 -position. 7 Monomer X acts as a compressed dinucleotide and forms base pairs with two adenines from the complementary strand in the centre of a DNA duplex. 7 After introducing also the monomer Y ( with an additional adenine in the 2 0 -position, 8 we established the formation of an additional Watson-Crick base pair between the additional thymine from monomer X and the additional adenine from monomer Y, when two modified nucleotides were placed in a so called (+1)-zipper arrangement. 8 In other words, the duplex was extended with an additional base pair on the same backbone. In the present study, we introduce a double headed nucleotide monomer Z with an additional thymine in the 2 0 -O-position of uridine attached through a methylene linker . This is hereby an oxa-analogue of W and the first example of a double-headed nucleoside with a ribose sugar. The behavior of Z is studied in the same duplexes and TWJ's as earlier studied with X and W, and furthermore, the behavior of X in the TWJ's is presented for the first time.
2004
by Pierre Tran Cis-syn cyclobutane pyrimidine dimer and the (6-4) photoproduct are the main photo-lesions formed following UV irradiation of skin cells. These lesions are responsible for cell death and development of skin cancers. Consequently, all organisms have developed DNA repair mechanisms to preserve the integrity of the genetic material. Crystallography, nuclear magnetic resonance, and spectroscopic analyses have shown that many enzymes “flip” the damaged base pairs out of the DNA duplex for repair. However, this process is poorly understood. The main problem with photodamaged DNA is the “flipped-out” structure, which cannot be resolved by any existing analytical method because of its small concentration. Additionally, the postulated “flipping” mechanism, now widely accepted and sometimes observed for other repair phenomenon, is a very fast process. The CPD in the “flipped-out” form is also not the most thermodynamically favorable product; its concentration is not measurable ...
A stable complex between homopyrimidine oligomers and the homologous regions of duplex DNAs
Nucleic Acids Research, 1988
When plasmid DNA duplexes carrying the regular homopurinehomopyrimidine inserts (dGA) * (dTC) and (dG) * (dC) are preincubated with homologous iRbeled o igo(dPy) (dTC) nand (dC) respectively) at acid pH, the label co-electrophoresgs with thg duplex DNA. Thus, a very strong complex is formed. Complementary oligo(dPu) does not form a complex under these conditions. No binding is observed for oligo(dPy) with non-homologous inserts as well as with vector plasmids without inserts. The complex is formed equally well with linear, nicked or superhelical DNA. The complex is not detected at pH > 6. Complex formation leads to very little, if any, unwinding of the duplex. The observed com-ple} appears to be the Py.Pu.Py triplex consisting of TAT and CGC base-triads with protonated cytosines. Two-dimensional gel electrophoresis patterns show that the presence of homologous oligo(dPy) destabilizes the formation of the H form. ©) I R L Press Limited, Oxford, England.
Tetrahedron, 1999
Protected forms of 1,2,3-propanetriol and cis, cis-l,3,5-cyclohexanetriol were incorporated onto solid supports which were exploited in the solid phase synthesis of 3'-3' linked oligodeoxyribonucleotides (ODNs), involving only nucleoside 3'-phosphoramidites as building blocks. UV thermal denaturation analysis showed the ability of ODNs with this inversion of polarity motif to cooperatively hybridize with duplexes of the type 5'-(Pu)m(PY)n-3' in an alternate strand recognition approach.
Biochemistry, 1999
DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G‚TA triplets, while CG base pairs are targeted with thymine, forming T‚CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1-propargylamino)dU (U P). For the target sequence (AAAT) 5 AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex-binding ligand, though stable triplexes were detected at the target site (AAAAAT) 3 AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT) 5 AA and (ATGA) 5 AA. In contrast, placing C + ‚GC triplets on the 5′-side of G‚TA, using the target (AGTA) 5 TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U P , and have shown that (TU P TG) 5 TT forms a more stable complex with target (AAAT) 5 AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA) 5 AA is improved by using the modified oligonucleotide (TCGU P) 5 TT, generating a complex in which the charged triplets C + ‚GC and U P ‚AT alternate with uncharged triplets. In contrast, placing U P ‚AT triplets adjacent to C + ‚GC, using the third strand oligonucleotide (U P CGT) 5 TT, reduces triplex formation, while the third strand with both substitutions, (U P CGU P) 5 TT, produces a complex with intermediate stability. It appears that, although adjacent U P ‚AT triplets form stable triplexes, placing U P ‚AT adjacent to C + ‚GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC) 3 AA were only detected in the presence of a triplex-binding ligand. Placing C + ‚GC on the 5′-side of T‚CG triplets also reduces triplex formation, while a 3′-C + ‚GC produces complexes with increased stability.
Chembiochem, 2003
The synthesis of new nucleoside derivatives, ara-uridine-2'-carbamates, and their incorporation into synthetic DNA oligomers is described. The modification directs ligands into the major groove of duplex DNA and somewhat destabilizes the duplexes of modified oligonucleotides with complementary DNA or RNA. In the case of pyrenemethyl carbamate modification in DNA ± DNA duplexes, the destabilization is considerably reduced. The pyrenemethyl derivative also shows remarkable spectral properties: a 'reversed' absorbance change for pyrene at 350 nm in the course of denaturation of the DNA duplex, as compared to the change seen in the nucleotide absorbance at 260 nm. This derivatization also causes pronounced sequence-dependent excimer formation in the major groove.