STRUCTURAL STUDIES OF THE 5'-PHENAZINIUM-TETHERED MATCHED AND G-A-MISMATCHED DNA DUPLEXES BY NMR-SPECTROSCOPY

The mechanism through which modified oligo-DNA analogues act as antise nse repressors at the transcriptional and translational level of gene expression is based on the information content in the nucleotide seque nce which is determined by the specific base pairing. The efficiency o f such action is largely determined by the stability of the duplex for med between the oligonucleotide reagent and the target sequence and al so by the mismatched base pairing, such as G-A, that occurs during rep lication or recombination. We herein report that the phenazinium (Pzn) -tethered matched duplex p(d(TGTTTGGC)):(Pzn)-p(d(CCAAACA)) (III) (T(m ) = 50-degrees-C) has a much larger stability than the parent matched duplex p(d(TGTTTGGC)): p(d(CCAAACA)) (I) (T(m) = 30-degrees-C). On the other hand, the Pzn-tethered G-A-mismatched duplex p(d(TGTTTGGC)):(Pz n)-p(d(ACAAACA)) (IV) (T(m) = 34-degrees-C) is only slightly more stab le than its parent mismatched duplex p(d(TGTTTGGC)): p(d(ACAAACA)) (T( m) = 25-degrees-C). A detailed 500 MHz NMR study and constrained MD re finements of NMR-derived structures have been undertaken for the DNA d uplexes (I), (II), (III) and (IV) in order to understand the structura l basis of stabilization of Pzn-tethered matched DNA duplex (DELTAT(m) = 20-degrees-C) compared to mismatched duplex (DELTA T(m) = 9-degrees -C). Assignment of the H-1-NMR (500 MHz) spectra of the duplexes has b een carried out by 2D NOESY, HOHAHA and DQF-COSY experiments. The tors ion angles have been extracted from the J-coupling constants obtained by simulation of most of the DQF-COSY cross-peaks using program SMART. The solution structure of the duplexes were assessed by an iterative hybride relaxation matrix method (MORASS) combined with NOESY distance s and torsion angles restrained molecular dynamics (MD) using program Amber 4.0. The standard Amber 4.0 force-field parameters were used for the oligonucleotide in conjunction with the new parameters for Pzn re sidue which was obtained by full geometry optimization using ab initio program (3-21G basis set). It has been shown that mismatched G-A base s are in the anti-anti conformation. The mismatched 7G-1A form stable base pairs through inter-strand hydrogen bonds (N7(A)... HN2(G) (1.92 angstrom) with a subtended angle of 176-degrees and N3(G)... HN6(A) (2 .01 angstrom) with a subtended angle of 153-degrees (the 'amino-type' hydrogen bond)) and a propeller twist of 36-degrees for 7G-1A residues . Observation of the nOe connectivities amongst the Pzn protons and Pz n-oligonucleotide protons in the spectrum of the duplex (III) has allo wed the unambiguous determination of stereochemical orientation of the Pzn residue in the matched duplex (III). It has been found that the P zn residue stacks with both residues of the neighbouring G-C base-pair in the matched duplex (III) and strongly stabilizes the matched G-C b ase-pair, while the Pzn residue in the mismatched duplex (IV) adopts a t least three different conformations in the NMR time scale, and it on ly partly stacks with the neighbouring G-A base-pair in one of these t hree conformations. Excellent stacked geometry of the Pzn residue perp endicular to the main axis of matched DNA duplex (III) may be partly r esponsible for the additional stability of the B-type DNA structure.