NMR and molecular modeling evidence for a G.A mismatch base pair in a purine-rich DNA duplex.

Abstract

ABSTRACT1H NMR experiments indicate that the oligomer 5'-d(ATGAGCGAATA) forms an unusual 10-base-pair duplex with 4 G-A base pairs (underlined) and a 3' unpaired adenosine. NMR results indicate that guanosine imino protons of the G-A mismatches are not hydrogen bonded but are stacked in the helix. A G -* I substitution in either G-A base pair causes a dramatic decrease in duplex stability and indicates that hydrogen bonding of the guanosine amino group is critical. Nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) results indicate that the overall duplex conformation is in the B-family. Cross-strand NOEs in two-dimensional NOESY spectra between a mismatched AH2 and an AHl' ofthe other mismatched base pair and between a mismatched GH8 and GNH1 of the other mismatch establish a purine-purine stacking pattern, adenosine over adenosine and guanosine over guanosine, which strongly stabilizes the duplex. A computer graphics molecular model of the unusual duplex was constructed with G-A base pairs containing A-NH2 to GN3 and G-NH2 to AN7 hydrogen bonds and B-form base pairs on both sides of the G'A pairs [5'-d(ATGAGC)J. The energy-minimized duplex satisfies all experimental constraints from NOESY and COSY results. A hydrogen bond from G-NH2 of the mismatch to a phosphate oxygen is predicted.Mismatches in DNA can arise, for example, in specific structures such as telomeres (1, 2), during replication, and in genetic recombination. If not corrected, the mismatches may lead to point mutations in subsequent replication (3-7). G-A mismatches are of particular interest because they are a common structural element in RNA folding (8)(9)(10)(11). Early studies have shown that a single G&A mismatch could be readily incorporated into DNA helices and the G-A mismatch was resistant to single-strand nucleases (12, 13). In neutral aqueous buffer with the normal base tautomers, there are four types of G&A mismatch base pairs (14), and these can be grouped by imino (Fig. 1, structures A and B) or amino hydrogen bond (Fig. 1, structures C and D) pairs with respect to guanosine. Several structural studies by single-crystal x-ray diffraction and NMR methods have shown that G-A mismatches are conformationally variable. Base pairs of types A and B but not C and D have been observed (7,(15)(16)(17)(18)(19)(20).In studies of the interaction of antisense oligonucleotides with 11-base analogs of ras p21 mRNA (21), we discovered a sequence, termed 2C, 5'-d(ATGAGCGAATA), that formed an unusually stable duplex (22). Eight of 11 bases in sequence 2C are purines and, from base-pairing possibilities, an unusual duplex with four G-A mismatch base pairs was proposed for this sequence (Fig. 2a) (22). We have undertaken NMR studies to define this unusual duplex in more detail and report here the observations of G-A mismatches oftype D and unusual purine purine base stacking in a B-form duplex.MATERIALS AND METHODS Oligonucleotides were synthesized as described (22). NMR experiments were performed on a Varian...

Thermodynamic stability and solution conformation of tandem G · A mismatches in RNA and RNA · DNA hybrid duplexes

“…A remaining possibility is the amino-paired G . A structures previously observed in DNA (Li et al, 1991Lane et al, 1992). A feature of this pairing in DNA is that the amino-pairing occurs only in the sequence context YGAR Cheng et al, 1992), and does not significantly destabilise the DNA duplex.…”

“…In DNA, the sequences that contain YGAR have been shown to have amino pairing of the G . A mismatches Lane et al, 1992;Chou et al, 1992) as first identified by Li et al (1991). All of the other sequences have the mismatched bases paired in the imino form.…”

“…A pairs form hydrogen bonds with the amino group of guanine rather than the N1H group. Further, the guanine residue stacks on the guanine residue of the opposite strand and the adenosine residue stacks on the other adenosine residue, leading to extensive base-base stacking (Li et al, 1991;Chou et al, 1992;Lane et al, 1992).…”

“…Heus and Pardi (1991a) have suggested that in conserved loops in rRNA, the loop region is closed by a G . A mismatch that uses the amino-pairing bonding pattern described by Li et al (1991). Recent studies (SantaLucia et al, 1992) indicate that hydrogen bonding between the mismatched bases contributes little to the thermodynamic stability of such loops and that the structure may be very sensitive to the exact nature of the bases closing the loop.…”

Rapid Detection of K-ras Mutations in Bile by Peptide Nucleic Acid-mediated PCR Clamping and Melting Curve Analysis: Comparison with Restriction Fragment Length Polymorphism Analysis

“…The competing wild-type PNA oligomer covered codons 10 -14. The antisense strand was chosen for the PNA oligomer and the detection probes because of its lower purine content and, therefore, more precise hybridization results (24 ). The primers, probes, and the PNA oligomer were from TIB MOLBIOL.…”