Self-association of short DNA loops through minor groove C:G:G:C tetrads

Abstract

In addition to the better known guanine-quadruplex, four-stranded nucleic acid structures can be formed by tetrads resulting from the association of Watson-Crick base pairs. When such association occurs through the minor groove side of the base pairs, the resulting structure presents distinctive features, clearly different from quadruplex structures containing planar G-tetrads. Although we have found this unusual DNA motif in a number of cyclic oligonucleotides, this is the first time that this DNA motif is found in linear oligonucleotides in solution, demonstrating that cyclization is not required to stabilize minor groove tetrads in solution. In this article, we have determined the solution structure of two linear octamers of sequence d(TGCTTCGT) and d(TCGTTGCT), and their cyclic analogue d<pCGCTCCGT>, utilizing 2D NMR spectroscopy and restrained molecular dynamics. These three molecules self-associate forming symmetric dimers stabilized by a novel kind of minor groove C:G:G:C tetrad, in which the pattern of hydrogen bonds differs from previously reported ones. We hypothesize that these quadruplex structures can be formed by many different DNA sequences, but its observation in linear oligonucleotides is usually hampered by competing Watson-Crick duplexes.

Scheme 1.

Sequences of the oligonucleotides studied in this work

Figure 1.

The 1D NMR spectra of d in D₂O at 5°C and different oligonucleotide concentrations (25 mM sodium phosphate buffer, pH 7, M stands for monomeric form) (A). CD (B) and TDS (C) spectra of d<pCGCTCCGT>, d(TGCTTCGT) and d(TCGTTGCT) (25 mM sodium phosphate buffer, pH 7, 100 mM NaCl, 10 mM MgCl₂).

Figure 2.

Top: 1D NMR spectra of d (A), d(TGCTTCGT) (B) and d(TCGTTGCT) (C) in H₂O at different temperatures (25 mM sodium phosphate buffer, pH 7, 100 mM NaCl, 10 mM MgCl₂). Bottom: CD spectra at different temperatures, 25 mM sodium phosphate buffer, pH 7, 100 mM NaCl, 10 mM MgCl₂.

Figure 3.

Two regions of the NOESY spectrum (T_m = 150 ms) of d<pCGCTCCGT> in H₂O (4 mM oligonucleotide concentration, 100 mM NaCl, T = 5°C, pH 7). Watson–Crick base pairing can be established from the H1G-H42C, H1G-H41C and H1G2-H5C cross-peaks for each of the GC base pairs. Sequential assignment pathways for non-exchangeable protons are shown. Cross-peaks are labelled according to the spin systems numbering shown in scheme 1.

Figure 4.

Superposition of the 10 refined structures of d, d(TGCTTCGT) and d(TCGTTGCT) (A), and stereoscopic views of the average structure of d<pCGCTCCGT> (B), d(TGCTTCGT) (C) and d(TCGTTGCT) (D). Red and blue indicate different molecules. The sugar-phosphate backbone is indicated in darker colors. Note that the perspective in (C) and (D) is not the same, but has been selected to show the two C:G:G:C tetrads in the same orientation.

Figure 5.

The three known minor groove tetrads involving GC base pairs. (A) Direct minor groove G:C:G:C tetrad observed in the dimeric solution structures of d<pTGCTCGCT>, (B) slipped minor groove tetrad found in the solution structure of d<pCCGTCCGT>, (C) C:G:G:C tetrad observed in d<pCGCTCCGT>, d(TGCTTCGT) and d(TCGTTGCT).