Proton exchange and local stability in a DNA triple helix containing a G.TA triad

Abstract

Recognition of a thymine-adenine base pair in DNA by triplex-forming oligonucleotides can be achieved by a guanine through the formation of a G.TA triad within the parallel triple helix motif. In the present work, we provide the first characterization of the stability of individual base pairs and base triads in a DNA triple helix containing a G.TA triad. The DNA investigated is the intramolecular triple helix formed by the 32mer d(AGATAGAACCCCTTCTATCTTATATCTGTCTT). The exchange rates of imino protons in this triple helix have been measured by nuclear magnetic resonance spectroscopy using magnetization transfer from water and real-time exchange. The exchange rates are compared with those in a homologous DNA triple helix in which the G.TA triad is replaced by a canonical C(+).GC triad. The results indicate that, in the G.TA triad, the stability of the Watson-Crick TA base pair is comparable with that of AT base pairs in canonical T.AT triads. However, the presence of the G.TA triad destabilizes neighboring triads by 0.6-1.8 kcal/mol at 1 degrees C. These effects extend to triads that are two positions removed from the site of the G.TA triad. Therefore, the lower stability of DNA triple helices containing G.TA triads originates, in large part, from the energetic effects of the G.TA triad upon the stability of canonical triads located in its vicinity.

Figure 1

(A) Structures of G.TA, C⁺.GC and T.AT triads (11,27). (B) Base sequence and folded conformation of the GTA triplex investigated (11). (C) Base sequence and folded conformation of the C⁺GC triplex investigated (24). (B and C) Watson–Crick hydrogen bonds are indicated by vertical bars, Hoogsteen hydrogen bonds are indicated by asterisks, and the bases containing hydrogen-bonded imino protons are shown in bold.

Figure 2

NMR resonances of imino protons in the GTA triplex (upper spectrum) and the C⁺GC triplex (lower spectrum) in 100 mM NaCl and 5 mM MgCl₂ at pH 5.3 and at 1°C.

Figure 3

(A) Selected spectra of the GTA triplex during experiments of transfer of magnetization from water at 1°C. The exchange delay, τ (s), for each spectrum is indicated. The labeled imino proton resonances show changes in intensity during transfer of magnetization from water. (B) Dependence of the intensity of the G₂₇ imino proton resonance on the exchange delay, τ, in experiments of transfer of magnetization from water.

Figure 4

Selected spectra of the GTA and C⁺GC triplexes during real-time exchange measurements at 1°C. The exchange time (min) is given for each spectrum. The imino proton resonances, which show changes in intensity during real-time exchange are indicated.