Unique condensation patterns of triplex DNA: physical aspects and physiological implications

Abstract

Triple-stranded DNA structures can be formed in living cells, either by native DNA sequences or following the application of antigene strategies, in which triplex-forming oligonucleotides are targeted to the nucleus. Recent studies imply that triplex motifs may play a role in DNA transcription, recombination and condensation processes in vivo. Here we show that very short triple-stranded DNA motifs, but not double-stranded segments of a comparable length, self-assemble into highly condensed and ordered structures. The condensation process, studied by circular dichroism and polarized-light microscopy, occurs under conditions that mimic cellular environments in terms of ionic strength, ionic composition and crowding. We argue that the unique tendency of triplex DNA structures to self-assemble, a priori unexpected in light of the very short length and the large charge density of these motifs, reflects the presence of strong attractive interactions that result from enhanced ion correlations. The results provide, as such, a direct experimental link between charge density, attractive interactions between like-charge polymers and DNA packaging. Moreover, the observations strongly support the notion that triple-stranded DNA motifs may be involved in the regulation of chromosome organization in living cells.

Figure 1

CD spectra exhibited by AT18 (1 and 3) and TAT18 (2 and 4) in a solution composed of 20 mM PIPES, 140 mM KCl, 1 mM MgCl₂ and 1 mM spermine, pH 7.0, in the absence (1 and 2) and presence (3 and 4) of 15% PEG. Samples were monitored at 25°C. DNA concentration in all experiments was 1.6 µM. (Insert) Spectra 1–3 at magnified scale.

Figure 2

CD spectra revealed by ‘AT20’ (1.6 µM) upon progressive addition of ‘T20’ (20 mM PIPES, 140 mM KCl, 1 mM MgCl₂, 1 mM spermine and 15% PEG, pH 7.0). The molar ratios between ‘T20’ and ‘AT20’ are: 0 (1), 0.2 (2), 0.4, (3), 0.6 (4), 0.8 (5) and 1.0 (6). Further increase in the molar ratio does not affect the CD spectrum. Samples were monitored at 25°C.

Figure 3

UV (A₂₆₀) melting curves of the triple-stranded motif ‘TAT20’ (1.6 µM, in 20 mM PIPES, 140 mM KCl, 1 mM MgCl₂, 1 mM spermine) in the absence (1) and presence (2) of 15% PEG. The low and high temperature transitions correspond to the triplex and duplex melting, respectively. Curve 3 is the melting curve of a 1:1 molar ratio mixture of ‘A20’ and ‘TAT20’, in both the absence and presence of PEG. Notably, whereas PEG is shown to stabilize the triplex motif, no such effect is observed for duplex DNA, as indeed previously reported (49). (Insert) Corresponding derivative curves dA₂₆₀/dT.

Figure 4

Liquid-crystalline phase of ‘TAT20’ observed by polarized light microscopy at 25°C. Samples consisted of DNA (16 µM) in 20 mM PIPES, 140 mM KCl, 1 mM MgCl₂, 1 mM spermine and 15% PEG, pH 7.0. Final magnification ×1000.

Figure 5

CD spectra of ‘TAT20’ (1.6 µM) at different KCl or spermine concentrations. (A) ‘TAT20’ in 20 mM PIPES, 1 mM MgCl₂, 1 mM spermine, 15% PEG and the following KCl concentrations (mM): 140 (1), 200 (2), 250 (3) and 300 (4). (B) ‘TAT20’ in 20 mM PIPES, 140 mM KCl, 1 mM MgCl₂, 15% PEG and the following spermine concentrations (mM): 0 (1), 0.5 (2), 1.0 (3), 1.5 (4), 2 (5), 2.5 (6), 3.0 (7) and 4.0 (8). The shift in ellipticity maxima exhibited upon the progressive addition of spermine is assigned to the very large sensitivity of non-conservative CD spectra to minute changes in the structural parameters of the cholesteric phase (14).