Formation of an intramolecular triple-stranded DNA structure monitored by fluorescence of 2-aminopurine or 6-methylisoxanthopterin

Abstract

The parallel (recombination) 'R-triplex' can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the fluorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or 6MI. The 2AP*(T.A) and 6MI*(C.G) triplets were monitored by their fluorescence emission and the thermal denaturation curves were analyzed with a quasi-two-state model. The fluorescence of 2AP introduced into an oligonucleotide sequence unable to form a triplex served as a negative control. We observed a remarkable similarity between the thermodynamic parameters derived from melting of the secondary structures monitored through absorption of all bases at 260 nm or from fluorescence of the single base analog. The similarity suggests that fluorescence of the 2AP and 6MI base analogs may be used to monitor the structural disposition of the third strand. We consider the data in the light of alternative 'branch migration' and 'strand exchange' structures and discuss why these are less likely than the R-type triplex.

Figure 1

(A) A*(T·A), 2AP*(T·A) and (B) G*(C·G) and 6MI*(C·G) base triplet schemes.

Figure 2

Oligonucleotide sequences. a, 2-aminopurine, (2AP); F, 6MI pteridine. Triplex-forming oligonucleotides: RCW, positive control for the triplex with no fluorophore; R^2APCW is the 2AP-containing triplex with a -GT₄C- loop connecting the third strand to the CW duplex; R^2APCW-2 is the 2AP-containing triplex with a -CT₄G- loop connecting the third strand to the CW duplex; R^6MICW, 6MI-containig triplex; NCW, non-triplex-forming control with no fluorophore; N^2APCW, 2AP-containing non-triplex-forming control. Hairpin-forming oligonucleotides: R^2APC, hairpin designed for detecting the fluorescence signal of the 2AP base analog involved in the 2AP*T base pair; CW, the hairpin representing the duplex part of the triplex; N^2APC, non-duplex-forming control. Single-stranded control oligonucleotides: R^2AP, R^6MI and N^2AP.

Figure 3

Schemes of temperature-dependent structural transitions for the R-triplex and alternative ‘strand exchange’ and ‘branch migration’ structures (shown in rectangles). The alternatives can involve multiple configurations, including the CW and the RC ‘strand exchange’ hairpins as well as the ‘branched’ structure (with various positions of the branch point). The open circles indicate positions of 2AP in various conformations.

Figure 4

Fluorescence emission and excitation spectra of oligonucleotides at 8°C. R^2APCW (filled circles), R^2APC (filled triangles) and R^2AP (crosses). The excitation wavelength for emission spectra was 310 nm, the emission wavelength for emission spectra was 370 nm. Samples contained 1 µM oligonucleotide, 0.5 M LiCl, 10 mM Tris–HCl buffer, pH 7.6.

Figure 5

Temperature dependence of intensity of the fluorescence emission at 370 nm of 2AP, incorporated in the oligonucleotides. The excitation wavelength was 310 nm. (A) oligonucleotides R^2APCW (filled circles), R^2APC (filled triangles) and R^2AP (crosses); (B) control N^2APCW (filled circles), ds N^2APC (filled triangles), ss N^2AP (crosses); (C) oligonucleotides R^2APCW-2 (filled circles) and N^2APCW (crosses). Solid curves are the best theoretical fits (see Materials and Methods); dashed curves are the smoothed experimental data. Derived thermodynamic parameters are given in Table 1. Solution conditions as in Figure 4.

Figure 5

Temperature dependence of intensity of the fluorescence emission at 370 nm of 2AP, incorporated in the oligonucleotides. The excitation wavelength was 310 nm. (A) oligonucleotides R^2APCW (filled circles), R^2APC (filled triangles) and R^2AP (crosses); (B) control N^2APCW (filled circles), ds N^2APC (filled triangles), ss N^2AP (crosses); (C) oligonucleotides R^2APCW-2 (filled circles) and N^2APCW (crosses). Solid curves are the best theoretical fits (see Materials and Methods); dashed curves are the smoothed experimental data. Derived thermodynamic parameters are given in Table 1. Solution conditions as in Figure 4.

Figure 5

Temperature dependence of intensity of the fluorescence emission at 370 nm of 2AP, incorporated in the oligonucleotides. The excitation wavelength was 310 nm. (A) oligonucleotides R^2APCW (filled circles), R^2APC (filled triangles) and R^2AP (crosses); (B) control N^2APCW (filled circles), ds N^2APC (filled triangles), ss N^2AP (crosses); (C) oligonucleotides R^2APCW-2 (filled circles) and N^2APCW (crosses). Solid curves are the best theoretical fits (see Materials and Methods); dashed curves are the smoothed experimental data. Derived thermodynamic parameters are given in Table 1. Solution conditions as in Figure 4.

Figure 6

Thermal denaturation of R^2APCW and control oligonucleotide N^2APCW monitored by absorption at 260 nm. (A) Melting curves of R^2APCW (filled circles) and of N^2APCW (open circles), left ordinate; melting curve of CW hairpin (filled triangles), right ordinate; (B) first derivatives of the melting curves of R^2APCW (filled circles) and N^2APCW (open circles); (C) determination of the thermodynamic parameters of R^2APCW triplex formation from the absorption thermal denaturation experiments. The triplex melting curve (filled circles) is a result of subtraction of the CW contribution to the melting profile of R^2APCW (see filled circle and filled triangle in curves Fig. 5A). The solid curve is the best theoretical fit for a two-state model (see Materials and Methods). The derived thermodynamic parameters are given in Table 1. The concentration of each oligonucleotide was 0.77 µM.

Figure 6

Thermal denaturation of R^2APCW and control oligonucleotide N^2APCW monitored by absorption at 260 nm. (A) Melting curves of R^2APCW (filled circles) and of N^2APCW (open circles), left ordinate; melting curve of CW hairpin (filled triangles), right ordinate; (B) first derivatives of the melting curves of R^2APCW (filled circles) and N^2APCW (open circles); (C) determination of the thermodynamic parameters of R^2APCW triplex formation from the absorption thermal denaturation experiments. The triplex melting curve (filled circles) is a result of subtraction of the CW contribution to the melting profile of R^2APCW (see filled circle and filled triangle in curves Fig. 5A). The solid curve is the best theoretical fit for a two-state model (see Materials and Methods). The derived thermodynamic parameters are given in Table 1. The concentration of each oligonucleotide was 0.77 µM.

Figure 6

Thermal denaturation of R^2APCW and control oligonucleotide N^2APCW monitored by absorption at 260 nm. (A) Melting curves of R^2APCW (filled circles) and of N^2APCW (open circles), left ordinate; melting curve of CW hairpin (filled triangles), right ordinate; (B) first derivatives of the melting curves of R^2APCW (filled circles) and N^2APCW (open circles); (C) determination of the thermodynamic parameters of R^2APCW triplex formation from the absorption thermal denaturation experiments. The triplex melting curve (filled circles) is a result of subtraction of the CW contribution to the melting profile of R^2APCW (see filled circle and filled triangle in curves Fig. 5A). The solid curve is the best theoretical fit for a two-state model (see Materials and Methods). The derived thermodynamic parameters are given in Table 1. The concentration of each oligonucleotide was 0.77 µM.

Figure 7

Thermal denaturation of R^2APCW at different oligonucleotide concentrations. (A) UV absorption melting profiles. R^2APCW concentrations were 0.97 (filled circles) and 12 µM (filled triangles). The 12 µM sample was measured in a 1 mm cell and the plotted OD values are calculated for a 1 cm path. (B) Temperature dependence of 2AP fluorescence in R^2APCW at 370 nm: oligonucleotide concentrations were 0.3 (filled circles) and 12 µM (filled triangles).

Figure 7

Thermal denaturation of R^2APCW at different oligonucleotide concentrations. (A) UV absorption melting profiles. R^2APCW concentrations were 0.97 (filled circles) and 12 µM (filled triangles). The 12 µM sample was measured in a 1 mm cell and the plotted OD values are calculated for a 1 cm path. (B) Temperature dependence of 2AP fluorescence in R^2APCW at 370 nm: oligonucleotide concentrations were 0.3 (filled circles) and 12 µM (filled triangles).

Figure 8

Temperature dependence of intensity of the fluorescence emission at 430 nm of 6MI, incorporated into the oligonucleotides. The excitation wavelength was 350 nm. The oligonucleotides were R^6MICW (filled circles) and R^6MI (crosses); solid curves are the best theoretical fits (see Materials and Methods). Derived thermodynamic parameters are given in the Table 1. Solution conditions as in Figure 4.

Figure 9

Probing the RCW and R^2APCW structures with EtBr. (A) Effect of EtBr binding on the thermal denaturation profiles of the oligonucleotides. RCW (triangles) and RCW in the presence of EtBr (circles). The concentration of bound EtBr calculated from binding isotherms was not more than one EtBr molecule per oligonucleotide, on average. (B) Temperature dependence of the relative hydrodynamic volume of the oligonucleotide–EtBr complexes: RCW (crosses) and R^2APCW (filled circles) compared to the hydrodynamic volume of CW under the same conditions.

Figure 9

Probing the RCW and R^2APCW structures with EtBr. (A) Effect of EtBr binding on the thermal denaturation profiles of the oligonucleotides. RCW (triangles) and RCW in the presence of EtBr (circles). The concentration of bound EtBr calculated from binding isotherms was not more than one EtBr molecule per oligonucleotide, on average. (B) Temperature dependence of the relative hydrodynamic volume of the oligonucleotide–EtBr complexes: RCW (crosses) and R^2APCW (filled circles) compared to the hydrodynamic volume of CW under the same conditions.