Controlling nucleic acid secondary structure by intercalation: effects of DNA strand length on coralyne-driven duplex disproportionation

Abstract

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)*poly(dT) into triplex poly(dA)*poly(dT)*poly(dT) and a poly(dA) self-structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)16*(dT)16 by coralyne reverts over the course of hours if the sample is maintained at 4 degrees C. Coralyne-disproportioned (dA)32. (dT)(32), on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)16*(dT)16 sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)16 self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)n self-structure] binds coralyne in a length-dependent manner.

Scheme 1. (A) Structural representations of the T·A·T base triplet and coralyne chloride. (B) Schematic representation of the disproportionation of a (dA)_n·(dT)_n duplex into coralyne-intercalated triplex (dA)_n·(dT)_n·(dT)_n and the (dA)_n self-structure.

Scheme 1. (A) Structural representations of the T·A·T base triplet and coralyne chloride. (B) Schematic representation of the disproportionation of a (dA)_n·(dT)_n duplex into coralyne-intercalated triplex (dA)_n·(dT)_n·(dT)_n and the (dA)_n self-structure.

Scheme 2. Schematic representation of the equilibrium secondary structure distribution at three temperatures for a sample containing an equal ratio of (dA)₁₆ and (dT)₁₆ in the presence of 0.25 molar equivalents of coralyne per base pair. The possibility of an alternative mode of coralyne binding to the (dA)₁₆·(dT)₁₆ duplex is depicted by the blue diagonal lines.

Scheme 2. Schematic representation of the equilibrium secondary structure distribution at three temperatures for a sample containing an equal ratio of (dA)₁₆ and (dT)₁₆ in the presence of 0.25 molar equivalents of coralyne per base pair. The possibility of an alternative mode of coralyne binding to the (dA)₁₆·(dT)₁₆ duplex is depicted by the blue diagonal lines.

Figure 1

CD spectra of triplex and duplex 16mer DNA samples demonstrating that complete triplex (dA)₁₆·(dT)₁₆·(dT)₁₆ formation requires heating of the DNA sample in the presence of coralyne. (A) Spectra of duplex (dA)₁₆·(dT)₁₆ and (dA)₁₆·(dT)₁₆ + (dT)₁₆ (unstable triplex) samples at 4°C. (B) Spectrum of (dA)₁₆·(dT)₁₆ + (dT)₁₆ in the presence of coralyne at 4°C. (C) Spectrum of (dA)₁₆·(dT)₁₆ + (dT)₁₆ at 30°C without coralyne and triplex (dA)₁₆·(dT)₁₆·(dT)₁₆ spectrum with coralyne at 30°C. (D) CD spectrum acquired at 4°C of (dA)₁₆·(dT)₁₆·(dT)₁₆ after being heated to 75°C with coralyne. (E) Spectra of triplex (dA)₁₆·(dT)₁₆·(dT)₁₆ at 4°C after heat cycling with 0.25 and 0.5 molar equivalents of coralyne, respectively. DNA concentrations were 55 µM base pair or base triplet, respectively. Coralyne concentration, in samples containing coralyne, was 0.25 molar equivalents per base pair or base triplet, respectively, except as stated in (E).

Figure 2

CD melting profiles for 16mer DNA samples at wavelengths selected to show structural transitions. (A) Duplex (dA)₁₆·(dT)₁₆ and (dA)₁₆·(dT)₁₆ + (dT)₁₆ melting curves exhibit a transition (T_m^2→1) at 37°C. (B) First and second heating of a triplex (dA)₁₆·(dT)₁₆·(dT)₁₆ sample after the addition of coralyne indicates triplex melting (T_m^3→1) at 46°C. The first heating of this sample after the addition of coralyne also shows a transition centered at ∼15°C, which is assigned to the reorganization of DNA strands from partial duplex and partial triplex to complete triplex. (C) First and second heating of duplex (dA)₁₆·(dT)₁₆ after the addition of coralyne; the duplex disproportionation transition is observed at ∼23°C during the first heating and triplex melting (T_m^3→1) is observed at 46°C in both the first and second heating of the sample. (D) (dA)₁₆ with coralyne exhibits the melting transition of the (dA)₁₆ self-structure at ∼25°C. The vertical ellipticity scale is the same for all samples. DNA concentrations were 55 µM nucleotide, base pair or base triplet, respectively. Coralyne concentration, in samples containing coralyne, was 0.25 molar equivalents per nucleotide, base pair or base triplet, respectively.

Figure 3

The 360–480 nm region of the UV-Vis absorption spectra of coralyne in the presence of triplex (dA)₁₆·(dT)₁₆·(dT)₁₆. Spectra at 4°C before and after heating illustrate the change in binding of coralyne to the 16mer triplex that is promoted by heat cycling above 36°C. DNA concentration was 142 µM base triplet. Coralyne concentration was 0.25 molar equivalents per base triplet.

Figure 4

CD spectra of duplex (dA)₁₆·(dT)₁₆ samples that illustrate duplex disproportionation. (A) Duplex (dA)₁₆·(dT)₁₆ sample with and without added coralyne at 4°C prior to heating. (B) Spectra of duplex (dA)₁₆·(dT)₁₆ samples with and without coralyne at 36°C. (C) Heat-cycled spectrum of disproportioned (dA)₁₆·(dT)₁₆ sample in the presence of coralyne at 4°C and composite spectrum {sum of spectra acquired at 4°C: [0.50 × triplex (dA)₁₆·(dT)₁₆·(dT)₁₆ with 0.25 molar equivalents of coralyne] + [0.50 × single stranded (dA)₁₆ with 0.25 molar equivalents of coralyne]}. (D) Spectra of (dA)₁₆ sample with and without coralyne at 4°C. DNA concentrations were 55 µM nucleotide or base pair, respectively. Coralyne concentration was 0.25 molar equivalents per nucleotide or base pair, respectively.

Figure 5

CD spectra of a (dA)₁₆·(dT)₁₆ sample with coralyne that illustrate the approach of this sample to equilibrium at 4 and 22°C after being heat cycled to 75°C. (A) Spectra of the (dA)₁₆·(dT)₁₆ sample with coralyne at 4°C. The dashed line is the spectrum acquired immediately after the addition of coralyne at 4°C, before heat cycling. The solid black line is the spectrum acquired immediately after the sample was heated to 75°C and cooled back to 4°C. Spectra drawn in gray were acquired at selected time intervals after the sample was heat cycled. After heat cycling, the sample was constantly maintained at 4°C. (Inset) A plot of the CD signal at 293 nm as a function of time after sample heat cycling for the sample maintained at 4°C. (B) Spectra of the (dA)₁₆·(dT)₁₆ sample with coralyne at 22°C. The dashed line is the spectrum acquired immediately after the addition of coralyne at 22°C, before heat cycling. The solid black line is the spectrum acquired immediately after the sample was heated to 75°C and cooled back to 22°C. Spectra drawn in gray were acquired at selected time intervals after the sample was heat cycled. After heat cycling, the sample was constantly maintained at 22°C. (Inset) A plot of the best fit exponential function of the CD signal at 293 nm for the (dA)₁₆·(dT)₁₆ sample with coralyne at 22°C as a function of time after heat cycling. DNA concentration was 55 µM base pair. Coralyne concentration was 0.25 molar equivalents per base pair for both samples.

Figure 6

CD spectra of (dA)₃₂·(dT)₃₂ and poly(dA)·poly(dT) samples with coralyne that illustrate the approach of these samples to equilibrium at 4°C after being heat cycled into the disproportioned state. (A) Spectra of the (dA)₃₂·(dT)₃₂ sample with coralyne at 4°C. The dashed line is the spectrum acquired immediately after the addition of coralyne at 4°C, before sample heat cycling. The solid black line is the spectrum acquired immediately after the sample was heated to 75°C and cooled back to 4°C. Spectra drawn in gray were acquired at selected time intervals after the sample was heat cycled. After heat cycling, the sample was constantly maintained at 4°C. (Inset) A plot of the best fit exponential function of the CD signal at 293 nm for the (dA)₃₂·(dT)₃₂ sample with coralyne at 4°C as a function of time after sample heat cycling. (B) Spectra of the poly(dA)·poly(dT) sample with coralyne at 4°C. The dashed line is the spectrum acquired immediately after the addition of coralyne at 4°C, before sample heat cycling. The solid black line is the spectrum acquired immediately after the sample was heated to 95°C and cooled back to 4°C. Spectra drawn in gray were acquired at selected time intervals after the sample was heat cycled. After heat cycling, the sample was constantly maintained at 4°C. (Inset) A plot of the best fit double exponential function of the CD signal at 300 for the poly(dA)· poly(dT) sample with coralyne at 4°C nm as a function of time after heat cycling. DNA concentration was 55 µM base pair. Coralyne concentration was 0.25 molar equivalents per base pair for both samples.