The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

Abstract

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

Figure 1

Bimolecular interaction between MR121 and guanosine. (a) Steady-state and time-resolved bimolecular Stern–Volmer plots of MR121 with dGMP in PBS, pH 7.4. F₀ (F) and τ₀ (τ) are the fluorescence intensity and longer lifetime component in the absence (presence) of dGMP. Squares represent the absolute intensity values F₀/F without any correction. Circles show static quenching, given by the quenching factor F₀/F divided by dynamic quenching τ₀/τ = 1 + 2.6M⁻¹ ·[dGMP] (as shown in inset). A linear fit of the static quenching curve yields the biomolecular association constant K_s = 75±2M⁻¹. (b) Formation of non-fluorescent MR121–dGMP complexes monitored by FCS. FCS was performed at 20°C in aqueous solution of MR121 (∼1 nM) and varying dGMP concentrations (0–30 mM). Association and dissociation rate constants k_as/dis were derived from parameters fitted according to Equation 3. The inset shows the variation of τ_as/dis = k_as/dis⁻¹ as a function of dGMP concentration. (c) Arrhenius plots ln k_as/dis = I−ΔH(RT)⁻¹ for the intermolecular interaction between MR121 and dGMP between 10 and 40°C. Closed squares (open circles) represent association (dissociation) rate constants. Arrhenius plots yield ΔH^dis = 31 ± 2 kJ mol⁻¹ and ΔH^as = 17 ± 2 kJ mol⁻¹.

Figure 2

FCS performed on DNA hairpin structures labeled with MR121 (F). (a) Schematic representation of conformational fluctuations of F-labeled DNA hairpin structures. MR121 is attached at the 5′ end and connected via a loop of polythymines·(dT) to the intrinsic quencher guanosine·(dG). The stem consists of 1 or 2 cytosine (dC, incorporated at the 5′ end) and dG (incorporated at the 3′ end) pairs. (b) Representative FCS data shown for F-(dT)_x–dG (lower curve) and F-(dC)_y–(dT)_x–(dG)_y with x = 5 and y = 1 (middle curve) or y = 2 (upper curve), where x is the number of loop bases and y the number of dC–dG pairs. Whereas FCS data for oligonucleotides without dC–dG pairs are well described by an exponential decay, those with dC–dG pairs can only be fitted with comparable accuracy using a stretched-exponential function (Equation 4). Stretch-exponents β, averaged over multiple measurements, are independent of temperature and presented as a function of dC–dG pairs with loop length x ranging from 4 to 9 (inset).

Figure 3

Opening (a) and closing (b) rate constants for F-(dT)_x–dG and F-(dC)_y–(dT)_x–(dG)_y oligonucleotides (x ranging from 3 to 9) as a function of dC–dG pairs (y ranging from 0 to 2). Average rate constants are calculated from stretched-exponential fits (according to Equation 5) to FCS data recorded at 20°C. Errors bars represent SDs.

Figure 4

Population of open (that is fluorescent) states P_open at 10°C (a), 20°C (b) and 30°C (c). P_open is derived from rate constants measured by FCS (following Equation 12). Whereas addition of the first dC–dG pair significantly reduces P_open for all hairpins, the effect of a second dC–dG pair depends strongly on the loop length: for hairpins with loops longer than five residues, P_open is further reduced; for structures with loops of 3, 4 and 5 residues, the effect of the second dC–dG pair is temperature dependent, showing less influence on P_open for shorter loops and lower temperature.

Figure 5

Arrhenius plots and activation enthalpies. Arrhenius plots of opening (open symbols) and closing (closed symbols) rate constants for (a) F-(dC)₂–(dT)₄–(dG)₂ and (b) F-(dC)₂–(dT)₅–(dG)₂ show perfect Arrhenius behavior for larger hairpins and significant deviation for those with a loop length ≤4. Arrhenius plots of oligonucleotides with loop length of 5 (squares) and 6 (circles) bases and 0–2 dC–dG pairs were analyzed to yield activation enthalpies and entropic contributions (insets) for the opening (c) and closing (d) process. Error bars represent SDs.