Monitoring the conformational fluctuations of DNA hairpins using single-pair fluorescence resonance energy transfer.

We present single-pair fluorescence resonance energy transfer (spFRET) observations of individual opening and closing events of surface-immobilized DNA hairpins. Two glass-surface immobilization strategies employing the biotin-streptavidin interaction and a third covalent immobilization strategy involving formation of a disulfide bond to a thiol-derivatized glass surface are described and evaluated. Results from image and time-trace data from surface-immobilized molecules are compared with those from freely diffusing molecules, which are unperturbed by surface interactions. Using a simple two-state model to analyze the open and closed time distributions for immobilized hairpins, we calculate the lifetimes of the two states. For hairpins with a loop size of 40 adenosines and a stem size of either seven or nine bases, the respective closed-state lifetimes are 45 +/- 2.4 and 103 +/- 6.0 ms, while the respective open-state lifetimes are 133 +/- 5.5 and 142 +/- 22 ms. These results show that the open state of the hairpin is favored over the closed state of the hairpin under these conditions, consistent with previous diffusion fluorescence correlation spectroscopy (FCS) experiments on poly(A)-loop hairpins. The measured open-state lifetime is about 30 times longer than the calculated 3 ms open-state lifetime for both hairpins based on a closing rate scaling factor derived from a previous FCS study for hairpins in diffusion with 12-30 thymidines in their loops. As predicted, the closed-state lifetime is dependent on the stem length and is independent of the loop characteristics. Our findings indicate that current models should consider sequence dependence in calculating ssDNA thermostability. The surface immobilization chemistries and other experimental techniques described here should prove useful for studies of single-molecule populations and dynamics.     

DNA confined in nanochannels: hairpin tightening by entropic depletion

A theory is presented of the elongation of double-stranded DNA confined in a nanochannel based on a study of the formation of hairpins. A hairpin becomes constrained as it approaches the wall of a channel which leads to an entropic force causing the hairpin to tighten. The DNA in the hairpin remains double-stranded. The free energy of the hairpin is significantly larger than what one would expect if this entropic effect were unimportant. As a result, the distance between hairpins or the global persistence length is often tens of micrometer long and may even reach millimeter sizes for 10 nm thin channels. The hairpin shape and size and the DNA elongation are computed for nanoslits and circular and square nanochannels. A comparison with experiment is given.     

Sensitivity and specificity of metal surface-immobilized "molecular beacon" biosensors

The separate developments of microarray patterning of DNA oligonucleotides, and of DNA hairpins as sensitive probes for oligonucleotide identification in solution, have had a tremendous impact on basic biological research and clinical applications. We have combined these two approaches to develop arrayable and label-free biological sensors based on fluorescence unquenching of DNA hairpins immobilized on metal surfaces. The thermodynamic and kinetic response of these sensors, and the factors important in hybridization efficiency, were investigated. Hybridization efficiency was found to be sensitive to hairpin secondary structure, as well as to the surface distribution of DNA hairpins on the substrate. The identity of the bases used in the hairpin stem as well as the overall loop length significantly affected sensitivity and selectivity. Surface-immobilized hairpins discriminated between two sequences with a single base-pair mismatch with high sensitivity (over an order of magnitude difference in signal) under identical assay conditions (no change in stringency). This represents a significant improvement over other microarray-based techniques.     

Long-distance radical cation transport in DNA: Horizontal charge hopping in a dimeric quadruplex

A series of DNA hairpins were synthesized and shown to associate to form quadruplexes formed by stacking five G-quartets in an antiparallel orientation. One of the hairpins in the quadruplex was linked covalently at the 5'-end to an anthraquinone (AQ) group and a 32P label was incorporated either at the 3'-terminus of the AQ-containing hairpin or on its partner hairpin in the quadruplex. Irradiation of the AQ group with UV light leads to the one-electron oxidation of the DNA and concomitant introduction of a radical cation into the DNA. Analysis by PAGE and autoradiography shows that the radical cation reacts at guanines both on the AQ-containing strand and with its partner hairpin in the quadruplex. This observation demonstrates that charge migration in DNA occurs vertically along a DNA chain and horizontally within a G-quartet.     

Charge-transfer and spin dynamics in DNA hairpin conjugates with perylenediimide as a base-pair surrogate

A perylenediimide chromophore (P) was incorporated into DNA hairpins as a base-pair surrogate to prevent the self-aggregation of P that is typical when it is used as the hairpin linker. The photoinduced charge-transfer and spin dynamics of these hairpins were studied using femtosecond transient absorption spectroscopy and time-resolved EPR spectroscopy (TREPR). P is a photooxidant that is sufficiently powerful to quantitatively inject holes into adjacent adenine (A) and guanine (G) nucleobases. The charge-transfer dynamics observed following hole injection from P into the A-tract of the DNA hairpins is consistent with formation of a polaron involving an estimated 3-4 A bases. Trapping of the (A 3-4) (+*) polaron by a G base at the opposite end of the A-tract from P is competitive with charge recombination of the polaron and P (-*) only at short P-G distances. In a hairpin having 3 A-T base pairs between P and G ( 4G), the radical ion pair that results from trapping of the hole by G is spin-correlated and displays TREPR spectra at 295 and 85 K that are consistent with its formation from (1*)P by the radical-pair intersystem crossing mechanism. Charge recombination is spin-selective and produces (3*)P, which at 85 K exhibits a spin-polarized TREPR spectrum that is diagnostic of its origin from the spin-correlated radical ion pair. Interestingly, in a hairpin having no G bases ( 0G), TREPR spectra at 85 K revealed a spin-correlated radical pair with a dipolar interaction identical to that of 4G, implying that the A-base in the fourth A-T base pair away from the P chromophore serves as a hole trap. Our data suggest that hole injection and transport in these hairpins is completely dominated by polaron generation and movement to a trap site rather than by superexchange. On the other hand, the barrier for charge injection from G (+*) back onto the A-T base pairs is strongly activated, so charge recombination from G (or even A trap sites at 85 K) most likely proceeds by a superexchange mechanism.     

Molecular assessment of S1 endonuclease-resistant snapback hairpin loops generated by DNA polymerase I during the in-vitro nick translation reaction

The in-vitro nick translation reaction used to label DNA to high specific activity also produces aberrant DNA structures known as “snapback” hairpin loops. Hairpin structures are precluded from participating in precise DNA-DNA hybridization interactions. Three nick translation systems were all found to yield significant quantities of snapback hairpins, as determined by their resistance to S1 endonuclease digestion following denaturation. The relative quantities of hairpins produced correlated with both the mass average size of the final DNA probe product synthesized as well as the overall rate of the nick translation reaction. Decreases in the amount of exogenous DNase I used in nick translation reactions produced significant decreases in the amount of hairpin loop structures formed. Hairpins could be effectively removed from nick-translated DNAs by employing hydroxylapatite column chromatography. Strategies to reduce hairpin formation during nick translation and the removal of hairpins from nick-translated DNAs are presented.     

Tethered DNA hairpins facilitate electrochemical detection of DNA ligation

A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.     

Structure and electronic spectra of DNA mini-hairpins with G(n):C(n) stems

The solution structure of a synthetic DNA mini-hairpin possessing a stilbenediether linker and three G:C base pairs has been obtained using (1)H NMR spectral data and constrained torsion angle molecular dynamics. Notable features of this structure include a compact hairpin loop having a short stilbene-guanine plane-to-plane distance and approximate B-DNA geometry for the three base pairs. Comparison of the electronic spectra of mini-hairpins having one-to-four G:C base pairs and stilbenediether or hexamethyleneglycol linkers reveals the presence of features in the UV and CD spectra of the stilbene-linked hairpins that are not observed for the ethyleneglycol-linked hairpins. Investigation of the electronic structure of a stilbene-linked hairpin having a single G:C base pair by means of time-dependent density functional theory shows that the highest occupied molecular orbital, but not the lowest unoccupied molecular orbital, is delocalized over the stilbene and adjacent guanine. The calculated UV and CD spectra are highly dependent upon hairpin conformation, but reproduce the major features of the experimental spectra. These results illustrate the utility of an integrated experimental and theoretical approach to understanding the complex electronic spectra of pi-stacked chromophores.     

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.     

Synthesis,structure, and photochemistry of exceptionally stable synthetic DNA hairpins with stilbene diether linkers

The structure and properties of 18 hairpin-forming bis(oligonucleotide) conjugates possessing stilbene diether linkers are reported. Conjugates possessing bis(2-hydroxyethyl)stilbene 4,4'-diether linkers form the most stable DNA hairpins reported to date. Hairpins with as few as two T:A base pairs or four noncanonical G:G base pairs are stable at room temperature. Increasing the length of the hydroxyalkyl groups results in a decrease in hairpin thermal stability. On the basis of the investigation of their circular dichroism spectra, all of the hairpins investigated adopt B-DNA structures, except for a hairpin with a short poly(G:C) stem which forms a Z-DNA structure. Both the strong fluorescence of the stilbene diether linkers and their trans-cis photoisomerization are totally quenched in hairpins possessing neighboring T:A and G:C base pairs. Quenching is attributed to an electron-transfer mechanism in which the singlet stilbene serves as an electron donor and T or C serves as an electron acceptor. In contrast, in denatured hairpins and hairpins possessing neighboring G:G base pairs the stilbene diether linkers undergo efficient photoisomerization.     

Probing the complete folding trajectory of a DNA hairpin using dual beam fluorescence fluctuation spectroscopy

The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.     

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.     

DNA as helical ruler: exciton-coupled circular dichroism in DNA conjugates

The structure and properties of oligonucleotide conjugates possessing stilbenedicarboxamide chromophores at both ends of a poly(dA):poly(dT) base-pair domain of variable length have been investigated using a combination of spectroscopic and computational methods. These conjugates form capped hairpin structures in which one stilbene serves as a hairpin linker and the other as a hydrophobic end-cap. The capping stilbene stabilizes the hairpin structures by ca. 2 kcal/mol, making possible the formation of a stable folded structure containing a single A:T base pair. Exciton coupling between the stilbene chromophores has little effect on the absorption bands of capped hairpins. However, exciton-coupled circular dichroism (EC-CD) can be observed for capped hairpins possessing as many as 11 base pairs. Both the sign and intensity of the EC-CD spectrum are sensitive to the number of base pairs separating the stilbene chromophores, as a consequence of the distance and angular dependence of exciton coupling. Calculated spectra obtained using a static vector model based on canonical B-DNA are in good agreement with the experimental spectra. Molecular dynamics simulations show that conformational fluctuations of the capped hairpins result in large deviations of the averaged spectra in both the positive and negative directions. These results demonstrate for the first time the ability of B-DNA to serve as a helical ruler for the study of electronic interactions between aligned chromophores. Furthermore, they provide important tests for atomistic theoretical models of DNA.     

Next generation hairpin polyamides with (R)-3,4-diaminobutyric acid turn unit

The characterization of a new class of pyrrole-imidazole hairpin polyamides with beta-amino-gamma-turn units for recognition of the DNA minor groove is reported. A library of eight hairpins containing ( R)- and ( S)-3,4-diaminobutyric acid (beta-amino-gamma-turn) has been synthesized, and the impact of the molecules on DNA-duplex stabilization was studied for comparison with the parent gamma-aminobutyric acid (gamma-turn) and standard ( R)-2,4-diaminobutyric acid (alpha-amino-gamma-turn)-linked eight-ring polyamides. For some, but not all, sequence compositions, melting temperature analyses have revealed that both enantiomeric forms of the beta-amino-gamma-turn increase the DNA-binding affinity of polyamides relative to the ( R)-alpha-amino-gamma-turn. The ( R)-beta-amine residue may be an attractive alternative for constructing hairpin polyamide conjugates. Biological assays have shown that ( R)-beta-amino-gamma-turn hairpins are able to inhibit androgen receptor-mediated gene expression in cell culture similar to hairpins bearing the standard ( R)-alpha-amino-gamma-turn, from which we infer they are cell-permeable.     

Single-molecule investigation of human telomeric G-quadruplex interactions with Thioflavin T

The interactions between human telomere sequence and a typical highly selective G-quadruplex ligand ThT were studied at the single-molecule level through α-hemolysin protein nanopore.     

Is hairpin formation in single-stranded polynucleotide diffusion-controlled?

An intriguing puzzle in biopolymer science is the observation that single-stranded DNA and RNA oligomers form hairpin structures on time scales of tens of microseconds, considerably slower than the estimated time for loop formation for a semiflexible polymer of similar length. To address the origin of the slow kinetics and to determine whether hairpin dynamics are diffusion-controlled, the effect of solvent viscosity (eta) on hairpin kinetics was investigated using laser temperature-jump techniques. The viscosity was varied by addition of glycerol, which significantly destabilizes hairpins. A previous study on the viscosity dependence of hairpin dynamics, in which all the changes in the measured rates were attributed to a change in solvent viscosity, reported an apparent scaling of relaxation times (tau(r)) on eta as tau(r) approximately eta(0.8). In this study, we demonstrate that if the effect of viscosity on the measured rates is not deconvoluted from the inevitable effect of change in stability, then separation of tau(r) into opening (tau(o)) and closing (tau(c)) times yields erroneous behavior, with different values (and opposite signs) of the apparent scaling exponents, tau(o) approximately eta(-0.4) and tau(c) approximately eta(1.5). Under isostability conditions, obtained by varying the temperature to compensate for the destabilizing effect of glycerol, both tau(o) and tau(c) scale as approximately eta(1.1+/-0.1). Thus, hairpin dynamics are strongly coupled to solvent viscosity, indicating that diffusion of the polynucleotide chain through the solvent is involved in the rate-determining step.     

Alpha- and beta-stilbenosides as base-pair surrogates in DNA hairpins

The synthesis, structure, and optical spectroscopy of hairpin oligonucleotide conjugates possessing synthetic stilbene C-nucleosides (stilbenosides) are reported. Synthetic methods for selective preparation of both the alpha- and beta-stilbenosides have been developed. Both anomers are effective in stabilizing hairpin structures when used as capping groups at the open end of the hairpin base-pair domain. However, only the beta-anomer effectively stabilizes the hairpin structure when located in the interior of the base-pair domain opposite an abasic site. Similar results are obtained for hairpins possessing two stilbenosides, either adjacent to each other or with one intervening base-pair. Molecular dynamics simulations are employed to obtain averaged structures for these conjugates. The calculated structures for the capped hairpins formed with either anomer show effective pi-stacking with the adjacent base-pair. The calculated structures for the internal stilbenosides show that the alpha- and beta-anomers form extrahelical and intrahelical structures, respectively. The relative orientations of the two stilbenes in the bis-stilbenosides have been studied using a combination of exciton-coupled circular dichroism spectroscopy and molecular modeling.     

Millisecond time-scale folding and unfolding of DNA hairpins using rapid-mixing stopped-flow kinetics

We report stopped-flow kinetics experiments to study the folding and unfolding of 5 base-pair stem and 21 nucleotide polythymidine loop DNA hairpins over various concentrations of NaCl. The reactions occurred on a time scale of milliseconds, considerably longer than the microsecond time scale suggested by previous kinetics studies of similar-sized hairpins. In comparison to a recent fluorescence correlation spectroscopy study (J. Am. Chem. Soc. 2006, 128, 1240-1249), we suggest the microsecond time-scale reactions are due to intermediate states and the millisecond time-scale reactions reported here are due to the formation of the fully folded DNA hairpin. These results support our view that DNA hairpin folding occurs via a minimum three-state mechanism.