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.     

Molecular dynamics simulation of the structure, dynamics, and thermostability of the RNA hairpins uCACGg and cUUCGg

Classical replica-exchange molecular dynamics simulations are performed to study structure, dynamics and thermostability of the 14-mer RNA hairpins uCACGg and cUUCGg. Despite of the different sequence and closing base pair of the two systems, recent NMR studies have shown that the tetraloop CACG is strikingly similar in overall geometry and hydrogen bonding to the canonical UUCG tetraloop. On the other hand, the two systems differ significantly in their functionality and thermostability. The simulations confirm the structural similarities of the two RNA hairpins at room temperature but also reveal that the UUCG loop is more flexible than the CACG loop. Concerning the functionality, the CACG loop shows a stronger attitude to donate hydrogens than the UUCG loop, although their global solvent accessible surface is quite similar. The simulations qualitatively reproduce the experimentally found difference in melting temperatures (20 K). In the case of the uCACGg hairpin, the thermal unfolding occurs cooperatively in an all-or-none fashion, while the cUUCGg hairpin shows less cooperativity but exhibits intermediate states during the unfolding process.     

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.     

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.     

Spectroscopic and structural impact of a stem base-pair change in DNA hairpins: GTTC-ACA-GAAC versus GTAC-ACA-GTAC

Successive investigations over the last decade have revealed and confirmed a stable loop closure in a family of d-[GTAC-5Pur6N7N-GTAC] hairpins, where 5Pur6N7N is a AAA, GAG and AXC loop (X being any nucleotide). The trinucleotide loop is characterized by a well defined 5Pur-7N mispairing mode, and by upfield chemical shifts for three sugar protons of the apical nucleotide 6N. The GTTC-ACA-GAAC DNA hairpin, of interest for its likely involvement in Vibrio cholerae genome mutations, has now been investigated. The GTAC-ACA-GTAC DNA hairpin has also been studied because it is intermediate between the other structures, as it contains the loop of the hairpin under consideration and the stem of the above family. The two hairpins with the ACA loop are stable. They show the same mispairing mode and similar upfield shifts as the previous family, but GTTC-ACA-GAAC seems to be slightly less compact than any other. GTTC-ACA-GAAC is remarkable in that it exhibits a B(II) character for the phosphate-ester conformation at 8Gp9A, together with a swing of the upper hairpin into the major groove that, in particular, brings 6CH1' roughly as close to 7AH2 as to 6CH6. These unexpected structural features are qualitatively deduced from (1)H and (31)P NMR spectra, and confirmed by Raman spectroscopy. This comparative study shows that not only the loop sequence but also the stem sequence may control hairpin structures.     

A three-state mechanism for DNA hairpin folding characterized by multiparameter fluorescence fluctuation spectroscopy

The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.     

Phosphorescence spectra and triplet state lifetimes of palladium octaethylporphyrin,palladium octaethylchlorin and palladium 2,3-dimethyloctaethylisobacteriochlorin at 77 K

The phosphorescence spectra and triplet state lifetimes of palladium octaethylporphyrin (PdOEP), palladium octaethylchlorin (PdOEC) and palladium 2,3-dimethyloctaethylisobacteriochlorin (PdOEiBC) in n-octane Shpolskii matrices at 77 K are reported. The lifetime and T(1)/S(0) origin energy of each complex are: PdOEP, 1.90+/-0.04 ms, 15162 cm(-1); PdOEC, 0.43+/-0.03 ms, 12547 cm(-1) and PdOEiBC, 0.59+/-0.03 ms, 12863 cm(-1).     

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.     

A novel RNA motif based on the structure of unusually stable 2',5'-linked r(UUCG) loops

We have recently shown that hairpins containing 2',5'-linked RNA loops exhibit superior thermodynamic stability compared to native hairpins comprised of 3',5'-RNA loops [Hannoush, R. N.; Damha, M. J. J. Am. Chem. Soc. 2001, 123, 12368-12374]. A remarkable feature of the 2',5'-r(UUCG) tetraloop is that, unlike the corresponding 3',5'-linked tetraloop, its stability is virtually independent of the hairpin stem composition. Here, we determine the solution structure of unusually stable hairpins of the sequence 5'-G(1)G(2)A(3)C(4)-(U(5)U(6)C(7)G(8))-G(9)(U/T(10))C(11)C(12)-3' containing a 2',5'-linked RNA (UUCG) loop and either an RNA or a DNA stem. The 2',5'-linked RNA loop adopts a new fold that is completely different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a). U5.G8 wobble base pairing, with both nucleotide residues in the anti-conformation, (b). extensive base stacking, and (c). sugar-base and sugar-sugar contacts, all of which contribute to the extra stability of this hairpin structure. The U5:G8 base pair stacks on top of the C4:G9 loop-closing base pair and thus appears as a continuation of the stem. The loop uracil U6 base stacks above U5 base, while the cytosine C7 base protrudes out into the solvent and does not participate in any of the stabilizing interactions. The different sugar pucker and intrinsic bonding interactions within the 2',5'-linked ribonucleotides help explain the unusual stability and conformational properties displayed by 2',5'-RNA tetraloops. These findings are relevant for the design of more effective RNA-based aptamers, ribozymes, and antisense agents and identify the 2',5'-RNA loop as a novel structural motif.     

Thermodynamic coupling of the loop and stem in unusually stable DNA hairpins closed by CG base pairs

For certain DNA hairpin loops, a CG closing base pair has enhanced stability over other closing base pairs, which cannot be explained by the current nearest-neighbor model. We report the use of three-carbon (C3) spacers to investigate the expandability of DNA hairpin loops and the coupling between the loop and closing base pair. Inserting the C3-spacers at most positions in these model loops produced only a modest stabilization or destabilization except for insertion between the 5' end of the loop and the CG closing base pair, which gave a large destabilization. Further investigation on tetraloops and triloops with other closing base pairs established that this destabilization is specific to the unusually stable CG closing base pair. Studies with the nucleotide analogues 2-aminopurine and 2,6-diaminopurine indicated that this stabilization may be due to coupling between functional groups on the first base of the loop and the CG closing base pair. The C3-spacers provide a simple way to interrupt potential interactions and thereby probe loop/stem coupling.     

A minimal hybridization chain reaction (HCR) system using peptide nucleic acids

The HCR represents a powerful tool for amplification in DNA-based circuitry and sensing applications, yet requires the use of long DNA sequences to grant hairpin metastability. Here we describe a minimal HCR system based on peptide nucleic acids (PNAs). A system comprising a 5-mer stem and 5-mer loop/toehold hairpins was found to be suitable to achieve rapid amplification. These hairpins were shown to yield >10-fold amplification in 2 h and be suitable for the detection of a cancer biomarker on live cells. The use of γ-peg-modified PNA was found to be beneficial.

A minimal peptide nucleic acid (PNA) HCR system based on a 5-mer stem and 5-mer loop/toehold hairpins was developed. The system was applied to the detection of a cancer biomarker on the surface of living cells.     

Metallation-Induced Heterogeneous Dynamics of DNA Revealed by Single-Molecule FRET

The metallation of nucleic acids is key to wide-ranging applications, from anticancer medicine to nanomaterials, yet there is a lack of understanding of the molecular-level effects of metallation. Here, we apply single-molecule fluorescence methods to study the reaction of an organo-osmium anticancer complex and DNA. Individual metallated DNA hairpins are characterised using Förster resonance energy transfer (FRET). Although ensemble measurements suggest a simple two-state system, single-molecule experiments reveal an underlying heterogeneity in the oligonucleotide dynamics, attributable to different degrees of metallation of the GC-rich hairpin stem. Metallated hairpins display fast two-state transitions with a two-fold increase in the opening rate to ≈2 s⁻¹, relative to the unmodified hairpin, and relatively static conformations with long-lived open (and closed) states of 5 to ≥50 s. These studies show that a single-molecule approach can provide new insight into metallation-induced changes in DNA structure and dynamics.     

Probing RNA hairpins with cobalt(III)hexammine and electrospray ionization mass spectrometry

In this work, electrospray ionization mass spectrometry (ESI MS) was employed to study the interactions of cobalt(III) hexammine, Co(NH3)6(3+), with five RNA hairpins representing the 790 loop of 16S ribosomal RNA and 1920 loop of 23S ribosomal RNA. The RNAs varied in mismatch identity (G.U versus A.C) and level of base modification (pseudouridine versus uridine). Co(NH3)6(3+) binding was observed with the four RNA hairpins that contained a G.U wobble pair in the stem region. ESI MS revealed 1:1 and 1:2 complex formation with all RNAs. Weaker binding was observed with the fifth RNA hairpin that contained an A.C wobble pair in the stem region. The effects of pH on Co(NH3)6(3+) binding were also examined.     

Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research

A new concept based on fluorescence lifetime correlation spectroscopy (FLCS) is presented allowing the simultaneous determination of diffusion coefficients of identical molecules located in different environments. The difference in fluorescence lifetimes, which is the main prerequisite for FLCS, is reached by locating one population of the dye close to a light-absorbing surface. Since such surfaces quench fluorescence, the fluorescence lifetime of chromophores located close to these surfaces can be tuned in a specific manner. This approach has been demonstrated for a BODIPY-tail-labeled lipid in supported phospholipid bilayers (SPBs) as well as in phospholipid multilayers adsorbed onto solid supports. In particular, the effect of the solid support type on the fluorescence lifetime as well as its dependence on the BODIPY-support distance has been characterized and verified by theoretical considerations based on precise determination of refractive indices of the used supports. While the fluorescence lifetime of BODIPY dye is 5.6 ns in small unilamellar vesicles (SUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), the lifetime is 1.8 ns in DOPC/DOPS SPBs adsorbed onto ITO-covered glass or 3.0 ns in a DOPC/DOPS monolayer adsorbed onto seven 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) layers on oxidized silicon. Using these particular systems, we demonstrated that FLCS enables one to characterize simultaneously two-dimensional lipid diffusion in the planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers using single dye labeling. The autocorrelation functions obtained by this new approach do agree with those obtained by standard FCS on isolated SPBs or vesicles. Possible applications of this virtual two-channel measurement using single dye labeling as well as one detection channel are discussed.     

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.     

Remarkable stability of hairpins containing 2',5'-linked RNA loops.

We report here the results of a comparative study of hairpin loops that differ in the connectivity of phosphodiester linkages (3',5'- versus 2',5'-linkages). In addition, we have studied the effect of changing the stem composition on the thermodynamic stability of hairpin loops. Specifically, we constructed hairpins containing one of six stem duplex combinations, i.e., DNA:DNA ("DD"), RNA:RNA ("RR"), DNA:RNA ("DR"), 2',5'-RNA:RNA ("RR"), 2',5'-RNA:DNA ("RD"), and 2',5'-RNA:2',5'-RNA ("RR"), and one of three tetraloop compositions, i.e., 2',5'-RNA ("R"), RNA ("R"), and DNA ("D"). All hairpins contained the conserved and well-studied loop sequence 5'-...C(UUCG)G...-3' [Cheong et al. Nature 1990, 346, 680-682]. We show that the 2',5'-linked loop C(UUCG)G, i.e.,...C(3'p5')U(2'p5')U(2'p5')C(2'p5')G(2'p5')G(3'p5')..., like its "normal" RNA counterpart, forms an unusually stable tetraloop structure. We also show that the stability imparted by 2',5'-RNA loops is dependent on base sequence, a property that is shared with the regioisomeric 3',5'-RNA loops. Remarkably, we find that the stability of the UUCG tetraloop is virtually independent of the hairpin stem composition (DD, RR, RR, etc.), whereas the native RNA tetraloop exerts extra stability only when the stem is duplex RNA (R:R). As a result, the relative stabilities of hairpins with a 2',5'-linked tetraloop, e.g. ggac(UUCG)gtcc (T(m) = 61.4 degrees C), are often superior to those with RNA tetraloops, e.g. ggac(UUCG)gtcc (T(m) = 54.6 degrees C). In fact, it has been possible to observe the formation of a 2',5'-RNA:DNA hybrid duplex by linking the hybrid's strands to a (UUCG) loop. These duplexes (RD), which are not stable enough to form in an intermolecular complex [Wasner et al. Biochemistry 1998, 37, 7478-7486], were stable at room temperature (T(m) approximately 50 degrees C). Thus, 2',5'-loops have potentially important implications in the study of nucleic acid complexes where structural data are not yet available. Furthermore, they may be particularly useful as structural motifs for synthetic ribozymes and nucleic acid "aptamers".     

Context-dependent photodimerization in isolated thymine-thymine steps in DNA

The effect of 280 nm irradiation on a family of synthetic DNA hairpins possessing an alkane linker connecting a six-base pair stem having a single T-T step located at different positions within the hairpin has been investigated. A single adduct assigned to the product of 2+2 dimerization is obtained except in the case of a T-T step located adjacent to the linker, in which case both 2+2 and 6-4 adducts are obtained. The efficiency of dimerization is similar for three hairpins having a T-T step located within the duplex interior. Lower efficiency is observed for a T-T step located at the open end of the hairpin and in T overhangs, whereas higher efficiency is observed for the T-T step adjacent to the linker and in a single T bulge. The context-dependence of dimerization efficiency is discussed.     

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.     

Structural and energetic consequences of expanding a highly cooperative stable DNA hairpin loop

Many hairpin loops are expanded versions of smaller, stable ones. Herein we investigate the extent to which the energetics and structure of d(cGNAg) hairpin loops will tolerate sequence variation. Changing the closing base pair from CG to GC was found to completely eliminate loop-loop interactions; in contrast, expanding the loop at the 3'-end resulted in similar energetics and nonadditivity parameters as the parent loop, suggesting that loop-loop interactions remain intact and highly coupled upon expansion. Together, these data suggest that the CG closing base pair forms an essential platform upon which a stable d(GNA) hairpin loop can fold and that this loop can undergo 3'-expansion with little effect to its structure or energetics.     

Configuration dependent critical nuclei in the self assembly of magic clusters

Evidence for the formation of various 2-D structures possessing different numbers of Co-Si magic clusters (size approximately 10.0 +/- 0.5 A), configurations and lifetimes are studied in real time on a Si(111)-(7 x 7) surface at elevated temperature in the STM. Observations of individual cluster diffusion, attachment and detachment dynamics resolve unequivocally the question of self assembly over surface reconstruction. The smallest stable structure consisting of seven individual Co-Si magic clusters arranged in a hexagonal closed packed formation (i = 7) is found to retain sufficient cohesive energy to avoid dissociation. A configuration dependent critical 2-D nuclei (i* = 6) is determined to exist in facilitating the self assembly dynamics.