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.     

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.     

Investigation of the pathways of excess electron transfer in DNA with flavin-donor and oxetane-acceptor modified DNA hairpins

Oxetane is a potential intermediate that is enzymatically formed during the repair of (6-4) DNA lesions by special repair enzymes (6-4 DNA photolyases). These enzymes use a reduced and deprotonated flavin to cleave the oxetane by single electron donation. Herein we report synthesis of DNA hairpin model compounds containing a flavin as the hairpin head and two different oxetanes in the stem structure of the hairpin. The data show that the electron moves through the duplex even over distances of 17 A. Attempts to trap the moving electron with N2O showed no reduction of the cleavage efficiency showing that the electron moves through the duplex and not through solution. The electron transfer is sequence dependent. The efficiency is reduced by a factor of 2 in GC rich DNA hairpins.     

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.     

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".     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Schiff Bases Derived from 6-Amino-2<Emphasis Type="Italic">H</Emphasis>-chromen-2-one. Synthesis and <Superscript>1</Superscript>H NMR Spectra

A number of 6-arylmethylideneamino-2H-chromen-2-ones were synthesized by reaction of 6-amino-2H-chromen-2-one with aromatic and heterocyclic aldehydes. A linear relation was revealed between the chemical shifts of the azomethine CH=N proton and protons in the chromene ring, on the one hand, and Hammett constants σ of the para substituents, on the other. Formation of intramolecular hydrogen bond in ortho-hydroxy derivatives induces a downfield shift of signals from protons in positions 3–5, 7, and 8 and CH=N proton (9-H) and upfield shift of the o-H signal (14-H).__________Translated from Zhurnal Organicheskoi Khimii, Vol. 41, No. 7, 2005, pp. 1085–1091.Original Russian Text Copyright © 2005 by Ganushchak, Kobrin, Bilaya, Mizyuk.     

Solution and solid-state models of peptide CH...O hydrogen bonds

Fumaramide derivatives were analyzed in solution by (1)H NMR spectroscopy and in the solid state by X-ray crystallography in order to characterize the formation of CH...O interactions under each condition and to thereby serve as models for these interactions in peptide and protein structure. Solutions of fumaramides at 10 mM in CDCl(3) were titrated with DMSO-d(6), resulting in chemical shifts that moved downfield for the CH groups thought to participate in CH...O=S(CD(3))(2) hydrogen bonds concurrent with NH...O=S(CD(3))(2) hydrogen bonding. In this model, nonparticipating CH groups under the same conditions showed no significant change in chemical shifts between 0.0 and 1.0 M DMSO-d(6) and then moved upfield at higher DMSO-d(6) concentrations. At concentrations above 1.0 M DMSO-d(6), the directed CH...O=S(CD(3))(2) hydrogen bonds provide protection from random DMSO-d(6) contact and prevent the chemical shifts for participating CH groups from moving upfield beyond the original value observed in CDCl(3). X-ray crystal structures identified CH...O=C hydrogen bonds alongside intermolecular NH...O=C hydrogen bonding, a result that supports the solution (1)H NMR spectroscopy results. The solution and solid-state data therefore both provide evidence for the presence of CH...O hydrogen bonds formed concurrent with NH...O hydrogen bonding in these structures. The CH...O=C hydrogen bonds in the X-ray crystal structures are similar to those described for antiparallel beta-sheet structure observed in protein X-ray crystal structures.     

β-Sheet 13C structuring shifts appear only at the H-bonded sites of hairpins

The (13)C chemical shifts measured for designed β-hairpins indicate that the structuring shifts (upfield for Cα and C', downfield for Cβ) previously reported as diagnostic for β-structuring in proteins appear only at the H-bonded strand residues. The resulting periodicity of structuring shift magnitudes is not, however, a consequence of H-bonding status; rather, it reflects a previously unrecognized alternation in the backbone torsion angles of β-strands. This feature of hairpins is also likely to be present in proteins. The study provides reference values for the expectation shifts for (13)C sites in β-structures that should prove useful in the characterization of the folding equilibria of β-sheet models.     

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.     

Folding of a stable DNA motif involves a highly cooperative network of interactions

Hairpins are structural elements that play important roles in the folding and function of RNA and DNA. The extent of cooperativity in folding is an important aspect of the RNA folding problem. We reasoned that an investigation into the origin of cooperativity might be best carried out on a stable nucleic acid system with a limited number of interactions, such as a stable DNA hairpin loop. The stable d(cGNAg) hairpin loop motif (closing base pair in lower case; loop in upper case; N = A, C, G, or T) is stabilized through only three interactions: two loop-loop hydrogen bonds in a sheared GA base pair and a loop-closing base pair interaction. Herein, we investigate this network of interactions and test whether the loop-loop and loop-closing base pair interactions communicate. Thermodynamic measurements of nucleotide analogue substituted oligonucleotides were used to probe the additivity of the interactions. On the basis of double mutant cycles, all interactions were found to be nonadditive and interdependent, suggesting that loop-loop and loop-closing base pair interactions form in a highly cooperative manner. When double mutant cycles were repeated in the absence of the other interaction, nonadditivity was significantly reduced suggesting that coupling is indirect and requires all three interactions in order to be optimal. A cooperative network of interactions helps explain the structural and energetic bases of stability in certain DNA hairpins and paves the way for similar studies in more complex nucleic acid systems.     

蛋白质全新设计：八残基序列形成发夹结构的圆二色谱

β-发夹是天然蛋白质中丰富的二级结构单元之一，在蛋白折叠和功能方面扮演着重要角色.文章报导了二条多肽序列（LTVd-PGLTV，n7和 LTVGDDTV， n5）的设计、合成和园二色谱研究结果.结果显示，n5在198 nm附近呈现负峰，表现为非规整结构特征；相反，n7表现为典型的发夹结构特征，在218 nm附近呈负峰,196 nm附近呈正峰，为β-转角与β-折叠的共同贡献.初步研究表明，β-转角、序列关系和氨基酸形成β-折叠结构倾向性是β-发夹结构形成和稳定的决定性因素.     

Effect of loop distortion on the stability and structural dynamics of DNA hairpin and dumbbell conjugates

The thermal stability and conformational dynamics of DNA hairpin and dumbbell conjugates having short A-tract base pair domains connected by tri- or hexa(ethylene glycol) linkers is reported. The formation of stable base-paired A-tract hairpins having oligo(ethylene glycol) linkers requires a minimum of four or five A-T base pairs. The formation of base-paired dumbbells having oligo(ethylene glycol) linkers by means of chemical ligation of nicked dumbbells requires a minimum of two A-T base pairs on either side of the nick. Molecular modeling indicates that the hexa(ethylene glycol) linker is sufficiently long to permit formation of strain-free loop regions and B-DNA base pair domains. In contrast, the tri(ethylene glycol) is too short to permit Watson-Crick base pairing between the bases attached to the linker. The shorter linker distorts the duplex, resulting in fluxional behavior in which the base pairs adjacent to the linker and at the open end of the hairpin dissociate on the nanosecond time scale. The loss of interstrand binding energy caused by these fluctuations leads to a difference of approximately 5 degrees C in melting temperature between EG3 and EG6 hairpins. An analysis of the fluxional behavior of the EG3 adjacent base-pair has been used to study the pathways for base flipping and base stacking, including the identification of rotated base (partially flipped) intermediates that have not been described previously for A-T base pairs.