Loop and stem dynamics during RNA hairpin folding and unfolding

2-Aminopurine (2AP) is a fluorescent adenine analog that probes mainly base stacking in nucleic acids. We labeled the loop or the stem of the RNA hairpin gacUACGguc with 2AP to study folding thermodynamics and kinetics at both loci. Thermal melts and fast laser temperature jumps detected by 2AP fluorescence monitored the stability and folding/unfolding kinetics. The observed thermodynamic and kinetic traces of the stem and loop mutants, though strikingly different at a first glance, can be fitted to the same free-energy landscape. The differences between the two probe locations arise because base stacking decreases upon unfolding in the stem, whereas it increases in the loop. We conclude that 2AP is a conservative adenine substitution for mapping out the contributions of different RNA structural elements to the overall folding process. Molecular dynamics (MD) totaling 0.6 μsec were performed to look at the conformations populated by the RNA at different temperatures. The combined experimental data, and MD simulations lead us to propose a minimal four-state free-energy landscape for the RNA hairpin. Analysis of this landscape shows that a sequential folding model is a good approximation for the full folding dynamics. The frayed state formed initially from the native state is a heterogeneous ensemble of structures whose stem is frayed either from the end or from the loop.     

Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence

2-Aminopurine (2AP) is an analogue of adenine that has been utilized widely as a fluorescence probe of protein-induced local conformational changes in DNA. Within a DNA strand, this fluorophore demonstrates characteristic decreases in quantum yield and emission decay lifetime that vary sensitively with base sequence, temperature, and helix conformation but that are accompanied by only small changes in emission wavelength. However, the molecular interactions that give rise to these spectroscopic changes have not been established. To develop a molecular model for interpreting the fluorescence measurements, we have investigated the effects of environmental polarity, hydrogen bonding, and the purine and pyrimidine bases of DNA on the emission energy, quantum yield, and intensity decay kinetics of 2AP in simple model systems. The effects of environmental polarity were examined in a series of solvents of varying dielectric constant, and hydrogen bonding was investigated in binary mixtures of water with 1,4-dioxane or N,N-dimethylformamide (DMF). The effects of the purine and pyrimidine bases were studied by titrating 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine (rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosphates ATP and GTP in neutral aqueous solution. The nucleosides and NTPs each quench the fluorescence of d2AP by a combination of static (affecting only the quantum yield) and dynamic (affecting both the quantum yield and the lifetime, proportionately) mechanisms. The peak wavelength and shape of the emission spectrum are not altered by either of these effects. The static quenching is saturable and has half-maximal effect at approximately 20 mM nucleoside or NTP, consistent with an aromatic stacking interaction. The rate constant for dynamic quenching is near the diffusion limit for collisional interaction (k(q) approximately 2 x 10(9) M(-1) s(-1)). Neither of these effects varies significantly between the various nucleosides and NTPs studied. In contrast, hydrogen bonding with water was observed to have a negligible effect on the emission wavelength, fluorescence quantum yield, or lifetime of 2AP in either dioxane or DMF. In nonpolar solvents, the fluorescence lifetime and quantum yield decrease dramatically, accompanied by significant shifts in the emission spectrum to shorter wavelengths. However, these effects of polarity do not coincide with the observed emission wavelength-independent quenching of 2AP fluorescence in DNA. Therefore, we conclude that the fluorescence quenching of 2AP in DNA arises from base stacking and collisions with neighboring bases only but is insensitive to base-pairing or other hydrogen bonding interactions. These results implicate both structural and dynamic properties of DNA in quenching of 2AP and constitute a simple model within which the fluorescence changes induced by protein-DNA binding or other perturbations may be interpreted.     

Excitation wavelength dependence of the fluorescence kinetics in Photosystem I particles from Synechocystis PCC 6803 and Synechococcus elongatus

The excitation-wavelength dependence of the excited-state dynamics of monomeric and trimeric Photosystem I (PSI) particles from Synechocystis PCC 6803 as well as trimeric PSI particles from Synechococcus elongatus has been studied at room temperature using time-resolved fluorescence spectroscopy. For aselective (400 nm), carotenoid (505 nm), and bulk chlorophyll (approximately 650 nm) excitation in all species, a downhill energy-transfer component is observed, corresponding to a lifetime of 3.4-5.5 ps. For selective red excitation (702-719 nm) in all species, a significantly faster, an approximately 1-ps, uphill transfer component was recorded. In Synechococcus PSI, an additional approximately 10-ps downhill energy-transfer component is found for all wavelengths of excitation, except 719 nm. Each of the species exhibits its own characteristic trap spectrum, the shape of which is independent of the wavelength of excitation. This trap spectrum decays in approximately 23 ps in both monomeric and trimeric Synechocystis PSI and in approximately 35 ps in trimeric Synechococcus PSI. The data were simulated based on the 2.5 A structural model of PSI of Synechococcus elongatus using the F?rster equation for energy transfer, and using the 0.6-1-ps charge-separation time and the value of 1.2-1.3 for the index of refraction that were obtained from the dynamics of a hypothetical PSI particle without red chls. The experimentally obtained lifetimes and spectra were reproduced well by assigning three of the chlorophyll-a (chla) dimers observed in the structure to the C708/C702RT pool of red chls present in PSI from both species. Essential for the simulation of the dynamics of Synechococcus PSI is the assignment of the single chla trimer in the structure to the C719/C708RT pool present in this species.     

Intramolecular recombination R-triplex in solution: stabilization by bis-intercalator YOYO

Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5'-GT-2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3' (R(2AP)CW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5'-CTCAGTCTACGC GAA GCGTAGACTGAG-3' (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R(2AP)CW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19 degrees C to 33 degrees C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R(2AP)CW triplex and up to six YOYO molecules to CW hairpin.     

Structure and dynamics in DNA looped domains: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence

The triplet repeat sequence (CAG)n and related triplet repeats are associated with dynamic DNA mutations implicated in a number of debilitating human diseases. To gain insight into the dynamics of the (CAG)n repeat, we have substituted a single 2-aminopurine (2AP) fluorescent base for adenine at select positions within the 18 base looped domain of a (GC)3(CAG)6(GC)3 hairpin oligonucleotide. Using temperature-dependent steady-state fluorescence measurements in combination with time correlated photon counting spectroscopy, we show the conformation and dynamics of the C2APG domains to be strongly dependent on the position of the probe in the looped region. In other words, rather than being a uniform, single stranded loop, the (CAG)6 triplet repeat looped domain exhibits order and dynamics that are position dependent. The 2AP fluorescence dynamics within the C2APG repeat are well described by a 4 component exponential decay model, with lifetimes ranging from 5 ps to 4 ns. Differences in global DNA conformation (duplex, hairpin, single strand), as well as the local position of the probe within the loop of a given hairpin, predominantly are reflected in the relative amplitude rather than the lifetime of the probe. The time dependent 2AP anisotropy in the hairpin (CAG)n loops is sensitive to the position of the fluorescent base, with the fluorescence depolarization of a centrally located 2AP probe within the loop proceeding significantly more slowly than 2AP positioned at the 5'- or 3'-end of the repeat sequence near the loop-stem junction. These results are consistent with segmental motions of the CAG repeat, while also suggesting that the 2AP probe is significantly stacked, possibly even hydrogen bonded, within the partially structured CAG looped domain. Our results characterize the position-dependent and conformation-dependent dynamics and order within (CAG)n triplet repeat DNAs, properties of relevance to the biological mechanisms by which such domains can lead to disease states.     

Energy transfer in spectrally inhomogeneous light-harvesting pigment-protein complexes of purple bacteria.

Energy transfer within the peripheral light-harvesting antenna of the purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris was studied by one- and two-color pump-probe absorption spectroscopy with approximately 100-fs tunable pulses at room temperature and at 77 K. The energy transfer from B800 to B850 occurs with a time constant of 0.7 +/- 0.05 ps at room temperature and 1.8 +/- 0.2 ps at 77 K and is similar in both species. Anisotropy measurements suggest a limited but fast B800 <--> B800 transfer time (tau approximately 0.3 ps). This is analyzed as incoherent hopping of the excitation in a system of spectrally inhomogeneous antenna pigment-protein complexes, by a master equation approach. The simulations show that the measured B800 dynamics is well described as energy transfer with a characteristic average nearest-neighbor pairwise transfer time of 0.35 ps among approximately 10 Bchl molecules in a circular arrangement, in good agreement with the recent high-resolution structure of LH2. The possible presence of fast intramolecular relaxation processes within the Bchl a molecule was investigated by measurement of time-resolved difference absorption spectra and kinetics of Bchl a in solution and in low-temperature glasses. From these measurements it is concluded that fast transients observed at room temperature are due mainly to solvation processes, whereas at 77 K predominantly slower (> 10-ps) relaxation occurs.     

Ultrafast fluorescence decay profiles reveal differential unstacking of 2-aminopurine from neighboring bases in single-stranded DNA-binding protein subsites

Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.     

Sequence dependence of energy transfer in DNA oligonucleotides

The sequence, temperature, concentration, and solvent dependence of singlet energy transfer from normal DNA bases to the 2-aminopurine base in synthesized DNA oligomers were investigated by optical spectroscopy. Transfer was shown directly by a variable fluorescence excitation band at 260-280 nm. Adenine (A) is the most efficient energy donor by an order of magnitude. Stacks of A adjacent to 2AP act as an antenna for 2AP excitation. An interposed G, C, or T base between A and 2AP effectively blocks transfer from A to 2AP. Base stacking facilitates transfer, while base pairing reduces energy transfer slightly. The efficiency is differentially temperature dependent in single- and double-stranded oligomers and is highest below 0 degrees C in samples measured. An efficiency transition occurs well below the melting transition of a double-stranded decamer. The transfer efficiency in the duplex decamer d(CTGA[2AP]TTCAG)(2) is moderately dependent on the sample and salt concentration and is solvent dependent. Transfer at physiological temperature over more than a few bases is improbable, except along consecutive A's, indicating that singlet energy transfer is not a major factor in the localization of UV damage in DNA. These results have features in common with recently observed electron transfer from 2AP to G in oligonucleotides.     

Dynamics of mismatched base pairs in DNA

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.     

Recognition of a flipped base in a hairpinloop DNA by a small peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258-259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 x 10(6) variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use pi-pi stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 x 10(6) M(-1) at 25 degrees C and the resulting binding free energy change at 25 degrees C (DeltaG degrees (25)) was -8.2 kcal mol(-1). The binding of the peptide to AP2 was also analyzed and the resulting binding constant and DeltaG degrees (25) were about 4.2 x 10(4) M(-1) and -6.3 kcal mol(-1), respectively. The difference in the binding free energy changes (DeltaDeltaG degrees (25)) of 1.9 kcal mol(-1) was comparable to the values reported in other systems and was considered a consequence of the loss of pi-pi stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Hairpins in a DNA site for topoisomerase II studied by 1H- and 31P-NMR.

1H- and 31P-NMR and UV-absorption studies were carried out with the oligonucleotide strands d(AGCT-TATC-ATC-GATAAGCT) (-ATC-) and d(AGCTTATC-GAT-GATAAGCT) (-GAT-) contained in the strongest and salt resistant cleavage site for topoisomerase II in pBR322 DNA. We found that the two oligonucleotides were stabilized under a hairpin structure characterized by a eight base pair stem and a three base loop at low DNA and salt concentrations. In such experimental conditions, only the -GAT- oligonucleotide displayed a partial homoduplex structure in slow equilibrium with its folded structure. Temperature dependencies of imino protons showed that the partial homoduplex of -GAT- melted at a lower temperature than the hairpin structure. It was suggested that the appearance of the partial homoduplex in -GAT- is related to the formation of two stabilizing (G.T) mismatched base pairs in the central loop of this structure. Finally, it was inferred from the dispersion of chemical shifts in the 31P-NMR spectra that the distortions affecting the backbone of the hairpin loop are larger in the case of -ATC- compared with -GAT-. At the same time NOEs proved that the base stacking was stronger within the loop of the -ATC- hairpin.     

Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine

Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.     

Prompt non-stacking binding of actinomycin D to hairpin oligonucleotide HP1 and slow redistribution from HP1 to DNA

Complexes of actinomycin D (AMD) and 7-amino-actinomycin D (7AAMD) with model hairpin oligonucleotide HP1 and various types of DNA in aqueous solutions were investigated by steady-state, polarized, time-resolved and stopped-flow fluorimetry, and photometry. Prompt non-stacking binding of the actinomycins inside HP1 was observed. No energy transfer from nucleotides to 7AAMD in the complex was detected, most likely because of the absence of stacking intercalation. Complex formation of AMD or 7AAMD and HP1 was followed by the transition from a random flexible conformation of the hairpin to a more compact rigid structure, and subsequently to hypochromism. Strong competition between AMD and 7AAMD for a cavity in HP1 was observed. The decrease in the 7AAMD emission after addition of DNA to the 7AAMD/HP1 complex indicates that actinomycins can be redistributed from HP1 to DNA, i.e. hairpin oligonucleotides can serve as molecular carriers of actinomycins.     

Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies

The self-complementary DNA decamer duplex d(CTGAATTCAG)2 and its modified counterpart d(CTGA[2AP]TTCAG)2, where the innermost adenine (6-aminopurine) has been replaced with the fluorescent analogue 2-aminopurine (2AP), have been studied by fluorescence and NMR spectroscopy and simulated by molecular dynamics. Both decamers are recognized and cleaved by the EcoRI restriction endonuclease. 2D NMR results show that both decamers have a standard B-type conformation below 20 degrees C, though a disturbance exists to the 5' side of the 2AP site which may originate from increased local mobility. The fluorescence and fluorescence anisotropy decays of both decamers, as well as the one containing 2AP in only one chain, were studied as a function of temperature. The data show that the 2AP base exists in a temperature-dependent distribution of states and shows rapid motions, suggesting interconversion among these states on a time scale of about 10(-10) s. The integrated fluorescence of the decamer with 2AP in both chains shows a large increase around the helix melting temperature whereas the decamer with one 2AP shows only a mild increase, showing that the mixed helix has a different structural transition as sensed by the 2AP base. The data suggest a model of conformational states which have distinct fluorescence decay times. The various states may differ in the degree of base stacking. Fluctuations in the degree of stacking of the A or 2AP base are supported by molecular dynamics simulations, which additionally show that the 2AP-T or A-T base pair hydrogen bonds remain intact during these large motions.     

Synthesis and properties of fluorescent NF-kappa B-recognizing hairpin oligodeoxyribonucleotide decoys

Intramolecular fluorescence quenching of cyanine dyes was investigated using a model hairpin oligonucleotide decoy encoding a NF-kappaB p50 subunit binding site. Two types of hairpin oligonucleotides were synthesized: (1) 5'-(6-aminohexyl)- and 3'-(3-aminopropyl)-linked (I); (2) 5'-(6-aminohexyl)- and 3'-[3-(3-hydroxypropyldithio)propyl]-linked (II). Oligonucleotide I was covalently modified using monofunctional either Cy3- or Cy5.5-N-hydroxysuccinimide esters. Using reverse-phase HPLC, mono-and dicyanineamide derivatives of I were isolated. Mono-Cy3-modified derivatives of I, but not the mono-Cy5.5-modified derivatives, showed a 2-fold higher Cy3 fluorescence intensity compared to the free dye. There was no detectable difference in fluorescence between the di-Cy3 derivative of I and the free dye at the same concentration. However, there was a 4-fold quenching of fluorescence in the case of the di-Cy5.5 derivative of the same hairpin oligonucleotide. The quenching of Cy5.5 fluorescence could not be explained by the interaction of Cy5.5 with nucleotide bases as demonstrated by incubating free Cy5.5 dye with oligonuclotides. The quenching effect was further investigated using an oligonucleotide bearing a cleavable 3'-amino-terminated linker bearing an S-S bond (III). After modification of the 5'- and 3'-end of oligonucleotide III with a Cy5.5 monofunctional hydroxysuccinimide ester, a 70-75% quenching of fluorescence was observed. Fluorescence was 100% dequenched after the reduction of S-S bond. The obtained result unequivocally demonstrates that the formation of intramolecular Cy5.5 dimers is the dominant mechanism of fluorescence quenching in symmetric dye-dye hairpin decoy beacons.     

Recognition of a Flipped Base in a Hairpinloop DNA by a Small Peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258–259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 × 10⁶ variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use π–π stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 × 10⁶ M^?1 at 25°C and the resulting binding free energy change at 25°C (ΔG°₂₅) was ?8.2 kcal mol^?1. The binding of the peptide to AP2 was also analyzed and the resulting binding constant and ΔG°₂₅ were about 4.2 × 10⁴ M^?1 and ?6.3 kcal mol^?1, respectively. The difference in the binding free energy changes (ΔΔG°₂₅) of 1.9 kcal mol^?1 was comparable to the values reported in other systems and was considered a consequence of the loss of π–π stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Intramolecular Recombination R-Triplex in Solution: Stabilization by bis-intercalator YOYO

Abstract

Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5′-GT- 2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3′ (R^2APCW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5′- CTCAGTCTACGC GAA GCGTAGACTGAG-3′ (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R^2APCW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19° C to 33° C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R^2APCW triplex and up to six YOYO molecules to CW hairpin.     

Drug recognition of a DNA single strand break: nogalamycin intercalation between coaxially stacked hairpins.

Two DNA hairpin motifs (5'-GCGAAGC-3' and 5'-ACGA AGT-3'), both stabilized by a 5'-GAA loop, have been used to design novel intramolecular double hairpin structures (5'-GCGAAGCACGAAGT-3' and 5'-ACGAAGTGCG AAGC-3') in which coaxial stacking of the two hairpin components generates a double-stranded stem region effectively with a single-strand break in the middle of the sequence at either the TG or CA step between unconnected 3' and 5' terminal bases. We have investigated by NMR the conformation and dynamics of the DNA at the strand break site. We show that mutual stacking significantly enhances the stability of each hairpin. Further, the anthracycline antibiotic nogalamycin binds cleanly to the 5'-TG (5'-CA) site formed by the mutually stacked hairpins despite the break in the sugar-phosphate backbone on one strand. The complex resembles the structure of nogalamycin-DNA complexes with the drug bound at 5'-TG sites in intact duplex sequences, with pi-stacking interactions probably the single dominant stabilizing interaction.     

Stacking-unstacking dynamics of oligodeoxynucleotide trimers

The structure and dynamics of DNA trimers are experimentally assessed using the fluorescent purine analogue 2-aminopurine (2AP), incorporating 2AP between purine and pyrimidine bases to form 5'dXp2APpY3' molecules. Circular dichroism and fluorescence quenching of the 2AP show that the bases are stacked; at the same time, fluorescence decay lifetimes are heterogeneous, indicative of conformational sampling. 2AP does not exhibit the long fluorescence decay time characteristic of the free nucleoside, suggesting that its motions in the trimers bring it into proximity of the neighboring bases, resulting in efficient charge transfer and average fluorescence lifetimes on the order of 1-2 ns.