Comparison of the B- and Z-form hairpin loop structures formed by d(CG)5T4(CG)5

The partially self-complementary synthetic DNA oligonucleotide d(CG)5T4(CG)5 has been studied by using 1H and 31P NMR and circular dichroism. Results show that, under low-salt conditions (120 mM NaCl buffer), an intramolecular hairpin loop exists in which the double-helical stem region is B-form and the thymidine loop residues have predominantly southern (C2'-endo) sugar conformations. The thymidine glycosidic torsion angles are intermediate between syn and anti or exist as an equilibrium mixture of residues in the two extremes. NOESY data indicate that the structure of the loop region is very similar to that found for d(CG)2T4(CG)2 [Hare, D. R., & Reid, B. R. (1986) Biochemistry 25, 5341-5350]. Under high-salt conditions (6 M NaClO4 buffer), the dominant form (approximately equal to 85%) is an intramolecular hairpin structure in which the stem region forms a Z-form double helix. As in the B-form, the loop thymidine residues are intermediate between the syn and anti conformations or exist as an equilibrium mixture of the two, but the thymidine sugar conformations differ in that they are biased toward northern (C3'-endo) conformations.     

Anomalous hairpin formation in an oligodeoxyribonucleotide.

An accurate method for deriving molar absorptivity-temperature profiles applied to a set of single-stranded oligodeoxyribonucleotides shows that the undecamer CGAGTTTGACGp exists in a hairpin conformation involving Watson-Crick base pairing between the two terminal CG dinucleotides. The hairpin, which has a transition midpoint of 40 degrees C in 0.115 M Na+, is unusually stable in comparison with previously reported hairpins. A non-linear least squares analysis of the undecamer's profile in terms of a two-state equilibrium model indicates that the hairpin-to-coil transition occurs with an enthalpy change about twice that expected if only combinations of Watson-Crick base-paired stacking interactions are considered. The analogous hairpin structure (containing an identical CG/CG stem) assignable to the complementary strand CGTCAAACTCGp does not form above 0 degrees C. Measurements on the two undecamers indicate that variation in non Watson-Crick interactions within the loops of two similar hairpins can produce a difference in stability of at least 2.2 kcal/mol (25 degrees C, 0.115 M Na+), roughly equal to the amount contributed to a double helix by a 5'-CG-3'/5'-CG-3' base-paired stacking interaction.     

Energetics of the hairpin to mismatched duplex transition of d(GCCGCAGC) on NaCl solution

We report Potential of Mean Force studies to describe the relative thermodynamic stabilities of d(GCCGCAGC) in a mismatched duplex and a hairpin monomer conformation in NaCl solution. The PMF calculations are combined with previous molecular mechanics and normal mode analysis in order to estimate the role of different components of the free energy in determining the relative stability of the duplex and hairpin structures. The high entropy associated with the loop region and the lack of minor groove phosphate-phosphate interactions in the hairpin compete against the gain in enthalpic contribution to the free energy due to base pairing in the mismatched duplex. The combined free energy calculations show that the hairpin is the most stable conformation at low salt and that a hairpin to duplex transition takes place at approximately 0.47 M NaCl. In addition, we studied the hairpin to partially stacked single helical conformation equilibrium at low salt. We found a small variation in transition temperature in salt concentration, delta Tm/delta log10(cs) approximately 2-3 degrees K/decade, in contrast to the duplex to hairpin or duplex to partially stacked single helix transition where the transition temperature exhibited marked dependence on salt concentration. This is in qualitative agreement with experimental data. Based on the Potential of Mean Force free energy calculation, the order of relative stability of the three-conformations studied varies with salt concentration. We observed the following orders of stability: stacked single helix greater than hairpin greater than duplex for cs less than 0.77 M NaCl; single helix greater than duplex greater than hairpin for 0.77 less than Cs less than 2.1 M; and duplex greater than hairpin greater than single strand for cs greater than 2.1 M. From the calculated PMF free energy curves in the NaCl concentration range, 0.012 less than cs less than 5.0 M, we can assign upper and lower bounds for the non-ionic differences in free energy between the duplex, hairpin, and stacked single helical states (at standard conditions: cs = 1.0 M, T = 25 degrees C, and 1 M oligomer concentration). We found that for delta G duplex single helix = G duplex - 2 x G single helix less than -7.38 Kcal/mol, the single helix is the least stable state. For the duplex-to-hairpin free energy difference in the range, -1.87 less than delta G duplex-hairpin less than 0.03 Kcal/mol, there will always be a salt-induced hairpin-to-duplex transition for 0.01 less than cs less than 1.6 M NaCl. If delta G duplex-hairpin less than -1.87, the duplex is always more stable than the hairpin; and for delta G duplex-hairpin greater than Kcal/mol, the hairpin state is always more stable than the duplex, for all salt concentrations.     

Conformational features of the four successive non-Watson-Crick base pairs in RNA duplex.

A tridecaribonucleotide, r(UGAGCUUCGGCUC) doesn't form hairpin or interior loop and forms a double helix of 12 base pairs including the four successive nonstandard base pairs, U.G-U.C-C.U-G.U, in the crystal. Non-Watson-Crick base pairs, G.U and U.C are nicely incorporated in RNA duplex maintaining the regular A-form backbone. There exist the good overlapping between base pairings, U.G and U.C, so as to stabilize the nonstandard base pair track. Hydrogen bond networks involving water molecules in the major and minor grooves to stabilize this mismatch base pairing array, are observed and its conformational features are described.     

NMR study of a synthetic DNA hairpin

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.     

Analysis of melting transitions of the DNA hairpins formed from the oligomer sequences d[GGATAC(X)4GTATCC] (X = A, T, G, C)

Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3 degrees C for the T4 loop hairpin to 55.3 degrees C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop formation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions. Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hairpin and duplex formation of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) in solution. An NMR study.

The partly self-complementary DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) was investigated by NMR spectroscopy in solution. It is demonstrated that this peculiar DNA fragment, under suitable conditions of concentration, salt and temperature, exclusively prefers to adopt a monomeric hairpin form with a stem of three Watson-Crick type base pairs and a loop of two residues. At high single strand concentration (8 mM DNA) and low temperature (i.e. below 295 K) the hairpin occurs in slow equilibrium with a B-dimer structure. At high ionic strength (greater than or equal to 100 mM Na+) and/or in the presence of methanol a third species appears, which is assigned to a Z-like dimer. In the B form, as well as in the Z dimer, the two central base pairs form G.T wobble pairs with the bases as major tautomers.     

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.     

Folding of a DNA hairpin loop structure in explicit solvent using replica-exchange molecular dynamics simulations

Hairpin loop structures are common motifs in folded nucleic acids. The 5'-GCGCAGC sequence in DNA forms a characteristic and stable trinucleotide hairpin loop flanked by a two basepair stem helix. To better understand the structure formation of this hairpin loop motif in atomic detail, we employed replica-exchange molecular dynamics (RexMD) simulations starting from a single-stranded DNA conformation. In two independent 36 ns RexMD simulations, conformations in very close agreement with the experimental hairpin structure were sampled as dominant conformations (lowest free energy state) during the final phase of the RexMDs ( approximately 35% at the lowest temperature replica). Simultaneous compaction and accumulation of folded structures were observed. Comparison of the GCA trinucleotides from early stages of the simulations with the folded topology indicated a variety of central loop conformations, but arrangements close to experiment that are sampled before the fully folded structure also appeared. Most of these intermediates included a stacking of the C(2) and G(3) bases, which was further stabilized by hydrogen bonding to the A(5) base and a strongly bound water molecule bridging the C(2) and A(5) in the DNA minor groove. The simulations suggest a folding mechanism where these intermediates can rapidly proceed toward the fully folded hairpin and emphasize the importance of loop and stem nucleotide interactions for hairpin folding. In one simulation, a loop motif with G(3) in syn conformation (dihedral flip at N-glycosidic bond) accumulated, resulting in a misfolded hairpin. Such conformations may correspond to long-lived trapped states that have been postulated to account for the folding kinetics of nucleic acid hairpins that are slower than expected for a semiflexible polymer of the same size.     

Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson–Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N4-ethyldeoxycytidine (d^4EtC), which hybridises specifically with natural dG to give a G:^4EtC base pair with reduced stability compared to the natural G:C base pair. Substitution by d^4EtC greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.     

Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution

The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com.     

The influence of single base triplet changes on the stability of a pur.pur.pyr triple helix determined by affinity cleaving.

The influence of sixteen base triplet changes at a single position within a pur.pur.pyr triple helix was examined by affinity cleaving. For the 15 base pair target site studied here, G.GC, A.AT and T.AT triplets stabilize a triple helix to a greater extent than the other 13 natural triplets (pH = 7.4, 25 degrees C). Weaker interactions were detected for the C.AT, A.GC and T.CG triplets. The absence of specific, highly stabilizing interactions between third strand bases and the CG or TA base pairs demonstrates a current sequence limitation to formation of this structure. Models for the two dimensional base triplet interactions for all possible 16 natural triplets are presented.     

Solution conformation of an RNA hairpin loop.

The hairpin conformation adopted by the RNA sequence 5'GCGAUUUCUGACCGCC3' has been studied by one- and two-dimensional NMR spectroscopy. Exchangeable imino spectra in 60 mM Na+ indicate that the hairpin has a stem of six base pairs (indicated by boldface type) and a loop of three nucleotides. NOESY spectra of nonexchangeable protons confirm the formation of the stem region. The duplex has an A-conformation and contains an A.C apposition; a G.U base pair closes the loop region. The stem nucleotides have C3'-endo sugar conformations, as expected of an A-form duplex, whereas the three loop nucleotides adopt C2'-endo sugar puckers. Stacking within the loop, C8 upon the sugar of U7, stabilizes the structure. The pH dependence of both the exchangeable and nonexchangeable NMR spectra is consistent with the formation of an A+.C base pair, protonated at the N1 position of adenine. The stability of the hairpin was probed by using absorbance melting curves. The hairpin structure with the A+.C base pair is about +2 kcal/mol less stable in free energy at 37 degrees C than the hairpin formed with an A.U pair replacing the A+.C pair.     

Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).     

Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.

RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments.     

31P NMR study of daunorubicin-d(CGTACG) complex in solution. Evidence of the intercalation sites

The interaction of daunorubicin with the self-complementary DNA fragment d(CGTACG) was studied by 31P NMR spectroscopy. The individual phosphates have been assigned for the nucleotide and the complex and signals from bound and free species in slow exchange at 19 degrees C were detected. In solution, the hexanucleotide binds two molecules of daunorubicin, which intercalate in the d(CG) sequence at both ends of the helix. Evidence for local deformations of the backbone at the sites of C5pG6, C1pG2 and G2pT3 phosphates is given. The binding constants for the stepwise equilibrium and the rate of dissociation of the intercalated duplex were also determined.     

Parallel-stranded DNA with mixed AT/GC composition: role of trans G.C base pairs in sequence dependent helical stability

Parallel-stranded (ps) DNAs with mixed AT/GC content comprising G.C pairs in a varying sequence context have been investigated. Oligonucleotides were devised consisting of two 10-nt strands complementary either in a parallel or in an antiparallel orientation and joined via nonnucleotide linkers so as to form 10-bp ps or aps hairpins. A predominance of intramolecular hairpins over intermolecular duplexes was achieved by choice of experimental conditions and verified by fluorescence determinations yielding estimations of rotational relaxation times and fractional base pairing. A multistate mode of ps hairpin melting was revealed by temperature gradient gel electrophoresis (TGGE). The thermal stability of the ps hairpins with mixed AT/GC content depends strongly on the specific sequence in a manner peculiar to the ps double helix. The thermodynamic effects of incorporating trans G.C base pairs into an AT sequence are context-dependent: an isolated G. C base pair destabilizes the duplex whereas a block of > or =2 consecutive G.C base pairs exerts a stabilizing effect. A multistate heterogeneous zipper model for the thermal denaturation of the hairpins was derived and used in a global minimization procedure to compute the thermodynamic parameters of the ps hairpins from experimental melting data. In 0.1 M LiCl at 3 degrees C, the formation of a trans G.C pair in a GG/CC sequence context is approximately 3 kJ mol(-)(1) more favorable than the formation of a trans A.T pair in an AT/TA sequence context. However, GC/AT contacts contribute a substantial unfavorable free energy difference of approximately 2 kJ mol(-)(1). As a consequence, the base composition and fractional distribution of isolated and clustered G.C base pairs determine the overall stability of ps-DNA with mixed AT/GC sequences. Thus, the stability of ps-DNA comprising successive > or =2 G.C base pairs is greater than that of ps-DNA with an alternating AT sequence, whereas increasing the number of AT/GC contacts by isolating G.C base pairs exerts a destabilizing effect on the ps duplex. Molecular modeling of the various helices by force field techniques provides insight into the structural basis for these distinctions.     

Mechanistic insights into temperature-dependent regulation of the simple cyanobacterial hsp17 RNA thermometer at base-pair resolution

The cyanobacterial hsp17 ribonucleicacid thermometer (RNAT) is one of the smallest naturally occurring RNAT. It forms a single hairpin with an internal 1×3-bulge separating the start codon in stem I from the ribosome binding site (RBS) in stem II. We investigated the temperature-dependent regulation of hsp17 by mapping individual base-pair stabilities from solvent exchange nuclear magnetic resonance (NMR) spectroscopy. The wild-type RNAT was found to be stabilized by two critical CG base pairs (C14-G27 and C13-G28). Replacing the internal 1×3 bulge by a stable CG base pair in hsp17^rep significantly increased the global stability and unfolding cooperativity as evidenced by circular dichroism spectroscopy. From the NMR analysis, remote stabilization and non-nearest neighbour effects exist at the base-pair level, in particular for nucleotide G28 (five nucleotides apart from the side of mutation). Individual base-pair stabilities are coupled to the stability of the entire thermometer within both the natural and the stabilized RNATs by enthalpy–entropy compensation presumably mediated by the hydration shell. At the melting point the Gibbs energies of the individual nucleobases are equalized suggesting a consecutive zipper-type unfolding mechanism of the RBS leading to a dimmer-like function of hsp17 and switch-like regulation behaviour of hsp17^rep. The data show how minor changes in the nucleotide sequence not only offset the melting temperature but also alter the mode of temperature sensing. The cyanobacterial thermosensor demonstrates the remarkable adjustment of natural RNATs to execute precise temperature control.     

Double-helix formation of self-complementary chimeric oligonucleotides r(CG)nd(CG)3-n (n = 0, 1, 2, 3).

Double-helix formations of self-complementary chimeric hexanucleotides, r(CGCGCG), r(CGCG)d(CG), r(CG)d(CGCG), and d(CGCGCG), have been studied spectrophotometrically an thermodynamically in 1 mol dm-3 NaCl buffer. CD (circular dichroism) spectra showed that r(CGCGCG), r(CGCG)d(CG), and r(CG)d(CGCG) formed A-type double helix, while d(CGCGCG) formed B-type double helix. The stabilization energies of these helices at 37 degrees C obtained from UV melting analyses were 9.2 kcal mol-1 for r(CGCGCG), 8.2 kcal mol-1 for r(CGCG)d(CG), 6.8 kcal mol-1 for r(CG)d(CGCG), and 8.5 kcal mol-1 for d(CGCGCG), respectively.     

NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.

One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.