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.     

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.     

RNA LEGO: magnesium-dependent formation of specific RNA assemblies through kissing interactions

The high affinity and specificity of nucleic acid base complementarity has been proven to be a powerful method for constructing specific molecular assemblies. On the other hand, recent structural studies of RNA have revealed the wide range of tertiary interactions utilized in RNA folding, which may potentially be used as tools for the design of specific macromolecular assemblies. Here, RNA building blocks containing two hairpin loops, based on the dimerization initiation site (DIS) of HIV RNA, connected by a short linker were used to construct large RNA assemblies through hairpin loop-loop ("kissing") interactions. We show that specific linear and circular assemblies can be constructed in a magnesium-dependent manner using several non-self-complementary loop-loop interactions designed in this study. These results show that the use of RNA tertiary interactions may broaden the repertoire of nucleic acid-based nanostructures.     

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.     

Formation of a Ligand‐Assisted Complex of Two RNA Hairpin Loops

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non‐structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand‐assisted formation of loop–loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G–G mismatches in double‐stranded DNA, we successfully demonstrated the formation of both inter‐ and intra‐molecular NCT6‐assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)₃ in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly‐labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6‐assisted loop–loop complex. Förster resonance energy‐transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.     

Visualisation of inter loop tertiary base pairing and stacking interactions in an unusual slipped loop DNA structure

The conformation of an unusual slipped loop DNA structure exhibited by the sequence d(GAATTCCCGAATTC)₂ is determined using a combination of geometrical and molecular mechanics methods. This sequence is known to form a B-DNA-like duplex with the central non-complementary cytosines extruded into single stranded loop regions. The unusual feature is that the interior guanine does not pair with the cytosine across, instead, it pairs with the cytosine upstream by skipping two cytosines, leading to a slipped loop DNA structure with the loops staggered by two base pairs. The two loops, despite being very small, can fold across minor or major groove symmetrically or asymmetrically disposed, with one of the loop bases partially blocking the major or minor groove. Most interestingly, for certain conformations, the loop bases approach one another at close proximity so as to engage even in base pairing as well as base stacking interactions across the major groove. While such pairing and stacking are common in the tertiary folds of RNA, this is the first time that such an interaction is visualized in a DNA. This observation demonstrates that a W-C pair can readily be accomplished in a typical slipped loop structure postulated for DNA. Such tertiary loop interaction may prevent access to regulatory proteins across the major groove of the duplex DNA, thus providing a structure-function relation for the occurrence of slipped loop structure in DNA. Contribution no. 839 from this department     

Synthesis of hairpin siRNA using 18β-glycyrrhetinic acid derivative as a loop motif

We synthesized a new phosphoramidite building block from 18β-glycyrrhetinic acid. This compound was introduced in the middle of the palindromic oligonucleotides and they formed hairpin structure. This 18β-glycyrrhetinic acid derivative excellently performed its role as hairpin loop. These hairpin ODNs had higher T_m values than the natural one and formed stable hairpin structure without any structural distortion. We found that stability of hairpin ODNs was changed according to the length of hairpin stem and modified hairpin ODNs, which had longer than seven base pair stem, were more stable than natural one based on T_m values. The shRNAs bearing similar modified loop were also synthesized. These modified shRNAs could suppress their target gene expression without losing their RNAi efficiency.     

Multiple proton-transfer reactions in DNA base pairs by coordination of pt complex

The possibility of multiple proton-transfer reactions in DNA base pairs because of coordination of cisplatin is theoretically elucidated by density functional theory (DFT) and by quantum mechanics/molecular mechanics (QM/MM) methods with an ONIOM method. From the energetics of two base pairs with the cisplatin, it is theoretically confirmed that the Pt complex is likely to bind in the order cis-(CG)-Pt-(GC), cis-(CG)-Pt-(AT), cis-(TA)-Pt-(AT), where G, C, A, and T are guanine, cytosine, adenine, and thymine, respectively, and the Pt atom bonds to the N7 site of G and A. This result supports the experimental evidence, where the structure cis-A-Pt-A is seldom observed at room temperature. The single proton-transfer reaction occurs in one of the two GC pairs. No simultaneous single proton-transfer reaction can occur in both base pairs. Two different single proton-transferred structures (cis-(CG*)(d)-Pt-(GC)(p) and cis-(CG)(d)-Pt-(G*C)(p), where the asterisk means a proton donor of G) are as stable as the original structures (CG)(d)-Pt-(GC)(p). The same tendency was observed with cis-(CG*)-Pt-(AT). In contrast to cisplatin, multiple single proton-transfer reactions occur in the system consisting of two base pairs with transplatin. The optimized structure agrees with the experimental data for Pt-G coordination except for the hydrogen-bonding length.     

RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.     

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.     

Analysis of stacking overlap in nucleic acid structures: algorithm and application

RNA contains different secondary structural motifs like pseudo-helices, hairpin loops, internal loops, etc. in addition to anti-parallel double helices and random coils. The secondary structures are mainly stabilized by base-pairing and stacking interactions between the planar aromatic bases. The hydrogen bonding strength and geometries of base pairs are characterized by six intra-base pair parameters. Similarly, stacking can be represented by six local doublet parameters. These dinucleotide step parameters can describe the quality of stacking between Watson–Crick base pairs very effectively. However, it is quite difficult to understand the stacking pattern for dinucleotides consisting of non canonical base pairs from these parameters. Stacking interaction is a manifestation of the interaction between two aromatic bases or base pairs and thus can be estimated best by the overlap area between the planar aromatic moieties. We have calculated base pair overlap between two consecutive base pairs as the buried van der Waals surface between them. In general, overlap values show normal distribution for the Watson–Crick base pairs in most double helices within a range from 45 to 50 Å² irrespective of base sequence. The dinucleotide steps with non-canonical base pairs also are seen to have high overlap value, although their twist and few other parameters are rather unusual. We have analyzed hairpin loops of different length, bulges within double helical structures and pseudo-continuous helices using our algorithm. The overlap area analyses indicate good stacking between few looped out bases especially in GNRA tetraloop, which was difficult to quantitatively characterise from analysis of the base pair or dinucleotide step parameters. This parameter is also seen to be capable to distinguish pseudo-continuous helices from kinked helix junctions.     

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

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

Face-to-face and edge-to-face pi-pi interactions in a synthetic DNA hairpin with a stilbenediether linker

Synthetic conjugates possessing bis(2-hydroxyethyl)stilbene-4,4'-diether linkers (Sd2) form the most stable DNA hairpins reported to date. Factors that affect stability are length and flexibility of the linkers and pi-stacking of the stilbene moiety on the adjacent base pair. The crystal structure of the hairpin d(GT(4)G)-Sd2-d(CA(4)C) was determined at 1.5 A resolution. The conformations of the two molecules in the asymmetric unit differ both in the linker and the stem portions. One of them shows a planar stilbene that is stacked on the adjacent G:C base pair. The other displays considerable rotation between the phenyl rings and an unprecedented edge-to-face orientation of stilbene and base pair. The observation of considerable variations in the conformation of the Sd moiety in the crystal structure allows us to exclude restriction of motion as the reason for the absence of Sd photoisomerization in the hairpins. Conformational differences in the stem portion of the two hairpin molecules go along with different Mg(2+) binding modes. Most remarkable among them is the sequence-specific coordination of a metal ion in the narrow A-tract minor groove. The crystal structure provides unequivocal evidence that a fully hydrated Mg(2+) ion can penetrate the narrow A-tract minor groove, causing the groove to further contract. Overall, the structural data provide a better understanding of the origins of hairpin stability and their photochemical behavior in solution.     

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.     

Phenanthrene‐Derived DNA Hairpin Mimics

Self‐complementary oligodeoxynucleotides containing 3,6‐disubstituted phenanthrenes adopt highly stable, hairpin‐like structures. The thermodynamic stability of the hairpin mimics depends on the overall length of the phenanthrene building block. Hairpin loops composed of a phenanthrene‐3,6‐dicarboxamide and ethylene linkers were found to be optimal. The hairpin mimics are more stable than the analogous hairpins containing either a dT₄ or dA₄ tetraloop. Model studies indicate that the thermodynamic stability of the hairpin mimics is primarily due to aromatic stacking of the phenanthrene‐3,6‐dicarboxamide onto the adjoining base pair of the DNA duplex.     

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 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.     

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".     

Enhanced hairpin stability through loop design: the case of the protein G B1 domain hairpin

A mutational study of the peptide corresponding to the second hairpin of the protein G B1 domain (GB1p) provided a series of mutants with significantly increased fold stability. Mutations focused on improvement of the direction-reversing loop and the addition of favorable Coulombic interactions at the sequence termini. The loop optimization was based on a database search for residues that occur with the greatest probability in similar hairpin loops in proteins. This search suggested replacing the native DDATKT sequence with NPATGK, which resulted in a 4.5 kJ/mol stabilization of the hairpin fold. The introduction of positively charged lysines at the N-terminus provided an additional 2.4 kJ/mol of stabilization, affording a GB1p mutant that is 86 +/- 3% folded at 25 degrees C with a melting temperature of 60 +/- 2 degrees C. The trpzip version of this peptide, in which three of the hydrophobic core residues were mutated to tryptophan, yielded a sequence that melted at 85 degrees C. Throughout, fold populations and melting temperatures were derived from the mutation and temperature dependence of proton chemical shifts and were corroborated by circular dichroism (CD) melts. The study also suggests that the wild-type GB1p sequence is significantly less stable than reported in some other studies: only 30% folded in water at 25 degrees C.