Motif prediction in ribosomal RNAs Lessons and prospects for automated motif prediction in homologous RNA molecules.

The traditional way to infer RNA secondary structure involves an iterative process of alignment and evaluation of covariation statistics between all positions possibly involved in basepairing. Watson-Crick basepairs typically show covariations that score well when examples of two or more possible basepairs occur. This is not necessarily the case for non-Watson-Crick basepairing geometries. For example, for sheared (trans Hoogsteen/Sugar edge) pairs, one base is highly conserved (always A or mostly A with some C or U), while the other can vary (G or A and sometimes C and U as well). RNA motifs consist of ordered, stacked arrays of non-Watson-Crick basepairs that in the secondary structure representation form hairpin or internal loops, multi-stem junctions, and even pseudoknots. Although RNA motifs occur recurrently and contribute in a modular fashion to RNA architecture, it is usually not apparent which bases interact and whether it is by edge-to-edge H-bonding or solely by stacking interactions. Using a modular sequence-analysis approach, recurrent motifs related to the sarcin-ricin loop of 23S RNA and to loop E from 5S RNA were predicted in universally conserved regions of the large ribosomal RNAs (16S- and 23S-like) before the publication of high-resolution, atomic-level structures of representative examples of 16S and 23S rRNA molecules in their native contexts. This provides the opportunity to evaluate the predictive power of motif-level sequence analysis, with the goal of automating the process for predicting RNA motifs in genomic sequences. The process of inferring structure from sequence by constructing accurate alignments is a circular one. The crucial link that allows a productive iteration of motif modeling and realignment is the comparison of the sequence variations for each putative pair with the corresponding isostericity matrix to determine which basepairs are consistent both with the sequence and the geometrical data.

Molecular dynamics of the frame-shifting pseudoknot from beet western yellows virus: the role of non-Watson-Crick base-pairing, ordered hydration, cation binding and base mutations on stability and unfolding.

Molecular dynamics simulations of the frame-shifting pseudoknot from beet western yellows virus (BWYV, NDB file UR0004) were performed with explicit inclusion of solvent and counterions. In all, 33 ns of simulation were carried out, including 10 ns of the native structure with protonation of the crucial cytosine residue, C8(N3+). The native structure exhibited stable trajectories retaining all Watson-Crick and tertiary base-pairs, except for fluctuations or transient disruptions at specific sites. The most significant fluctuations involved the change or disruption of hydrogen-bonding between C8(N3+) and bases G12, A25, and C26, as well as disruption of the water bridges linking C8(N3+) with A25 and C26. To increase sampling of rare events, the native simulation was continued at 400 K. A partial, irreversible unfolding of the molecule was initiated by slippage of C8(N3+) relative to G12 and continued by sudden concerted changes in hydrogen-bonding involving A23, A24, and A25. These events were followed by a gradual loss of stacking interactions in loop 2. Of the Watson-Crick base-pairs, only the 5'-terminal pair of stem 1 dissociated at 400 K, while the trans sugar-edge/sugar-edge A20.G4 interaction remained surprisingly stable. Four additional room-temperature simulations were carried out to obtain insights into the structural and dynamic effects of selected mutations. In two of these, C8 was left unprotonated. Considerable local rearrangements occurred that were not observed in the crystal structure, thus confirming N3-protonation of C8 in the native molecule. We also investigated the effect of mutating C8(N3+) to U8, to correlate with experimental and phylogenetic studies, and of changing the G4 x C17 base-pair to A4 x U17 to weaken the trans sugar-edge interaction between positions 4 and 20 and to test models of unfolding. The simulations indicate that the C8 x G12 x C26 base-triple at the junction is the most labile region of the frame-shifting pseudoknot. They provide insights into the roles of the other non-Watson-Crick base-pairs in the early stages of unfolding of the pseudoknot, which must occur to allow readthrough of the message by the ribosome. The simulations revealed several critical, highly ordered hydration sites with close to 100 % occupancies and residency times of individual water molecules of up to 5 ns. Sodium cation coordination sites with occupancies above 50 % were also observed.

Geometric nomenclature and classification of RNA base pairs.

Non-Watson-Crick base pairs mediate specific interactions responsible for RNA-RNA self-assembly and RNA-protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson-Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 2'-OH and which has also been referred to as the Shallow-groove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson-Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.

TectoRNA: modular assembly units for the construction of RNA nano-objects.

Structural information on complex biological RNA molecules can be exploited to design tectoRNAs or artificial modular RNA units that can self-assemble through tertiary interactions thereby forming nanoscale RNA objects. The selective interactions of hairpin tetraloops with their receptors can be used to mediate tectoRNA assembly. Here we report on the modulation of the specificity and the strength of tectoRNA assembly (in the nanomolar to micromolar range) by variation of the length of the RNA subunits, the nature of their interacting motifs and the degree of flexibility of linker regions incorporated into the molecules. The association is also dependent on the concentration of magnesium. Monitoring of tectoRNA assembly by lead(II) cleavage protection indicates that some degree of structural flexibility is required for optimal binding. With tectoRNAs one can compare the binding affinities of different tertiary motifs and quantify the strength of individual interactions. Furthermore, in analogy to the synthons used in organic chemistry to synthesize more complex organic compounds, tectoRNAs form the basic assembly units for constructing complex RNA structures on the nanometer scale. Thus, tectoRNA provides a means for constructing molecular scaffoldings that organize functional modules in three-dimensional space for a wide range of applications.

Solution conformation of a bulged adenosine base in an RNA duplex by relaxation matrix refinement.

Bulges are common structural motifs in RNA secondary structure and are thought to play important roles in RNA-protein and RNA-drug interactions. Adenosine bases are the most commonly occurring unpaired base in double helical RNA secondary structures. The solution conformation and dynamics of a 25-nucleotide RNA duplex containing an unpaired adenosine, r(GGCAGAGUGCCGC): r(GCGGCACCUGCC) have been studied by NMR spectroscopy and MORASS iterative relaxation matrix structural refinement. The results show that the bulged adenosine residue stacks into the RNA duplex with little perturbation around the bulged region. Most of the bases in the RNA duplex adopt C(3)'-endo conformation, exhibiting the N-type sugar pucker as found in the A form helices. The sugars of the bulged residue and the 5' flanking residue to it are found to exhibit C(2)'-endo conformation. None of the residues are in syn conformation.

Self-assembled complexes of oligopeptides and metalloporphyrins: measurements of the reorganization and electronic interaction energies for photoinduced electron-transfer reactions.

Cationic porphyrins form ground state electrostatically associated complexes with anionic oligo-electrolytes such as those formed by a series of glutamic acid (E) residues. Temperature dependencies were measured of the rate constants for intra-complex electron transfer to the triplet state of Pd(II)TMPyP4+ from a tyrosine (tyr, Y) or tryptophan (trp, W) moiety connected to a glutamic acid tetramer. In complexes such as YE4, E2YE2, YE4G10E (G, glycine), and WE4 these data were used to estimate the reorganization energy (lambda) and electronic interaction energy (HDA) relevant to the process. For all tyr-peptide complexes, lambda values were found to be large (lambda approximately 1.60 +/- 0.06 eV), reflecting a relatively high medium polarity in the vicinity of tyr residues. It further indicates that the tyr residues in all oligo-peptides are exposed to the aqueous medium in a similar way irrespective of the position of the aromatic moiety in the peptide chain. A significantly lower lambda value (lambda = 1.08 eV) was derived for the tryptophan-containing peptide complex, indicating a relatively higher hydrophobic character of trp compared to tyr. The electronic coupling matrix elements (HDA) derived for tyr-peptide complexes (5.1 meV for YE4, 5.4 meV for YE4G10E and 7.5 meV for E2YE2) were larger than that found for WE4 (1.1 meV). Molecular dynamics calculations were employed to obtain structural features of the porphyrin-peptide complexes. These showed average distances between the center of mass (COM) of the porphyrin ring and the center of mass of the amino acid aromatic ring of 816 +/- 140 pm (YE4), 800 +/- 80 pm (E2YE2), 900 +/- 130 pm (YE4G10E) and 970 +/- 160 pm (WE4). The molecular dynamics calculations were shown to be in good agreement with the experimentally determined electronic interaction energies, strongly suggesting that HDA is primarily responsible for the dependence of the electron-transfer rate constant (KET) on the donor-acceptor separation distance and relative orientation. The higher HDA (7.55 meV) derived for tyr incorporated into the middle of the peptide backbone (E2YE2) was presumed to be associated with a higher degree of orbital overlap due to a more favorable ring-ring orientation. Overlap parameters (beta derived for all peptide-porphyrin complexes were similar (approximately 0.95 +/- 0.06 A-1), being in good agreement with most literature values for similar systems. Finally, the intra-complex electron-transfer ratio (ktrp/ktyr) derived from flash photolysis experiments and the corresponding ratio derived from Marcus' theory combined with experimental data from the temperature-dependence investigations and electrochemical measurements were found to be in excellent agreement. This same consistency was found for the couple E4Y and E2YE2. The empirical expression (Moser and Dutton) governing the intraprotein electron-transfer rate constant in native systems combined with our experimental data (kET, lambda, delta G0) yielded tunneling pathway distances in excellent agreement with those arising from the molecular modeling studies. The exception was for the long peptide YE4G10E, for which the Quenched Molecular Dynamic (QMD) sampling technique was complicated and is probably inadequate.

Cationic 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin fully intercalates at 5'-CG-3' steps of duplex DNA in solution.

The interaction of 5,10,15, 20-tetrakis(N-methylpyridinium-4-yl)porphyrin (T4MPyP(4+)) with the oligonucleotide DNA duplex [d(GCACGTGC)](2) was studied by two-dimensional (1)H NMR spectroscopy, optical absorbance, circular dichroism, and molecular dynamics simulation employing particle mesh Ewald methods. T4MPyP(4+) is one of the largest aromatic molecules for which intercalative binding to DNA has been proposed, although this has been called into question by recent X-ray crystallographic work [Lipscomb et al. (1996) Biochemistry 35, 2818-2823]. T4MPyP(4+) binding to [d(GCACGTGC)](2) produced a single set of (mostly) upfield-shifted DNA resonances in slow exchange with the resonances of the free DNA. Intra- and intermolecular NOEs observed in the complex showed that the porphyrin intercalates at the central 5'-CG-3' step of the DNA duplex without disrupting the flanking base pairs. Absorption and circular dichroism spectra of the complex also support intercalative binding. Molecular dynamics simulations (using explicit solvent and PME methods), carried out for fully and partially intercalated complexes, yielded stable trajectories and plausible structures, but only the symmetrical, fully intercalated model agreed with NOESY data. Stable hydrogen bonding was observed during 600 ps of MD simulation for the base pairs flanking the binding site.

Hybrid-hybrid matrix structural refinement of a DNA three-way junction from 3D NOESY-NOESY.

Homonuclear 3D NOESY-NOESY has shown great promise for the structural refinement of large biomolecules. A computationally efficient hybrid-hybrid relaxation matrix refinement methodology, using 3D NOESY-NOESY data, was used to refine the structure of a DNA three-way junction having two unpaired bases at the branch point of the junction. The NMR data and the relaxation matrix refinement confirm that the DNA three-way junction exists in a folded conformation with two of the helical stems stacked upon each other. The third unstacked stem extends away from the junction, forming an acute angle (approximately 60 degrees) with the stacked stems. The two unpaired bases are stacked upon each other and are exposed to the solvent. Helical parameters for the bases in all three strands show slight deviations from typical values expected for right-handed B-form DNA. Inter-nucleotide imino-imino NOEs between the bases at the branch point of the junction show that the junction region is well defined. The helical stems show mobility (+/- 20 degrees) indicating dynamic processes around the junction region. The unstacked helical stem adjacent to the unpaired bases shows greater mobility compared to the other two stems. The results from this study indicate that the 3D hybrid-hybrid matrix MORASS refinement methodology, by combining the spectral dispersion of 3D NOESY-NOESY and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. Our results also indicate that the 3D MORASS method gives higher quality structures compared to the 2D complete relaxation matrix refinement method.

A common motif organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs.

Phylogenetic and chemical probing data indicate that a modular RNA motif, common to loop E of eucaryotic 5 S ribosomal RNA (rRNA) and the alpha-sarcin/ricin loop of 23 S rRNA, organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs. The motif occurs in the 3' domain of 16 S rRNA at positions 1345-1350/1372-1376 (Escherichia coli numbering), within the three-way junction loop, which binds ribosomal protein S7, and which contains nucleotides that help to form the binding site for P-site tRNA in the ribosome. The motif also helps to structure a three-way junction within domain I of 23 S, which contains many universally conserved bases and which lies close in the primary and secondary structure to the binding site of r-protein L24. Several other highly conserved hairpin, internal, and multi-helix loops in 16 S and 23 S rRNA contain the motif, including the core junction loop of 23 S and helix 27 in the core of 16 S rRNA. Sequence conservation and range of variation in bacteria, archaea, and eucaryotes as well as chemical probing and cross-linking data, provide support for the recurrent and autonomous existence of the motif in ribosomal RNAs. Besides its presence in the hairpin ribozyme, the loop E motif is also apparent in helix P10 of bacterial RNase P, in domain P7 of one sub-group of group I introns, and in domain 3 of one subgroup of group II introns.

The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure.

A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings. Thus, each non-Watson-Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings. In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur. The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.

Structure and dynamics of ribosomal RNA.

Over the past two years, progress in X-ray crystallography, NMR spectroscopy and electron microscopy has begun to reveal the complex structure of the RNA within the ribosome. The structures of ribosomal proteins L11 and S15, among others, show how RNA-protein interactions organize the conformation of the junctions between ribosomal RNA helices. Genetic and biochemical methods have also identified a three base-pair switch within the 16S rRNA that is linked to mRNA decoding.

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.

Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.

Refinement of the solution structure of a branched DNA three-way junction.

We have refined the structure of the DNA Three-Way Junction complex, TWJ-TC, described in the companion paper by quantitative analysis of two 2D NOESY spectra (mixing times 60 and 200 ms) obtained in D2O solution. NOESY crosspeak intensities extracted from these spectra were used in two kinds of refinement procedure: 1) distance-restrained energy minimization (EM) and molecular dynamics (MD) and 2) full relaxation matrix back calculation refinement. The global geometry of the refined model is very similar to that of a published, preliminary model (Leontis, 1993). Two of the helical arms of the junction are stacked. These are Helix 1, defined by basepairs S1-G1/S3-C12 through S1-C5/S3-G8 and Helix 2, which comprises basepairs S1-C6/S2-G5 through S1-G10/S2-G1. The third helical arm (Helix 3), comprised of basepairs S2-C6/S3-G5 through S2-C10/S3-G1 extends almost perpendicularly from the axis defined by Helices 1 and 2. The bases S1-C5 and S1-C6 of Strand 1 are continuously stacked across the junction region. The conformation of this strand is close to that of B-form DNA along its entire length, including the S1-C5 to S1-C6 dinucleotide step at the junction. The two unpaired bases S3-T6 and S3-C7 lie outside of the junction along the minor groove of Helix 1 and largely exposed to solvent. Analysis of the refined structure reveals that the glycosidic bond of S3-T6 exists in the syn conformation, allowing the methyl group of this residue to contact the hydrophobic surface of the minor groove of Helix 1, at S3-G11. The helical parameters of the three helical arms of the structure exhibit only weak deviations from typical values for right-handed B-form DNA. Unusual dihedral angles are only observed for the sugarphosphate backbone joining the "hinge" residues, S2-G5 and S2-C6, and S3-G5 through S3-G8. The glycosidic bond of S3-G8also lies within the syn range, allowing favorable Watson-Crick base-pairing interactions with Si -C5. The stability of this structure was checked in 39 ps molecular dynamic simulation at 330 K in water. The structure of TWJ-TC retained the geometrical features mentioned above at the end of the simulation period. The final R(1/6)-factor of the refined structure is 5%.

The thermodynamics of formation of a three-strand, DNA three-way junction complex.

Isothermal titration calorimetry (ITC) is used to study the thermodynamics of assembly of the three DNA oligonucleotides S1 (5'-GCCTGCCACCGC), S2 (5'-GCGGTGCGTCCG), and S3AA (5'-CGGACGAAGCAGGC) to form a three-way junction (TWJ) complex consisting of three double-helical arms radiating from a junction region having two unpaired adenosines in one strand (S3AA). The thermodynamics of assembly were measured for three different orders of addition of the component oligonucleotides at four temperatures between 10 and 25 degrees C. At each temperature studied, the overall values of delta H, delta S degrees, and delta G degrees for assembly of the complex from the component single strands were found to be independent of the order of addition. The enthalpy of binding, delta H, was found to be linearly dependent on temperature. From the temperature dependence of delta H, the change in heat capacity delta Cp, for the overall assembly of three strands to form the junction complex was calculated and found to be -1.6 kcal mol-1K-1. This work represents the first attempt to evaluate the thermodynamics of DNA TWJ formation by ITC.

Structure-specific binding and photosensitized cleavage of branched DNA three-way junction complexes by cationic porphyrins.

The interactions of cationic porphyrins with DNA oligonucleotides that form branched, three-way junction complexes (TWJ) were investigated using native gel electrophoresis, absorption spectroscopy and photochemical probing using DNA sequencing techniques. Meso-tetra(para-N- trimethylaniliniumyl)porphine (TMAP), meso-tetra(4-N-methylpyridiniumyl)porphine (T4MPyP) and meso-tetra(3-N-methylpyridiniumyl)porphine(T3MPyP) were found to bind more tightly to DNA TWJ than to DNA duplexes. The binding to the junction DNA persists at high ionic strength, conditions that greatly decrease porphyrin binding affinity to duplex DNA. THe TWJ DNA binding sites of TMAP and T4MPyP were localized to the junction region based on the observation of site- and structure-specific, porphyrin-sensitized photodamage to guanosine residues flanking the junction region.

Effects of unpaired bases on the conformation and stability of three-arm DNA junctions.

Three-arm DNA junctions, in which three double helices intersect at a branch, have unique structure and reactivity of bases at and near the branch. Their solution conformation is asymmetric in the presence of Mg2+, while bases at the branch are sensitive to single-strand-specific agents. Following the surprising report that unpaired bases at the branch stabilize three-arm junctions, we have investigated the geometry and thermodynamics of three-arm junctions containing pendant T and A bases. The results are consistent with additional structure formation in junctions containing up to four pendant bases at the branch: relative to the tight junction, the thermal stability of junctions with two T's or A's at the branch increases; bases near the branch become less reactive to single-strand-reactive probes; and the enthalpy of formation is more negative. The interaction of ethidium observed at the branch in three-arm junctions is enhanced in junctions with unpaired bases at the branch. The geometry of three-arm junctions is perturbed by the presence of pendant bases, as seen by measuring the electrophoretic mobility of junctions to which long duplex arms are appended pairwise.

A model for the solution structure of a branched, three-strand DNA complex.

The solution structure of a DNA three-way junction (TWJ) containing two unpaired thymidines was elucidated using two- and three-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. TWJs with unpaired nucleotides are ubiquitous structural motifs of complex single-stranded nucleic acids. In the presence of Mg2+, the TWJ complex adopts a unique conformation in which the bases of one of the oligonucleotides ("strand 1") are continuously stacked across the junction. Guanosine 8 of strand 3 (S3-G8), which pairs with S1-C5, stacks on S2-G5, which is paired to S1-C6. The unpaired thymidine bases (S3-T6 and S3-T7) are exposed to the solvent, whereas the sugar of S3-G8 is largely buried. S3-T6 also interacts with the sugar residue of S3-G11. All three stems conform to B-type DNA.