132 Impact of sticky end length on the diffraction of self-assembled DNA crystals

Our laboratory has reported a self-assembled 3-D crystal based on a DNA tensegrity triangle. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry consisting of three helices whose axes are directed along three linearly independent directions (1). The triangles form a crystalline lattice stabilized via sticky ends (2). The length of the sticky ends reported previously was two nucleotides (nt) GA:TC. Although diffracting to 4 Å resolution at the APS-ID19 beam line, they diffract only to 4.9 Å at the NSLS-X25 beam line. In the current study, we have analysed the effect of sticky end length and sequence on crystal formation and the resolution of the X-ray diffraction pattern on NSLS-X25. Tensegrity triangle motifs having 1-, 2-, and 3-nt sticky ends have all formed crystals. X-ray diffraction data from the same beam line revealed that the crystal resolution was somewhat better for the 2-nt sticky end having an AA:TT base pair (4.75 Å) than GA:CT and CC:GG (8.0 Å). Moreover, the 1-nt sticky end (C:G) yielded a diffraction pattern whose resolution (3.5 Å) compared favorably with all the three 2-nt sticky end systems. However, the triangle motif having a 1-nt sticky end with an A:T base pair did not yield any crystals. For motifs with 3-nt sticky ends, the sequence GAG:CTC produced small crystals (10–20?μm), while larger crystals (150?μm) were obtained with the sequences TAG:ATC and TAT:ATA. Our results indicate that not only do the lengths and sequences of the sticky ends define the interactions between motifs, but they also have an impact on the resulting resolution. We expect redesigned assemblies to form 3-D crystals with better resolution that can aid in the scaffolding of biological macromolecules for crystallographic structure determination. Applications in many areas of DNA nanotechnology are expected to benefit from a complete analysis of the effects of sticky end length, sequence, and free energy.     

133 Designed DNA crystals with a triple-helix veneer

DNA has been used as a tool for the self-assembly of nano-sized objects and arrays in two and three-dimensions. Triplex-forming oligonucleotides (TFOs) can be exploited to recognize and introduce functionality at precise duplex regions within these DNA nanostructures (Rusling et al., 2012). Here we have examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry, consisting of three helices directed along three linearly independent directions (Liu et al., 2004). The triangles form a three-dimensional crystalline lattice stabilized via sticky-end cohesion (Zheng et al., 2009). The TFO 5′-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine–oligopyrimidine binding site. Formation of DNA triplex in the motif was characterized by an electrophoretic mobility shift assay (EMSA), UV melting studies and FRET analysis. Non-denaturing gel analysis of annealed DNA motifs showed a band with slower mobility only in the presence of TFO and only when the DNA motif contained the triplex binding site. Experiments were undertaken at pH 5.0, since the formation of a triplex with cytidine-containing TFOs requires slightly acidic conditions (pH<?6.0). TFOs with modified C-analogs and T-analogs having a higher pK _a worked at a more neutral pH, also evidenced by EMSA. UV melting studies revealed that the melting point of the 3-turn triangle was 64?°C and the TFO binding increased the melting point to 80?°C. FRET analysis was done by labeling the triangle with fluorescein and the TFO with a cyanine dye (Cy5). The FRET melting curve revealed that a signal was observed only when the TFO was bound to the DNA motif and the results were consistent with UV melting studies. These results indicate that a TFO can be specifically targeted to the tensegrity triangle motif.     

Polymorphic crystal structures of an all‐AT DNA dodecamer

In this work, we explore the influence of different solvents and ions on the crystallization behavior of an all‐AT dodecamer d(AATAAATTTATT)₂ In all cases, the oligonucleotides are found as continuous columns of stacked duplexes. The spatial organization of such columns is variable; consequently we have obtained seven different crystal forms. The duplexes can be made to crystallize in either parallel or crossed columns. Such versatility in the formation of a variety of crystal forms is characteristic for this sequence. It had not been previously reported for any other sequence. In all cases, the oligonucleotide duplexes have been found to crystallize in the B form. The crystallization conditions determine the organization of the crystal, although no clear local interactions have been detected. Mg²⁺ and Ni²⁺ can be used in order to obtain compact crossed structures. DNA–DNA interactions in the crystals of our all‐AT duplexes present crossovers which are different from those previously reported for mixed sequence oligonucleotides. Our results demonstrate that changes in the ionic atmosphere and the crystallization solvent have a strong influence on the DNA–DNA interactions. Similar ionic changes will certainly influence the biological activity of DNA. Modulation of the crystal structure by ions should also be explored in DNA crystal engineering. Liquid crystals with a peculiar macroscopic shape have also been observed. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 123–133, 2015.     

Cytosine, the double helix and DNA self-assembly

DNA self-assembly has crucial implications in reading out the genetic information in the cell and in nanotechnological applications. In a recent paper, self-assembled DNA crystals displaying spectacular triangular motifs have been described (Zheng et al., 2009). The authors claimed that their data demonstrate the possibility to rationally design well-ordered macromolecular 3D DNA lattice with precise spatial control using sticky ends. However, the authors did not recognize the fundamental features that control DNA self-assembly in the lateral direction. By analysing available crystallographic data and simulating a DNA triangle, we show that the double helix geometry, sequence-specific cytosine–phosphate interactions and divalent cations are in fact responsible for the precise spatial assembly of DNA.     

Crystallography of polysaccharides: Current state and challenges

Polysaccharides are the most abundant class of biopolymers, holding an important place in biological systems and sustainable material development. Their spatial organization and intra- and intermolecular interactions are thus of great interest. However, conventional single crystal crystallography is not applicable since polysaccharides crystallize only into tiny crystals. Several crystallographic methods have been developed to extract atomic-resolution structural information from polysaccharide crystals. Small-probe single crystal diffractometry, high-resolution fiber diffraction and powder diffraction combined with molecular modeling brought new insights from various types of polysaccharide crystals, and led to many high-resolution crystal structures over the past two decades. Current challenges lie in the analysis of disorder and defects by further integrating molecular modeling methods for low-resolution diffraction data.     

Crystal structure of SUMO-3-modified thymine-DNA glycosylase

Modification of cellular proteins by the small ubiquitin-like modifier SUMO is important in regulating various cellular events. Many different nuclear proteins are targeted by SUMO, and the functional consequences of this modification are diverse. For most proteins, however, the functional and structural consequences of modification by specific SUMO isomers are unclear. Conjugation of SUMO to thymine-DNA glycosylase (TDG) induces the dissociation of TDG from its product DNA. Structure determination of the TDG central region conjugated to SUMO-1 previously suggested a mechanism in which the SUMOylation-induced conformational change in the C-terminal region of TDG releases TDG from tight binding to its product DNA. Here, we have determined the crystal structure of the central region of TDG conjugated to SUMO-3. The overall structure of SUMO-3-conjugated TDG is similar to the previously reported structure of TDG conjugated to SUMO-1, despite the relatively low level of amino acid sequence similarity between SUMO-3 and SUMO-1. The two structures revealed that the sequence of TDG that resembles the SUMO-binding motif (SBM) can form an intermolecular beta-sheet with either SUMO-1 or SUMO-3. Structural comparison with the canonical SBM shows that this SBM-like sequence of TDG retains all of the characteristic interactions of the SBM, indicating sequence diversity in the SBM.     

Energetics of the lattice: Packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice—these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine · cytosine⁺ (C · C⁺) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other—for example, when folded in more complicated higher order structures, such as mRNA and chromatin. © 1998 John Wiley & Sons, Inc. Biopoly 44: 257–267, 1997     

Comparison of energy-minimized crystal structures of 2,3-dilauroyl-D-glycerol, 3-palmitoyl-DL-glycerol-1-phosphorylethanolamine and 1,2-dilauroyl-DL-phosphatidylethanolamine:acetic acid

Computational procedures have been developed by which the total energy of a lipid multibilayer can be calculated and minimized. The energy is expressed as a sum of non-bonded, electrostatic, hydrogen bonded and torsional energy terms and includes intramolecular and intermolecular components. Calculations were carried out on three lipid crystals for which structural data are available from X-ray diffraction analysis. For each crystal, the energy was minimized as a function of all bond rotations, molecular rotations and translations and the lattice constants. The minimized structures differed by only small amounts from the experimental structures, which confirms the validity of the current set of energy functions and parameters for use with lipids. The intermolecular energy of each crystal is analyzed in terms of lateral interactions, interactions between the two monolayers of the same bilayer and interactions between bilayers. The intermolecular non-bonded energy per CH2 or CH3 group in the acyl chains is also given.     

Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.     

Is counterion delocalization responsible for collapse in RNA folding?

Although energetic and phylogenetic methods have been very successful for prediction of nucleic acid secondary structures, arrangement of these secondary structure elements into tertiary structure has remained a difficult problem. Here we explore the packing arrangements of DNA, RNA, and DNA/RNA hybrid molecules in crystals. In the conventional view, the highly charged double helix will be pushed toward isolation by favorable solvation effects; interactions with other like-charged stacks would be strongly disfavored. Contrary to this expectation, we find that most of the cases analyzed ( approximately 80%) exhibit specific, preferential packing between elements of secondary structure, which falls into three categories: (i) interlocking of major grooves of two helices, (ii) side-by-side parallel packing of helices, and (iii) placement of the ribose-phosphate backbone ridge of one helix into the major groove of another. The preponderance of parallel packing motifs is especially surprising. This category is expected to be maximally disfavored by charge repulsion. Instead, it comprises in excess of 50% of all packing interactions in crystals of A-form RNA and has also been observed in crystal structures of large RNA molecules. To explain this puzzle, we introduce a novel model for RNA folding. A simple calculation suggests that the entropy gained by a cloud of condensed cations surrounding the helices more than offsets the Coulombic repulsion of parallel arrangements. We propose that these condensed counterions are responsible for entropy-driven RNA collapse, analogous to the role of the hydrophobic effect in protein folding.     

Sister chromatid interactions in bacteria revealed by a site-specific recombination assay

The process of Sister Chromosome Cohesion (SCC), which holds together sister chromatids upon replication, is essential for chromosome segregation and DNA repair in eukaryotic cells. Although cohesion at the molecular level has never been described in E. coli, previous studies have reported that sister sequences remain co-localized for a period after their replication. Here, we have developed a new genetic recombination assay that probes the ability of newly replicated chromosome loci to interact physically. We show that Sister Chromatid Interaction (SCI) occurs exclusively within a limited time frame after replication. Importantly, we could differentiate sister cohesion and co-localization since factors such as MatP and MukB that reduced the co-localization of markers had no effect on molecular cohesion. The frequency of sister chromatid interactions were modulated by the activity of Topo-IV, revealing that DNA topology modulates cohesion at the molecular scale in bacteria.     

The ‘n’ effect in molecular recognition

The cooperativity which exists in crystal melting and many biological molecular recognition phenomena arises from extended arrays of weak interactions. We present a correlation between the melting temperature of a crystal and the intermolecular energy (which is evident only when compounds possessing several or many internal rotors are excluded). The correlation is used as the basis for a model of crystal melting which is capable of estimating the melting temperature of crystals. This model provides the basis for an understanding of the sharpness of the crystal melting transition for organic and inorganic substances. The cooperativity illustrated by the extended arrays of weak interactions, or the ‘n’ effect, is extended to analogous order/disorder transitions in biological systems, such as the ‘melting’ of DNA and RNA duplexes, providing insights into the physical properties of these structures.     

Metallic nanoparticles used to estimate the structural integrity of DNA motifs

Branched DNA motifs can be designed to assume a variety of shapes and structures. These structures can be characterized by numerous solution techniques; the structures also can be inferred from atomic force microscopy of two-dimensional periodic arrays that the motifs form via cohesive interactions. Examples of these motifs are the DNA parallelogram, the bulged-junction DNA triangle, and the three-dimensional-double crossover (3D-DX) DNA triangle. The ability of these motifs to withstand stresses without changing geometrical structure is clearly of interest if the motif is to be used in nanomechanical devices or to organize other large chemical species. Metallic nanoparticles can be attached to DNA motifs, and the arrangement of these particles can be established by transmission electron microscopy. We have attached 5 nm or 10 nm gold nanoparticles to every vertex of DNA parallelograms, to two or three vertices of 3D-DX DNA triangle motifs, and to every vertex of bulged-junction DNA triangles. We demonstrate by transmission electron microscopy that the DNA parallelogram motif and the bulged-junction DNA triangle are deformed by the presence of the gold nanoparticles, whereas the structure of the 3D-DX DNA triangle motif appears to be minimally distorted. This method provides a way to estimate the robustness and potential utility of the many new DNA motifs that are becoming available.     

Hydrogen bond connectivity patterns and hydrophobic interactions in crystal structures of small, acyclic peptides.

Crystal structures of all available unblocked linear peptides with two to five residues were retrieved from the Cambridge Structural Database and their intermolecular contacts and packing modes studied using molecular graphics. This survey reveals that interactions between hydrophobic portions of the molecules are critically important in determining the overall features of their crystal packing patterns. Distinct hydrophobic columns or layers are observed in almost all crystal structures. Analyses of the relationships between these interactions and crystal growth properties of small peptides are given. It is suggested that needle growth is promoted by hydrophobic packing, usually along a short crystallographic axis (4.6-6.0 angstroms). Also contributing to these morphologic characteristics are entropic factors associated with hydrophobic aggregation as well as tightly bound water molecules on hydrophobic faces. The paper also provides a comprehensive overview of hydrogen bond patterns in acyclic peptide crystals. It is demonstrated that one of their primary roles is to provide a scaffolding within which hydrophobic groups can aggregate. Even though there is a high density of hydrogen bonds in the crystals, often with complex patterns and networks, certain motifs are found to recur in a number of structures indicating specific hydrogen bond preferences. Water, for example, is an integral part of the hydrogen bond networks in these crystals, usually acting as the primary donor for main-chain carboxylate groups in peptide hydrates.     

Structure and interactions in benzamide molecular crystals

In this study, we resolved discrepancies concerning the experimentally determined structure of benzamide molecular crystals from dispersion-corrected density functional calculations. A clear energy ranking was obtained for the two candidates of the stable (P1) modification of benzamide. This was rationalised by subtle differences of the molecular interactions in the molecular crystal. The potential energy of the different structures was dominated by the interplay of intermolecular attraction and molecular torsion/deformation to accommodate favourable hydrogen-bonded networks. Using suitable proxies arranged in pseudo-crystalline set-ups, we discriminated the contribution of electrostatics, π–π interactions and intra-molecular interactions to the lattice energies.     

Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding

Though the molecular architecture of many native RNA structures has been characterized, the structures of folding intermediates are poorly defined. Here, we present a nucleotide-level model of a highly structured equilibrium folding intermediate of the specificity domain of the Bacillus subtilis RNase P RNA, obtained using chemical and nuclease mapping, circular dichroism spectroscopy, small-angle X-ray scattering and molecular modeling. The crystal structure indicates that the 154 nucleotide specificity domain is composed of several secondary and tertiary structural modules. The structure of the intermediate contains modules composed of secondary structures and short-range tertiary interactions, implying a sequential order of tertiary structure formation during folding. The intermediate lacks the native core and several long-range interactions among peripheral regions, such as a GAAA tetraloop and its receptor. Folding to the native structure requires the local rearrangement of a T-loop in the core in concert with the formation of the GAAA tetraloop-receptor interaction. The interplay of core and peripheral structure formation rationalizes the high degree of cooperativity observed in the folding transition leading to the native structure.     

Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein with increased processivity

In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins at the replication fork. Single-stranded DNA binding protein (SSB) is an essential protein that binds tightly and cooperatively to single-stranded DNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase with its cognate SSB via a short six amino acid linker increases affinity for primer-template DNA by sixfold and subsequently increases processivity by sevenfold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.