Design of immobile nucleic acid junctions

Nucleic acids that interact to generate structures in which three or more double helices emanate from a single point are said to form a junction. Such structures arise naturally as intermediates in DNA replication and recombination. It has been proposed that stable junctions can be created by synthesizing sets of oligonucleotides of defined sequence that can associate by maximizing Watson-Crick complementarity (Seeman N. C., 1981, Biomolecular Stereodynamics. Adenine Press, New York. 1: 269-278; Seeman, N. C., 1982, J. Theor. Biol. 99:237-247.) To make it possible to design molecules that will form junctions of specific architecture, we present here an efficient algorithm for generating nucleic acid sequences that optimize two fundamental properties: fidelity and stability. Fidelity refers to the relative probability of forming the junction complex relative to all alternative paired structures. Calculations are described that permit approximate prediction of the melting curves for junction complexes.     

正在崛起的RNA纳米技术

RNA纳米技术得益于纽约大学西曼(Nadrian C.Seeman)教授开创的DNA纳米技术,RNA是由腺嘌呤(A)、尿嘧啶(U)、鸟嘌呤(G)和胞嘧啶(C)构成的一种核糖核酸高分子,与DNA的Watson-Crick碱基配对(A-T,G-C)的双螺旋链的结构不完全一样,RNA的二级结构里经常出现一些非传统的碱基配对如环环相互作用,这些非传统配对促使RNA分子折叠成刚性结构。本文综述了正在崛起的RNA纳米技术,列举了一些著名的实验,如郭培宣(Peixuan Guo)等从自然界的phi29噬菌体中发现的pRNA纳米马达是由六个小RNA分子构成的六环结构,Jaeger等发展了RNA构造术(RNA-tectonics),根据已知的RNA分子的碱基和非传统配对,他们设计利用小RNA分子构造二聚体、一维线性多聚体、和二维网状的七巧板迷宫(jigsaw puzzle)等图案,用tRNA分子或设计用几条RNA分子来构建多面体如立方体和八面体等立体结构等。RNA纳米技术正在崛起,它将在医学、生物技术、合成生物学和纳米技术领域扮演重要的角色。     

Variability in the Geometry of RNA Double Helices Generated from Dinucleoside Building-Blocks

Abstract

A graphical method is presented for the generation of helical parameters from single-crystal structures of RNA nucleic acid fragments that are minimally dinucleosides. The method is compared with other published procedures, for a number of text examples. The RNA double helices generated from three different salts of the dinucleoside monophosphate GpC are examined in relation to the variations in helix morphology that are produced. It is shown that small differences between these GpC salts can be amplified to very distinct helix characteristics.     

An affinity scale for the interaction of amino acid residues with nucleic acids

The affinity of amino acid residues to nucleic acids is probed by measurements of melting temperatures t_m for the helix–coil transition at various concentrations of amino acid amides. The increase of t_m on addition of ligand is described by the equation t_m = t*_m + αlog(1+K_tc_λ), where t*_m is the melting temperature in the absence of ligand, c_λ the ligand concentration, and K_t the “t_m-onset” constant, which is analogous to an equilibrium constant. It is shown that K_t is closely related to the affinity of the ligands to the double helix, whereas the slope α mainly reflects the preference of the ligand binding to the helix versus the coil form. In the case of the amino acid amides, α is found to be virtually independent of the nature of the side chain with few exceptions, e. g., aromatic amides. The t_m-onset constant, however, strongly depends on the nature of the amino acid side chain. For simple aliphatic amino acids, the relative free energy of binding decreases with increasing hydrophobic free energy, e.g., a high affinity is found for Gly-amide and a low affinity for Leu-amide. This relation is modified by functional groups like OH in Ser-amide. The helices poly[d(A-T)], ploy[d(I-C)]. and poly[d(A-C)]·poly[d(G-T)] exhibit similar affinity scales with relatively small variations. Our results demonstrate that the hydrophilic character of double helices at their surface disfavors binding of hydrophobic ligands unless special contacts can be formed. From our results we establish an affinity scale for the binding of amino acids to double helices.     

Crystal structure of a poly(rA) staggered zipper at acidic pH: evidence that adenine N1 protonation mediates parallel double helix formation

We have solved at 1.07 Å resolution the X-ray crystal structure of a polyriboadenylic acid (poly(rA)) parallel and continuous double helix. Fifty-nine years ago, double helices of poly(rA) were first proposed to form at acidic pH. Here, we show that 7-mer oligo(rA), i.e. rA₇, hybridizes and overlaps in all registers at pH 3.5 to form stacked double helices that span the crystal. Under these conditions, rA₇ forms well-ordered crystals, whereas rA₆ forms fragile crystalline-like structures, and rA₅, rA₈ and rA₁₁ fail to crystallize. Our findings support studies from ∼50 years ago: one showed using spectroscopic methods that duplex formation at pH 4.5 largely starts with rA₇ and begins to plateau with rA₈; another proposed a so-called ‘staggered zipper’ model in which oligo(rA) strands overlap in multiple registers to extend the helical duplex. While never shown, protonation of adenines at position N1 has been hypothesized to be critical for helix formation. Bond angles in our structure suggest that N1 is protonated on the adenines of every other rAMP−rAMP helix base pair. Our data offer new insights into poly(rA) duplex formation that may be useful in developing a pH sensor.     

Are there structural analogies between amino acids and nucleic acids?

Space-filling molecular models have been used to examine structural analogies between amino acids and nucleic acids. The three-dimensional structures of amino acid R groups appear to be stereochemically related to cavities formed by removal of single bases in double helical nucleic acids. The common L amino acids may thus be complementary to their codons.     

Nucleic acid crystallography: current progress

Fifty years after the publication of the DNA double helix model by Watson and Crick, new nucleic acid structures keep emerging at an ever-increasing rate. The past three years have brought a flurry of new oligonucleotide structures, including those of a Hoogsteen-paired DNA duplex, Holliday junctions, DNA-drug complexes, quadruplexes, a host of RNA motifs and various nucleic acid analogues. Major advances were also made in terms of the structure and function of catalytic RNAs. These range from improved models of the phosphodiester cleavage reactions catalyzed by the hairpin and hepatitis delta virus ribozymes to the visualization of a complete active site of a group I self-splicing intron with bound 5'- and 3'-exons. These triumphs are complemented by a refined understanding of cation-nucleic-acid interactions and new routes to the generation of derivatives for phasing of DNA and RNA structures.     

Experimental support for a right-handed vertical double helix

The experimentally observed geometry of a miniature double helix of high anti (χCN fixed ? 120°) nucleic acid structure indicates substantial interstrand base stacking with little intrastrand base stacking. This geometry is consistent with the right-handed vertical double helix in which the base planes are parallel to helical axis proposed by Olson [(1977) Proc. Natl. Acad. Sci. USA 74 , 1775] from theoretical calculations. The experimental data do not agree with the left-handed model in which the base planes are perpendicular to helical axis that has been proposed by Yathindra and Sundaralingam [(1976) Nucl. Acids Res. 3 , 729]. The theoretically computed chemical shift changes for the various double-helical configurations show that for the system examined, only the vertical model can explain the experimentally observed shift trends. The melting curve for the helix–coil transition for high anti dinucleoside monophosphate has been observed for the first time. Even though the experimental data support vertical double helices when χCN is fixed at 120°, data on naturally occurring nucleic acid structures indicate that they have no proclivity to enter into vertically stabilized double-helical arrays.     

On the mechanism of interaction between histone II B1 and DNA and histone II B2 and DNA

A mechanism is suggested at the molecular level whereby histone IIB₂ can act as a cross-link between two (or possibly three) adjacent and parallel strands of DNA double helix some 40 Å apart. Application of Prothero's rule and the Lewis probability functions indicate the probable locations of three a-helices and a number of β-turns. This, coupled with the requirement that the tertiary conformation of the histone be complementary to the DNA molecules and for as many basic groups as possible to bind to phosphate oxygens, allows us to suggest, on the basis of model building using accurate space-filling (CPK) models, a complex conformation that achieves this.A similar process applied to histone IIB₁, whose complete amino acid sequence is also known, shows the location of five probable a-helices, a number of β-turns, and a segment of β-pleated sheet. The basic amino acids are gathered in four groupings. Model building experiments suggest that histone IIB₁ forms a complex strut joining four parallel strands of DNA double helix that form a diamond with diameters 100 and 40 Å. In both these models the purpose and function of a fair proportion of the individual amino acids can be specified.This paper is the third and last of a series in this Journal in which models are presented for the tertiary conformation and function of all five histones of known (in whole or in part) amino acid sequence. This suggests that all five are concerned in packing the long DNA double helix, which may be in a “square helix” form, into the confined space of the chromosome. The hypotheses may be tested by a direct investigation of nucleoprotein in situ to see if these 40, 70, and 100 Å interhelical distances can be detected by biophysical methods.     

An ancient anion‐binding structural module in RNA and DNA helicases

RNA and DNA helicases manipulate or translocate along single strands of nucleic acids by grasping them using a conserved structural motif. We have examined the available crystal structures of helicases of the two principal superfamilies, SF1 and SF2, and observed that the most conserved interactions with the nucleic acid occur between the phosphosugar backbone of a trinucleotide and the three strand‐helix loops within a (β‐strand/α‐helix)₃ structural module. At the first and third loops is a conserved hydrogen‐bonded feature called a thr‐motif, often seen at α‐helical N‐termini, with the threonine as the N‐cap residue. These loops can be aligned with few insertions or deletions, and their main chain atoms are structurally congruent amongst the family members and between the two modules found as tandem pairs in all SF1 and SF2 proteins. The other highly conserved interactions with nucleic acid involve mainchain NH groups, often at the helical N‐termini, interacting with phosphate groups. We comment on how the sequence motifs that are commonly used to identify helicases map to locations on the module and discuss the implications of the conserved orientation of nucleic acid on the surface of the module for directional stepping along DNA or RNA. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis and properties of novel psoralen derivatives

We have synthesized a set of new trimethylpsoralen derivatives that are characterized by a chain extending from the 4'-position of the furan ring and linked to this ring by an aminomethylene group. The nature of the side chain can be varied widely. In these derivatives, the chains contain either amino or ethylene oxide units for enhanced water solubility and allow the introduction of a thiol or amine group to nucleic acids. These compounds represent the first set of thiolated psoralen derivatives, and their usefulness is demonstrated in several nucleic acid cross-linking experiments. The reagents can be used to create both intraduplex reversible cross-links between the two single-strand partners in a DNA double helix and interduplex reversible cross-links between two DNA double helices.     

Stability decrease of RNA double helices by phenylalanine-, tyrosine- and tryptophane-amides. Analysis in terms of site binding and relation to melting proteins.

The amides of L-phenylalanine, L-tyrosine and L-tryptophane decrease the melting temperatures tm of poly(A)*poly(U) and poly(I)*poly(C) double helices at low concentrations (1 mM), whereas high concentrations finally lead to an increase of tm. This dependence of the tm-values upon the ligand concentration can be represented quantitatively by a simple site binding model, providing binding parameters for the interaction between the amides and the nucleic acids both in the double- and the single-stranded conformation. According to these data the affinity to the single strands is higher than that to the double strands and increases in the series Phe less than Tyr less than Trp. The binding constants decrease with increasing salt concentration as expected for an interaction driven by electrostatic attraction. However, part of the interaction is also due to stacking between the aromatic amides and the nucleic acid bases. The present results indicate a direct correlation between the presence of aromatic amino acids at the binding site of helix destabilising proteins and the properties of simple derivatives of these amino acids. Furthermore the results suggest that very simple peptides containing aromatic amino acids served as a starting point for the evolution of helix destabilising proteins.     

Variability in the geometry of RNA double helices generated from dinucleoside building-blocks

A graphical method is presented for the generation of helical parameters from single-crystal structures of RNA nucleic acid fragments that are minimally dinucleosides. The method is compared with other published procedures, for a number of text examples. The RNA double helices generated from three different salts of the dinucleoside monophosphate GpC are examined in relation to the variations in helix morphology that are produced. It is shown that small differences between these GpC salts can be amplified to very distinct helix characteristics.     

A Proposal For A Specific Double-Helical Structure In Which The Polynucleotide Strands Intercalate Instead of Forming Base-pairs

Abstract

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen- bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5′-dCMPNa₂ which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3Å. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30° and a base separation of 6.6Å along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5Å, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.     

Progress in designing nucleic acid structures and fine-tuning their interactions

The prediction of the structure of biological macromolecules at the atomic level and the design of new meta-stable structures and secondary interactions are critical tests of our understanding of the structures and the inter-atomic forces that underlie molecular biology. The capacity to accurately predict and design new structures and interactions will allow us to create nucleic acid sequences that will fold in new and useful ways. Here, we present some results to demonstrate the progress we have made in designing and assembling new nucleic acid structures that will make an increasingly important contribution to biology and medicine. We call the reaction cycle that exemplifies our approach 'A handshake from a hairpin on the way to a double helix.'     

Mechanisms of DNA binding determined in optical tweezers experiments

The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments.     

Influence of the incorporation of a cyclohexenyl nucleic acid (CeNA) residue onto the sequence d(CGCGAATTCGCG)

Cyclohexene nucleic acids (CeNA), which are characterized by the presence of a cyclohexene moiety instead of a natural (deoxy)ribose sugar, are known to increase the thermal and enzymatic stability when incorporated in RNA oligonucleotides. As it has been demonstrated that even a single cyclohexenyl nucleoside, when incorporated in an oligonucleotide, can have a profound effect on the biological activity of the oligonucleotide, further research is warranted to study the complex of such oligonucleotides with target proteins. In order to analyse the influence of CeNA residues onto the helix conformation and hydration of natural nucleic acid structures, a cyclohexenyl-adenine building block (xAr) was incorporated into the Dickerson sequence CGCGA(xAr)TTCGCG. The crystal structure of this sequence determined to a resolution of 1.90 Å. The global helix belongs to the B-type family and shows a water spine, which is partially broken up by the apolar cyclohexene residue. The cyclohexene ring adopts the ²E-conformation allowing a better incorporation of the residue in the dodecamer sequence. The crystal packing is stabilized by cobalt hexamine residues and belongs to space group P222₁, never before reported for nucleic acids.     

The DNA Phosphate orientation. Infrared data and energetically favorable structures

The conformation of nucleic acids can be probed reliably by the geometrical arrangement of the PO₂- groups in the backbone. The phosphate arrangement is described by two angles, θ_OO and θ_OPO, which can be determined by several methods. In the present paper, the two angles obtained for the A, B, and C forms of DNA, experimentally by ir linear dichroism (LD) of oriented films as well as theoretically from energy-minimized structures. The methodology of the phosphate angle determination from the ir dichroism spectra is presented in detail, elucidating the potentialities and limitations of this method. Advantageously, tilt and twist angles of the DNA bases, found by theoretical calculations, were used in the ir data processing. The phosphate angles were compared with the corresponding x-ray and nmr results from the literature. Regarding the rather high flexibility of the double helix, as well as the sequence-dependent variation of the conformational angles, a fairly good agreement between our results and the majority of all discussed data can be established. Thus ir LD provides reliable data on the orientation of phosphate groups in DNA. Reasons for discrepancies with some literature data from x-ray fiber-diffraction analysis, published several years ago, will be discussed.     

Are there structural analogies between amino acids and nucleic acids?

Space-filling molecular models have been used to examine structural analogies between amino acids and nucleic acids. The three-dimensional structures of amino acidR groups appear to be stereochemically related to cavities formed by removal of single bases in double helical nucleic acids. The common L amino acids may thus be complementary to their codons.Their publications are indicated by an asterisk in the references at the end of this paper.