Hairpin and duplex forms of a self-complementary dodecamer, d-AGATCTAGATCT, and interaction of the duplex form with the peptide KGWGK: can a pentapeptide destabilize DNA?

Ordered forms of a synthetic dodecamer, d-AGATCTAGATCT, a direct repeat of the BglII recognition sequence, have been investigated using UV, CD, and fluorescence spectroscopy. Complex hairpin-duplex equilibria are manifest in UV thermal transitions, which are monophasic in the presence of very low or high NaCl concentrations but distinctly biphasic at intermediate ionic strengths. In 100 mM NaCl, the 1/Tm vs 1n C curve has a reasonable positive slope, which yields delta H and delta S for duplex formation as -66.2 kcal/mol and -190 cal/mol, respectively. Interaction of the dodecamer in duplex form with a tryptophan-containing peptide, KGWGK, has also been investigated to test the "bookmark" hypothesis (Gabbay et al., 1976) under the uniform structural constraint of the oligonucleotide of defined sequence. CD spectra of the peptide (P), the oligonucleotide (N), and their mixtures at different P/N ratios show a dramatic change in peptide spectrum but little change in nucleic acid dichroism with peptide binding. The Tm of P-N complexes decreases with an increase in peptide binding and levels off at saturation binding of P/N = 2.0. The data are interpreted in terms of a groove-cum-intercalation mode of binding, where intercalation to the tryptophan side chain destabilizes the double helix. A Scatchard plot of the binding data is nonlinear, with best-fit values for an overall association constant K = 4.33 x 10(5) M-1, and the number of binding sites n = 3.23 when fitted to the site-exclusion model of binding.     

Unusual conformation of a 3′-thioformacetal linkage in a DNA duplex

Summary The DNA·DNA duplex ·d(GCGCAAAACGCG) (designated duplex III) containing a 3-thioformacetal (3-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH₂O) linkage at the central T^T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3-TFMA-modified T6 sugar adopts an O4-endo form (an intermediate structure between the usual C2-endo and C3-endo forms). This change is accompanied by a change in the (C4–C3–S3–CH₂) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6–T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3-TFMA linkages is merely in the substitution of the T6(O3) in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.Supplementary material available from the authors: One table containing J₁₂, J₁₂ and J₃₄ of duplexes I, II and III.     

Recognition of RNA duplex by a neomycin–Hoechst 33258 conjugate

DNA minor groove binding drugs such as Hoechst 33258 have been shown to bind to a number of RNA structures. Similarly, RNA binding ligands such as neomycin have been shown by us to bind to a number of A-form DNA structures. A neomycin–Hoechst 33258 conjugate was recently shown to bind B-DNA, where Hoechst exhibits high affinity for the minor groove of A/T tract DNA and neomycin docks into the major groove. Further studies now indicate that the Hoechst moiety of the conjugate can be driven to bind RNA duplex as a consequence of neomycin binding in the RNA major groove. This is the first example of Hoechst 33258 binding to RNA duplex not containing bulges or loop motifs.     

Sequence-dependent stability test of a left-handed β-helix motif

The left-handed β-helix (LHBH) is an intriguing, rare structural pattern in polypeptides that has been implicated in the formation of amyloid aggregates. We used accurate all-atom replica-exchange molecular dynamics (REMD) simulations to study the relative stability of diverse sequences in the LHBH conformation. Ensemble-average coordinates from REMD served as a scoring criterion to identify sequences and threadings optimally suited to the LHBH, as in a fold recognition paradigm. We examined the repeatability of our REMD simulations, finding that single simulations can be reliable to a quantifiable extent. We find expected behavior for the positive and negative control cases of a native LHBH and intrinsically disordered sequences, respectively. Polyglutamine and a designed hexapeptide repeat show remarkable affinity for the LHBH motif. A structural model for misfolded murine prion protein was also considered, and showed intermediate stability under the given conditions. Our technique is found to be an effective probe of LHBH stability, and promises to be scalable to broader studies of this and potentially other novel or rare motifs. The superstable character of the designed hexapeptide repeat suggests theoretical and experimental follow-ups.     

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with k_cat ≈ 8 x 10⁻³ s⁻¹ and K_M < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower k_cat and a K_M ≈ 300 nM. The rate of ligation increased with addition of Mn²⁺, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.     

4′-C-Aminomethyl-2′-deoxy-2′-fluoroarabinonucleoside increases the nuclease resistance of DNA without inhibiting the ability of a DNA/RNA duplex to activate RNase H

An antisense oligonucleotide is expected as an innovative drug for cancer and hereditary diseases. In this paper, we designed and synthesized DNAs containing a novel nucleoside analog, 1-(4-C-aminomethyl-2-deoxy-2-fluoro-β-d-arabinofuranosyl)thymine, and evaluated their properties. It was revealed that the analog slightly decreases the thermal stability of the DNA/RNA duplex but significantly increases the stability of DNA in a buffer containing bovine serum. Furthermore, it turned out that the DNA/RNA duplex containing the analog is a good substrate for Escherichia coli RNase H. Thus, DNAs containing the nucleoside analog would be good candidates for the development of therapeutic antisense oligonucleotides.     

Crystal structure of RNase H3–substrate complex reveals parallel evolution of RNA/DNA hybrid recognition

RNases H participate in the replication and maintenance of genomic DNA. RNase H1 cleaves the RNA strand of RNA/DNA hybrids, and RNase H2 in addition hydrolyzes the RNA residue of RNA–DNA junctions. RNase H3 is structurally closely related to RNases H2, but its biochemical properties are similar to type 1 enzymes. Its unique N-terminal substrate-binding domain (N-domain) is related to TATA-binding protein. Here, we report the first crystal structure of RNase H3 in complex with its RNA/DNA substrate. Just like RNases H1, type 3 enzyme recognizes the 2′-OH groups of the RNA strand and detects the DNA strand by binding a phosphate group and inducing B-form conformation. Moreover, the N-domain recognizes RNA and DNA in a manner that is highly similar to the hybrid-binding domain of RNases H1. Our structure demonstrates a remarkable example of parallel evolution of the elements used in the specific recognition of RNA and DNA.     

Analysis of the conformational features of Watson–Crick duplex fragments by molecular mechanics and quantum mechanics methods

It is generally accepted that important features of the Watson–Crick duplex (WCD) originate from the molecular structure of its subunits. However, it is still unclear what properties of each subunit are responsible for significant features of the WCD structure. Computations of deoxydinucleoside monophosphate (dDMP) complexes with Na ions on the basis of the density functional theory (DFT) have shown that conformational properties of minimal single-stranded fragments of DNA play a pivotal role in the origin of the unique features of the WCD. The directionality of the sugar-phosphate backbone (SPB) and preferable ranges of its torsion angles combined with the difference in geometry between purines and pyrimidines have been found to define the nucleotide sequence dependence of the WCD three-dimensional structure. In this work, density functional theory computations were extended to minimal duplex fragments, that is, complementary dDMP (cdDMP) complexes with Na ions. Using several computational methods and various functionals, energy minima were searched for the BI conformation of cdDMP complexes with different nucleotide sequences. Two sequences were optimized using an ab initio method at the MP2/6-31++G** level of theory. An analysis of the SPB torsion angles, sugar-ring puckering, and mutual base positions in the optimized structures showed that the conformational features of cdDMP complexes with Na ions remained within the BI ranges and become more similar to the corresponding features that WCDs display in a crystal. Qualitatively, the main features of each cdDMP complex were invariant with different computational methods, although the values of certain conformational parameters could vary, but still within the limits that are typical of the corresponding family. Common functionals that are employed in DFT calculations were observed to overestimate the distance between base pairs, while MP2 computations and new complex functionals yielded structures with atom–atom contacts that are too close. Several energy minima that correspond to the BI conformation have been proven to exist for certain cdDMP complexes with Na ions, indicating that the topography of the potential energy surface is complex. This circumstance accounts for the variation of conformational parameters among duplex fragments with the same nucleotide sequence. The common AMBER and CHARMM molecular mechanics force fields reproduce many conformational characteristics of dDMPs and their complementary complexes with Na ions, but fail to reproduce certain details of the nucleotide sequence dependence of the WCD conformation.     

The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.     

The conformational states of Mg·ATP in water

The conformational equilibria of Mg·ATP in solution is studied using molecular dynamics (MD) augmented with umbrella sampling methods. Free energy comparisons show that the Mg²⁺ ion is equally likely to coordinate the oxygens of the two end phosphates, or of all three phosphates. The MD trajectories reveal two major degrees of freedom of the Mg·ATP molecule in solution, and we compute the free energy as a function of these variables, and determine its elastic properties. Comparing the free energy function with several crystallographic structures of ATP analogs, we find that the crystal structures correspond to states where ATP would be elastically strained. The average water density around Mg·ATP is investigated to show the average number of hydrogen bonds and the hydrophobicity.     

Structure determination of DNA methylation lesions N1-meA and N3-meC in duplex DNA using a cross-linked protein–DNA system

N¹-meA and N³-meC are cytotoxic DNA base methylation lesions that can accumulate in the genomes of various organisms in the presence of S_N2 type methylating agents. We report here the structural characterization of these base lesions in duplex DNA using a cross-linked protein–DNA crystallization system. The crystal structure of N¹-meA:T pair shows an unambiguous Hoogsteen base pair with a syn conformation adopted by N¹-meA, which exhibits significant changes in the opening, roll and twist angles as compared to the normal A:T base pair. Unlike N¹-meA, N³-meC does not establish any interaction with the opposite G, but remains partially intrahelical. Also, structurally characterized is the N⁶-meA base modification that forms a normal base pair with the opposite T in duplex DNA. Structural characterization of these base methylation modifications provides molecular level information on how they affect the overall structure of duplex DNA. In addition, the base pairs containing N¹-meA or N³-meC do not share any specific characteristic properties except that both lesions create thermodynamically unstable regions in a duplex DNA, a property that may be explored by the repair proteins to locate these lesions.     

Can recA protein promote homologous pairing between duplex regions of DNA?

RecA protein has been shown to promote the formation of joint molecules between intact duplex DNA and homologous gapped DNA. When examined by electron microscopy, such joint molecules display a junction that is, in most cases, distant from the site of the gap. This led us to test whether the observed location of the joint was due to pairing at the gap followed by branch migration, or whether recA-promoted pairing could also take place between duplex homologous regions away from the gap. To test the latter possibility, intact duplex DNA was incubated with DNA which contained a gap in a region of non-homology. Joint molecules were detected by filter binding assay and by electron microscopy at about one-third of the yield observed for fully homologous molecules. These results indicate that initial homologous pairing promoted by recA protein is not restricted to the single-stranded region in the gap but can also take place in regions where both molecules are duplex.     

Leeches as a source of mammalian viral DNA and RNA—a study in medicinal leeches

Surveillance of wild vertebrates can be challenging in remote and inaccessible areas such as tropical rainforests. Blood-feeding parasites, such as leeches, can facilitate wild vertebrate monitoring by targeting residual DNA from the animals the leeches feed on. Successes in detecting host DNA from leeches suggest that host viruses may also be detectable. To systematically test this hypothesis, we performed a proof of concept study using quantitative PCR (qPCR) to detect DNA viruses (bovine herpesvirus [BHV], human adenovirus [HAdV]) and RNA viruses (influenza A [InfA] and measles morbillivirus [MeV]) from nucleic acids extracted from medicinal leeches fed with blood spiked with each virus. All viruses except BHV showed a gradual decline in concentration from day 1 to 50, and all except BHV were detectable in at least half of the samples even after 50 days. BHV exhibited a rapid decline at day 27 and was undetectable at day 50. Our findings in medicinal leeches indicate that leeches collected in the wild might be an untapped resource for detecting vertebrate viruses and could provide new opportunities to study wildlife viral diseases of rare species in challenging environments, where capturing and handling of animals is difficult.     

Case for an RNA–prion world: a hypothesis based on conformational diversity

Prions and other misfolded proteins can impart their structure and functions to normal molecules. Based upon a thorough structural assessment of RNA, prions and misfolded proteins, especially from the perspective of conformational diversity, we propose a case for co-existence of these in the pre-biotic world. Analyzing the evolution of physical aspects of biochemical structures, we put forward a case for an RNA–prion pre-biotic world, instead of, merely, the “RNA World”.     

Is DNA a nonlinear dynamical system where solitary conformational waves are possible?

DNA is considered as a nonlinear dynamical system in which solitary conformational waves can be excited. The history of the approach, the main results, and arguments in favour and against are presented. Perspectives are discussed pertaining to studies of DNA’s nonlinear properties.     

Is there a glycosaminoglycan-related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus.     

Structural consequences of a 3′ → 3′ DNA interstrand cross-link by a trinuclear platinum complex: unique formation of two such cross-links in a 10-mer duplex

Combined multidimensional nuclear magnetic resonance spectroscopy and electrospray mass spectrometry was used to analyze the platinated DNA adduct of the phase II anticancer drug [{trans-PtCl(NH₃)₂}₂-μ-{trans-Pt(NH₃)₂(NH₂(CH₂)₆NH₂)₂}](NO₃)₄ (BBR3464) with [5′-d(ACG*TATACG*T)-3′]₂. Two 1,2-interstrand cross-links were formed by concomitant binding of two trinuclear moieties to the oligonucleotide. The four DNA-bound platinum atoms coordinated in the major groove at N7 positions of guanines in the 3′ → 3′ direction and the central platinum unit is expected to lie in the DNA minor groove. This is the first report of such a DNA lesion. The melting temperature of the adduct is 76 °C and is 42 °C higher than that of the unplatinated DNA. The sugar residues of the platinated bases are in the N-type conformation and the G9 nucleoside is in the syn orientation, while the G3 nucleoside appears to retain the anti configuration. The secondary structure of DNA was significantly changed upon cross-linking of the two BBR3464 molecules. Base destacking occurs between A1/C2 and C2/G3 and weakened stacking is seen for the C8/G9 and G9/T10 bases. The lack of Watson–Crick base pairing is also seen for A1–T10 and C2–G9 base pairs, whereas Watson–Crick base pairs in the central sequence of the DNA (T4 → A7) are well maintained. While DNA repair proteins may “see” different platinated adducts as bulky “lesions”, the subtle differences involved in base pairing and stacking, as summarized here, may extend to their role as a substrate for repair enzymes. Thus, differences in protein recognition and repair efficiency among the various interstrand cross-links are likely and a subject worthy of detailed exploration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence-specific cleavage of the RNA strand in DNA–RNA hybrids by the fusion of ribonuclease H with a zinc finger

Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA–RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA–RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA–RNA hybrids. The optimization of the fusion enzyme’s specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.