Repertoires of the Nucleosome-Positioning Dinucleotides

It is generally accepted that the organization of eukaryotic DNA into chromatin is strongly governed by a code inherent in the genomic DNA sequence. This code, as well as other codes, is superposed on the triplets coding for amino acids. The history of the chromatin code started three decades ago with the discovery of the periodic appearance of certain dinucleotides, with AA/TT and RR/YY giving the strongest signals, all with a period of 10.4 bases. Every base-pair stack in the DNA duplex has specific deformation properties, thus favoring DNA bending in a specific direction. The appearance of the corresponding dinucleotide at the distance 10.4 xn bases will facilitate DNA bending in that direction, which corresponds to the minimum energy of DNA folding in the nucleosome. We have analyzed the periodic appearances of all 16 dinucleotides in the genomes of thirteen different eukaryotic organisms. Our data show that a large variety of dinucleotides (if not all) are, apparently, contributing to the nucleosome positioning code. The choice of the periodical dinucleotides differs considerably from one organism to another. Among other 10.4 base periodicities, a strong and very regular 10.4 base signal was observed for CG dinucleotides in the genome of the honey bee A. mellifera. Also, the dinucleotide CG appears as the only periodical component in the human genome. This observation seems especially relevant since CpG methylation is well known to modulate chromatin packing and regularity. Thus, the selection of the dinucleotides contributing to the chromatin code is species specific, and may differ from region to region, depending on the sequence context.     

An integrated,structure- and energy-based view of the genetic code

The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon–anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson–Crick pairs in the codon–anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon–anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids.     

SinoDuplex: An Improved Duplex Sequencing Approach to Detect Low-frequency Variants in Plasma cfDNA Samples

Accurate detection of low frequency mutations from plasma cell-free DNA in blood using targeted next generation sequencing technology has shown promising benefits in clinical settings. Duplex sequencing technology is the most commonly used approach in liquid biopsies. Unique molecular identifiers are attached to each double-stranded DNA template, followed by production of low-error consensus sequences to detect low frequency variants. However, high sequencing costs have hindered application of this approach in clinical practice. Here, we have developed an improved duplex sequencing approach called Sino Duplex, which utilizes a pool of adapters containing pre-defined barcode sequences to generate far fewer barcode combinations than with random sequences, and implemented a novel computational analysis algorithm to generate duplex consensus sequences more precisely. Sino Duplex increased the output of duplex sequencing technology, making it more cost-effective. We evaluated our approach using reference standard samples and cell-free DNA samples from lung cancer patients. Our results showed that Sino Duplex has high sensitivity and specificity in detecting very low allele frequency mutations. The source code for Sino Duplex is freely available at https://github.com/Sin Oncology/sinoduplex.     

Topological testing of the mechanism of homology search promoted by RecA protein

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA–single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA–ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA–ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA–ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson–Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA–ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.     

Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.     

Energetics of the hairpin to mismatched duplex transition of d(GCCGCAGC) on NaCl solution

We report Potential of Mean Force studies to describe the relative thermodynamic stabilities of d(GCCGCAGC) in a mismatched duplex and a hairpin monomer conformation in NaCl solution. The PMF calculations are combined with previous molecular mechanics and normal mode analysis in order to estimate the role of different components of the free energy in determining the relative stability of the duplex and hairpin structures. The high entropy associated with the loop region and the lack of minor groove phosphate-phosphate interactions in the hairpin compete against the gain in enthalpic contribution to the free energy due to base pairing in the mismatched duplex. The combined free energy calculations show that the hairpin is the most stable conformation at low salt and that a hairpin to duplex transition takes place at approximately 0.47 M NaCl. In addition, we studied the hairpin to partially stacked single helical conformation equilibrium at low salt. We found a small variation in transition temperature in salt concentration, delta Tm/delta log10(cs) approximately 2-3 degrees K/decade, in contrast to the duplex to hairpin or duplex to partially stacked single helix transition where the transition temperature exhibited marked dependence on salt concentration. This is in qualitative agreement with experimental data. Based on the Potential of Mean Force free energy calculation, the order of relative stability of the three-conformations studied varies with salt concentration. We observed the following orders of stability: stacked single helix greater than hairpin greater than duplex for cs less than 0.77 M NaCl; single helix greater than duplex greater than hairpin for 0.77 less than Cs less than 2.1 M; and duplex greater than hairpin greater than single strand for cs greater than 2.1 M. From the calculated PMF free energy curves in the NaCl concentration range, 0.012 less than cs less than 5.0 M, we can assign upper and lower bounds for the non-ionic differences in free energy between the duplex, hairpin, and stacked single helical states (at standard conditions: cs = 1.0 M, T = 25 degrees C, and 1 M oligomer concentration). We found that for delta G duplex single helix = G duplex - 2 x G single helix less than -7.38 Kcal/mol, the single helix is the least stable state. For the duplex-to-hairpin free energy difference in the range, -1.87 less than delta G duplex-hairpin less than 0.03 Kcal/mol, there will always be a salt-induced hairpin-to-duplex transition for 0.01 less than cs less than 1.6 M NaCl. If delta G duplex-hairpin less than -1.87, the duplex is always more stable than the hairpin; and for delta G duplex-hairpin greater than Kcal/mol, the hairpin state is always more stable than the duplex, for all salt concentrations.     

The NMR Structure of Estrone (Es)-tethered Tandem DNA Duplex: [d(5′pCAGCp3′)-Es] + [Es-d(5′ pTCCA3′)]: d(5′ pTGGAGCTG3′)

Abstract

The solution structure of an estrone (Es)-tethered tandem DNA duplex consisting of two Estethered tetranucleotides and a target octameric DNA sequence is reported. The structure of this Es-tethered tandem duplex has been compared with a corresponding natural tandem duplex without estrones. The T_m of the 3′-Es-tethered tetranucleotide part of the tandem duplex increases by 5°C, whereas the T_m of the 5′-Es-tethered tetranucleotide part increases by 7°C, compared with the corresponding natural counterpart. The NMR structures of both the Es-tethered tandem duplex and the natural counterpart have been based on 24 experimental NMR constraints per residue. Despite the fact that there is considerable distortion at the junction of two Es-tethered tetranucleotides in the major groove of the Es-tethered DNA duplex compared to the natural counterpart, both duplexes do take up B-type DNA structures. It is likely that the spatial proximity of two Es residues, and the resulting hydrophobic interaction between them might be responsible for the increase of the thermal stability of the Es-tethered tandem duplex in comparison with the natural counterpart.     

The Mechanical Properties of RNA-DNA Hybrid Duplex Stretched by Magnetic Tweezers

RNA can anneal to its DNA template to generate an RNA-DNA hybrid (RDH) duplex and a displaced DNA strand, termed R-loop. RDH duplex occupies up to 5% of the mammalian genome and plays important roles in many biological processes. The functions of RDH duplex are affected by its mechanical properties, including the elasticity and the conformation transitions. The mechanical properties of RDH duplex, however, are still unclear. In this work, we studied the mechanical properties of RDH duplex using magnetic tweezers in comparison with those of DNA and RNA duplexes with the same sequences. We report that the contour length of RDH duplex is ～0.30 nm/bp, and the stretching modulus of RDH duplex is ～660 pN, neither of which is sensitive to NaCl concentration. The persistence length of RDH duplex depends on NaCl concentration, decreasing from ～63 nm at 1 mM NaCl to ～49 nm at 500 mM NaCl. Under high tension of ～60 pN, the end-opened RDH duplex undergoes two distinct overstretching transitions; at high salt in which the basepairs are stable, it undergoes the nonhysteretic transition, leading to a basepaired elongated structure, whereas at low salt, it undergoes a hysteretic peeling transition, leading to the single-stranded DNA strand under force and the single-stranded RNA strand coils. The peeled RDH is difficult to reanneal back to the duplex conformation, which may be due to the secondary structures formed in the coiled single-stranded RNA strand. These results help us understand the full picture of the structures and mechanical properties of nucleic acid duplexes in solution and provide a baseline for studying the interaction of RDH with proteins at the single-molecule level.     

The sense strand pre-cleaved RNA duplex mediates an efficient RNA interference with less off-target and immune response effects

RNA interference is an appealing and promising therapeutic approach in cancer and other diseases. Designing novel strategies aiming to increase the efficiency, duration, and reduce the off-target silencing by sense strand is of great significance for its future application clinically. Here, we report that RNA duplex with the sense strand pre-cleaved at the base between base 10 and 11 relative to the 5′ end of the antisense strand induced a target-specific RNA silencing effectively. Furthermore, different from the canonical RNA duplex, this novel RNA duplex rarely inhibits the luciferase activity in the reporter, bearing the target sequence corresponding to the sense strand, suggesting a less off-target effects of this novel strategy. Furthermore, the immune response of the novel RNA duplex induced a much milder immune response as seen from the NFkappaB activity. In addition, our newly designed RNA duplex should be easier for preservation than the asymmetric RNA duplex. Our results establish a novel method to design a new class of RNA duplex for improved RNA interference.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔH_ITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex.     

Kissing complex-mediated dimerisation of HIV-1 RNA: coupling extended duplex formation to ribozyme cleavage

Initiation of retroviral genomic RNA dimerisation is mediated by the mutual interaction of the dimerisation initiation site (DIS) stem–loops near to the 5′ end of the RNA. This process is thought to involve formation of a transient ‘kissing’ complex over the self-complementary loop bases, which then refolds into a more stable extended interaction. We have developed a novel experimental system that allows us to clearly detect the extended duplex in vitro. Ribozyme sequences were incorporated into or adjacent to the type 1 human immunodeficiency virus DIS stem, leading to the formation of a functional ribozyme only in the extended duplex conformer. Here we show that extended duplex formation results in ribozyme cleavage, thus demonstrating the double-stranded nature of the extended complex and confirming that refolding occurs via melting of the DIS stems. Loop complementarity is essential for extended duplex formation but no sequence requirements for the loops were observed. Efficiency of extended duplex formation is dependent on the strength of the loop–loop interaction, temperature, the magnesium concentration and is strongly accelerated by the viral nucleocapsid protein NCp7. Our ribozyme-coupled approach should be applicable to the analyses of other refolding processes involving RNA loop–loop interactions.     

Discriminating duplex and hairpin oligonucleotides using chemical shifts: application to the anticodon stem–loop of Escherichia coli tRNAPhe

A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G·U wobble base pair formed between a ¹⁵N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D ¹⁵N–¹H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin–duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of ~20 µM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem–loop of Escherichia coli tRNA^Phe and suggests that this hairpin has a 3 nt loop.     

Characterization of vesicular stomatitis virus mRNA species synthesized in vitro.

The smallest size class of mRNA (12S) synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed.     

2D 1H and 31P NMR Spectra and Distorted A-DNA-Like Duplex Structure of a Phosphorodithioate Oligonucleotide

Abstract

Assignment of the ¹H and ³¹P NMR spectra of a phosphorodithioate modified oligonucleotide decamer duplex, d(CGCTTpS^? ₂AAGCG)₂ (10-mer-S; a site of dithioate substitution is designated with the symbols pS^? ₂), was achieved by two-dimensional homonuclear TOCSY, NOES Y and ¹H-³¹P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. In contrast to the parent palindromic decamer sequence (1) which has been shown to exist entirely in the duplex B-DNA conformation under comparable conditions (100 mM KCI), the dithiophosphate analogue forms a hairpin loop. However, the duplex form of the dithioate oligonucleotide can be stabilized at lower temperatures, higher salt and strand concentration. The solution structure of the decamer duplex was calculated by an iterative hybrid relaxation matrix method (MORASS) combined with 2D NOESY-distance restrained molecular dynamics. These backbone modified compounds, potentially attractive antisense oligonucleotide agents, are often assumed to possess similar structure as the parent nucleic acid complex. Importantly, the refined structure of the phosphorodithioate duplex shows a significant deviation from the parent unmodified, phosphoryl duplex. An overall bend and unwinding in the phosphorodithioate duplex is observed. The structural distortion of the phosphorodithioate duplex was confirmed by comparison of helicoidal parameters and groove dimensions. Especially, the helical twists of the phosphorodithioate decamer deviate significantly from the parent phosphoryl decamer. The minor groove width of phosphorodithioate duplex 10-mer-S varies between 8.4 and 13.3 Å which is much wider than those of the parent phosphoryl decamer d(CGCTTAAGCG)₂ (4.2～9.4Å). The larger minor groove width of 10-mer-S duplex contributes to the unwinding of the backbone and indicates that the duplex has an overall A-DNA-like conformation in the region surrounding the dithiophosphate modification.     

Kinetic and thermodynamic characterization of DNA duplex–hairpin interconversion for two DNA decamers: d(CAACGGGTTG) and d(CAACCCGTTG)

The duplex–hairpin interconversion of two DNA decamers, d(CAACGGGTTG) and d(CAACCCGTTG), has been characterized thermodynamically and kinetically by using uv-melting and nmr relaxation methods. Separately, each decamer shows slow exchange between hairpin and duplex conformations. The hairpin conformations have melting points of 47 and 50°C, respectively, and exhibit similar thermodynamic stabilities. The enthalpies of duplex formation, measured by nmr, were found to be very similar (ΔH_DH = 26 ± 3 kcal/mole) for both decanters at low salt concentrations (< 50 mM NaCl). However, as the salt concentration was increased the behavior of ΔH_DH, and kinetics is significantly different for each decamer. The d(CAACGGGTTG) decamer forms a duplex containing two central G·G mismatches at high salt and DNA concentration. Based upon the measurement of high interconversion activation energies and a decrease in hairpin formation rate with increasing salt, the interconversion between hairpin and duplex was concluded to proceed by complete strand dissociation. In contrast, the d(CAAC-CCGTTG) decamer was determined to form a duplex with two centrally located C·C mismatches at pH values less than 6.2, consistent with the formation of a hemiprotonated C⁺·C mismatch. At pH values greater than 6.4, the hairpin–duplex equilibrium is almost completely shifted toward the hairpin conformation at DNA concentrations of 0.5–7.0 mM and salt concentrations of 10–100 mM. The interconversion of duplex and hairpin conformations was ascertained by means of both kinetic and thermodynamic measurements to proceed by a slightly different mechanism than its complementary decamer. Although the interconversion proceeds by complete strand separation as suggested by high duplex-hairpin interconversion activation enthalpies, the increasing hairpin formation rate with increasing ionic strength as well as the ΔH_DH, dependence on sail indicate that an intermediate internally bulged duplex (no C⁺·C formation) is stabilized by increasing ionic strength. These data support an interconversion mechanism where an intermediate internally bulged duplex may be the rate limiting step before strand separation. © 1995 John Wiley & Sons, Inc.     

Resolution of a structural competition involving dimeric G-quadruplex and its C-rich complementary strand

The resolution of the dimeric intermolecular G-quadruplex/duplex competition of the telomeric DNA sequence 5′-TAG GGT TAG GGT-3′ and of its complementary 5′ ACC CTA ACC CTA-3′ is reported. To achieve this goal, melting experiments of both sequences and of the mixtures of these sequences were monitored by molecular absorption, molecular fluorescence and circular dichroism spectroscopies. Molecular fluorescence measurements were carried out using molecular beacons technology, in which the 5′-TAG GGT TAG GGT-3′ sequence was labelled with a fluorophore and a quencher at the ends of the strand. Mathematical analysis of experimental spectroscopic data was performed by means of multivariate curve resolution, allowing the calculation of concentration profiles and pure spectra of all resolved structures (dimeric antiparallel and parallel G-quadruplexes, Watson–Crick duplex and single strands) present in solution. Our results show that parallel G-quadruplex is more stable than antiparallel G-quadruplex. When the complementary C-rich strand is present, a mixture of both G-quadruplex structures and Watson–Crick duplex is observed, the duplex being the major species. In addition to melting temperatures, equilibrium constants for the parallel/antiparallel G-quadruplex equilibrium and for the G-quadruplex/duplex equilibrium were determined from the concentration profiles.