What is the structure of the RecA-DNA filament?

The bacterial RecA protein has been a model system for understanding how a protein can catalyze homologous genetic recombination. RecA-like proteins have now been characterized from many organisms, from bacteriophage to humans. Some of the RecA-like proteins, including human RAD51, appear to function as helical filaments formed on DNA. However, we currently have high resolution structures of inactive forms of the protein, and low resolution structures of the active complexes formed by RecA-like proteins on DNA in the presence of ATP or ATP analogs. Within a crystal of the E. coli RecA protein, a helical polymer exists, and it has been widely assumed that this polymer is quite similar to the active helical filament formed on DNA. Recent developments have suggested that this may not be the case.     

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

Inhibition of Escherichia coli RecA by rationally redesigned N-terminal helix

Bacterial RecA promotes the development and transmission of antibiotic resistance genes by self-assembling into an ATP-hydrolyzing filamentous homopolymer on single-stranded DNA. We report the design of a 29mer peptide based on the RecA N-terminal domain involved in intermonomer contact that inhibits RecA filament assembly with an IC50 of 3 microM.     

Conductive metal nanowires templated by the nucleoprotein filaments, complex of DNA and RecA protein

Development of preprogrammable conductive nanowires is a requisite for the future fabrication of nanoscale electronics based on molecular assembly. Here, we report the synthesis of conductive metal nanowires from nucleoprotein filaments, complexes of single- or double-stranded DNA and RecA protein. A genetically engineered RecA derivative possessing a reactive and surface accessible cysteine residue was reacted with functionalized gold particles, resulting in nucleoprotein filaments with gold particles attached. The template-based gold particles were enlarged by chemical deposition to form uniformly metallized nanowires. The programming information can be encoded in DNA sequences so that an intricate electrical circuit can be constructed through self-assembly of each component. As the RecA filament has higher degree of stiffness than double-stranded DNA, it provides a robust scaffold that allows us to fabricate more reliable and well-organized electrical circuitry at the nanoscale. Furthermore, the function of homologous pairing provides sequence-specific junction formation as well as sequence-specific patterning metallization.     

Solid-phase synthesis, thermal denaturation studies, nuclease resistance, and cellular uptake of (oligodeoxyribonucleoside)methylborane phosphine-DNA chimeras

The major hurdle associated with utilizing oligodeoxyribonucleotides for therapeutic purposes is their poor delivery into cells coupled with high nuclease susceptibility. In an attempt to combine the nonionic nature and high nuclease stability of the P-C bond of methylphosphonates with the high membrane permeability, low toxicity, and improved gene silencing ability of borane phosphonates, we have focused our research on the relatively unexplored methylborane phosphine (Me-P-BH(3)) modification. This Article describes the automated solid-phase synthesis of mixed-backbone oligodeoxynucleotides (ODNs) consisting of methylborane phosphine and phosphate or thiophosphate linkages (16-mers). Nuclease stability assays show that methylborane phosphine ODNs are highly resistant to 5' and 3' exonucleases. When hybridized to a complementary strand, the ODN:RNA duplex was more stable than its corresponding ODN:DNA duplex. The binding affinity of ODN:RNA duplex increased at lower salt concentration and approached that of a native DNA:RNA duplex under conditions close to physiological saline, indicating that the Me-P-BH(3) linkage is positively charged. Cellular uptake measurements indicate that these ODNs are efficiently taken up by cells even when the strand is 13% modified. Treatment of HeLa cells and WM-239A cells with fluorescently labeled ODNs shows significant cytoplasmic fluorescence when viewed under a microscope. Our results suggest that methylborane phosphine ODNs may prove very valuable as potential candidates in antisense research and RNAi.     

Probing DNA Mismatched and Bulged Structures by Using 19F NMR Spectroscopy and Oligodeoxynucleotides with an 19F‐Labeled Nucleobase

In this study, DNA local structures with bulged bases and mismatched base pairs as well as ordinary full‐matched base pairs by using ¹⁹F NMR spectroscopy with ¹⁹F‐labeled oligodeoxynucleotides (ODNs) were monitored. The chemical shift change in the ¹⁹F NMR spectra allowed discrimination of the DNA structures. Two types of ODNs possessing the bis(trifluoromethyl)benzene unit (F‐unit) at specified uridines were prepared and hybridized with their complementary or noncomplementary strands to form matched, mismatched, or bulged duplexes. By using ODN F1, in which an F‐unit was connected directly to a propargyl amine‐substituted uridine, three local structures, that is, full‐matched, G–U mismatch, and A‐bulge could be analyzed, whereas other structures could not be discriminated. A molecular modeling study revealed that the F‐unit in ODN F1 interacted little with the nucleobases and sugar backbone of the opposite strand because the linker length between the F‐unit and the uridine base was too short. Therefore, the capacity of ODN F1 to discriminate the DNA local structures was limited. Thus, ODN F2 was designed to improve this system; aminobenzoic acid was inserted between the F‐unit and uridine base so the F‐unit could interact more closely with the opposite strand. Eventually, the G‐bulge and T–U mismatch and the three aforementioned local structures could be discriminated by using ODN F2. In addition, the dissociation processes of these duplexes could be monitored concurrently by ¹⁹F NMR spectroscopy.     

Characterization of labeled oligonucleotides using enzymatic digestion and tandem mass spectrometry

A simple and powerful method for the determination of labeling sites on oligodeoxynucleotides (ODN) has been developed. The method is based on the finding that nuclease P1 (NP1) digestions of label-containing ODNs produce site-specific products: 5′-labeled ODNs produce label-nucleotide (L-N); 3′-labeled ODN produces phosphorylated label (pL); and a label in between the ODN termini produces pL-N. Mass spectrometry spectra of these products from the digestion mixture can be easily utilized for structural verification of labeled ODNs such as DNA probes. We also developed a method for the determination of the labeling sites of ODNs with unknown label structures. In this method, NP1 digestion products generate site-specific fragmentation patterns upon collision-induced dissociation. These patterns can be easily recognized and used for the identification of labeling sites of ODNs with unknown label structures. When an ODN is internally labeled, phosphodiesterase digestion may be used to determine the exact labeling site (sequence location). It was demonstrated that these methods can be applied for ODNs with single or multiple labels, and for ODNs with the same or different labels within an ODN.     

Synthesis and hybridization properties of oligonucleotide-perylene conjugates: influence of the conjugation parameters on triplex and duplex stabilities

We report here the synthesis of oligo-2'-deoxyribonucleotides (ODNs) conjugated with perylene. Introduction of perylene, coupled either directly or via a propyl linker to the anomeric position of a 2'-deoxyribose residue, induces the formation of two anomers. Single incorporations of each pure anomer of these sugar-perylene units have been performed at either the 5'-end or an internal position of a pyrimidic pentadecamer. The binding properties of these modified ODNs with their single- and double-stranded DNA targets were studied by absorption spectroscopy. Double incorporations of the sugar-perylene unit most efficient at stabilizing the triplex and duplex structures (the beta-anomer involving the propyl linker) have been performed at both the 5'-end and at an internal position (or both the 5'- and 3'-ends) of the ODN chain. Comparison has been made with ODN-perylene conjugates involving either one or two perylenes attached via a longer polymethylene chain to either the 5'- or 3'- (or both the 5'-and 3'-) terminal phosphate groups. The ODNs involving two perylenes are more efficient at stabilizing the triplex and the duplex structures than the ODNs involving only one perylene and, among these, the ODN-perylene conjugate involving two sugar-perylene units attached at both termini is the most efficient. The results of the fluorescence studies have shown an important increase in the intensity of the fluorescent signal upon hybridization of the ODNs involving two perylenes with either the single- or the double-stranded targets. This increase in the intensity of the fluorescent signal could be used as proof of the hybridization.     

2′,4′-BNA/LNA with 9-(2-Aminoethoxy)-1,3-diaza-2-oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance**

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex-forming abilities and enhanced nuclease resistance. 2′-O,4′-C-Methylene-bridged nucleic acid/locked nucleic acid (2′,4′-BNA/LNA) with 1,3-diaza-2-oxophenoxazine (BNAP ( B^H )) was previously reported. Herein, a novel B^H analogue, 2′,4′-BNA/LNA with 9-(2-aminoethoxy)-1,3-diaza-2-oxophenoxazine (G-clamp), named BNAP-AEO ( B^AEO ), was designed. The B^AEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing B^AEO possessed up to 10⁴-, 152-, and 11-fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2′-deoxycytidine ( C ), 2′,4′-BNA/LNA with 5-methylcytosine ( L ), or 2′-deoxyribonucleoside with G-clamp ( P^AEO ), respectively. Moreover, duplexes formed by ODN bearing B^AEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing B^AEO was more resistant to 3′-exonuclease than ODNs with phosphorothioate linkages.     

Template directed reversible photochemical ligation of oligodeoxynucleotides

We demonstrated that 5-vinyldeoxyuridine ((V)U) and 5-carboxyvinyldeoxyuridine ((CV)U) can be used to photoligate a longer oligonucleotide (ODN) from smaller ODNs on a template. By performing irradiation at 366 nm, these artificial nucleotides make photoligated ODNs with high efficiency without any side reactions. Moreover, by performing irradiation at 312 nm, these photoligated ODNs were reversed to the original ODN. (V)U needs to be irradiated 366 nm for 6 h, but (CV)U needs to be irradiated at 366 nm for 15 min. Finally, we made a self-assembled structure with an ODN containing (CV)U and observed the photoligated ODN by photoirradiation.     

Atomic force microscopic study of low temperature induced disassembly of RecA-dsDNA filaments

The assembly and disassembly of RecA-DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are important steps for homologous recombination and DNA repair. The assembly and disassembly of the nucleoprotein filaments are sensitive to the reaction conditions. In this work, we investigated different morphologies of the formed nucleoprotein filaments at low temperature under different solution conditions by atomic force microscopy (AFM). We found that low temperature and long keeping time could induce the incomplete disassembly of the formed nucleoprotein filaments. In addition, when the formed filaments were kept at -20 degrees C for 20 h with 1,4-dithiothreitol (DTT), the integrated filaments disassembled. It was similar to the case under the same condition without anything added. However, when glycerol was used as a substitute for DTT, there was no obvious disassembly at the same condition. Oppositely, when the formed filaments were kept at 4 degrees C for 20 h, the disassembly with additional DTT was not as obvious as the case at -20 degrees C for 20 h, whereas the case with additional glycerol disassembled. The experiments indicated the effect of cold denaturation on the interaction of DNA and RecA. Meanwhile, the study of these phenomena can supply guidelines for the property and stability of RecA as well as the relevant roles of influencing factors to RecA and DNA in further theoretical studies.     

Modulation of pyrene fluorescence in DNA probes depends upon the nature of the conformationally restricted nucleotide

The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.     

A cysteine-appended deoxyuridine for the postsynthetic DNA modification using native chemical ligation

A new deoxyuridine derivative 6 bearing a cysteine group at the C5 position was synthesized and incorporated into oligodeoxynucleotide (ODN) by phosphoramidite chemistry. The postsynthetic DNA modification of the cysteine-containing ODN using native chemical ligation with thioesters of biotin and green fluorescent protein variant was successfully demonstrated.     

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

Background

Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase.

Results

We have cloned and purified SSB from Bacillus anthracis (SSB_BA). In the absence of DNA, at concentrations ??100 ??g/ml, SSB_BA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSB_BA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)₇₀ binding suggested that SSB_BA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure.

Conclusions

Unlike other prokaryotic SSB proteins, SSB_BA from Bacillus anthracis appeared to be monomeric at concentrations ??100 ??g/ml as determined by SE-HPLC. SSB_BA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSB_BA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSB_BA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.     

快速蛋白质液相色谱法分离鉴定脱氧寡核苷酸片段

由机器合成的反义核酸药物需要有效的纯度鉴定方法。用ＭＯＮＯ Ｑ柱在ｐＨ１２时以ＮａＣｌ的浓度梯度洗脱,可将１９至２１Ｎｔｓ的小片段寡核苷酸很好分开,因此快速蛋白质液相色谱法可用来分离鉴定反义核酸药物。     

<Emphasis Type="Italic">Escherichia coli</Emphasis> single-strand binding protein–DNA interactions on carbon nanotube-modified electrodes from a label-free electrochemical hybridization sensor

An electrochemical hybridization biosensor based on the intrinsic oxidation signals of nucleic acids and proteins has been designed, that makes use of the unique binding event between Escherichia coli single-strand binding protein (SSB) and single-stranded DNA (ssDNA). The voltammetric signal from guanine oxidation significantly decreased upon binding of SSB to single-stranded oligonucleotides (probe), anchored on a single-walled carbon nanotube (SWCNT) -modified screen-printed carbon electrode (SPE). Simultaneously, oxidation of the tyrosine (Tyr) and tryptophan (Trp) residues of the SSB protein increased upon binding of the SSB protein to ssDNA and ss-oligonucleotides. After the hybridization, SSB did not bind to the double helix form, and the guanine signal could be observed along with the disappearance of the oxidation signal of the protein. The amplification of intrinsic guanine and protein oxidation signals by SWCNT, and a washing step with sodium dodecylsulfate, enabled the specific detection of a point mutation. Monitoring the changes in the guanine and protein signals upon hybridization greatly simplified the detection procedure. The detection limit of 0.15 g/ml target DNA can be applied to genetic assays. To the best of our knowledge, this is the first work that utilizes the monitoring of SSB–DNA interactions on a solid transducer for the electrochemical detection of DNA hybridization by using intrinsic oxidation signals.     

Highly selective and sensitive template-directed photoligation of DNA via 5-carbamoylvinyl-2'-deoxycytidine

We describe a highly efficient template-directed photoligation of oligodeoxynucleotides (ODNs) through 5-carbamoylvinyl-2'-deoxycytidine ((CV)C). When an ODN containing (CV)C at the 5' end was photoirradiated with an ODN containing a pyrimidine base at the 3' end in the presence of template DNA, efficient photoligation was observed without any byproduct formation. Single nucleotide differences can be successfully distinguished by using photoligation-based DNA chip assay. [structure: see text]     

Synthesis and hybridization properties of oligonucleotides containing polyamines at the C-2 position of purines: a pre-synthetic approach for the incorporation of spermine into oligodeoxynucleotides containing 2-(4,9,13-triazatridecyl)-2'-deoxyguanosine

We have developed a synthesis of spermine-containing oligonucleotides (ODN-sper) which allows incorporation of multiple polyamine residues. This approach was based on the pertrifluoroacetylated 5'DMT-dGsper phosphoramidite synthon. Its coupling yield with resin-bound ODN decreased dramatically when close to the 3'-end. Optimization of the coupling conditions allowed 22-mer ODNs containing up to six spermine residues to be synthesized. Several ODNs of different sequences with 1-4 pendent spermines could be purified and their hybridization properties were evaluated. Duplex melting temperatures increased linearly with the number of polyamine residues (deltaTm/sper = 3.0 +/- 0.2 degrees C in 100mM NaCl). This compares very favorably with values reported for duplexes of similar initial stability containing other cation-substituted bases. Moreover, the stability increase was neither sequence nor position-dependent, and even contiguous spermine residues did not cross-talk. Extrapolation based on these findings leads to the conclusion that a duplex formed with a 22-mer oligonucleotide containing seven spermine residues would be as stable as genomic DNA, which highlights its potential for DNA strand invasion.