Synthesis and properties of a new cleavable nucleic acid-protein crosslinking reagent

A new compound, dithiobis[9-(2-ethylenecarbamoylethylamino)-2,3-dimethoxy-6-azido-acridine], was synthesized and used in some preliminary experiments to form cleavable complexes between nucleic acids and proteins. In a first step the azidoacridine moiety of the reagent intercalates between the bases of nucleic acids and is then bound by reaction of the azido group. The disulfide group of the reagent is simultaneously converted under reducing conditions into a thiol which, in a second step, can be bound by oxidation to -SH groups of a vicinal protein (additional -SH groups can be inserted in the protein using 2-iminothiolane). The nucleic acid-protein complexes thus formed can be redissociated by reduction. The potential applications of this new cleavable crosslinking reagent could be extended to topographical investigations of any biological structure composed of nucleic acids and proteins.     

Biotin-streptavidin-labeled oligonucleotides as probes of helicase mechanisms

Helicases use the energy from ATP hydrolysis to catalyze formation of single-stranded nucleic acids by unwinding double-stranded nucleic acids. The ATP-dependent reaction can be broken down into at least two steps: melting of the duplex and translocation of the enzyme along the nucleic acid lattice. Each step presents difficulties for study because clear end points for the reactions are not always available. For example, translocation involves the movement of the enzyme from one point along the lattice to a new position, with no net change in chemical structure of the nucleic acid. Hence, new assays have been developed in which the nucleic acid is modified to contain a "protein block" that impedes translocation of the enzyme. To prepare such protein blocks, biotin-streptavidin has been used due to the ease with which the biotin can be incorporated into nucleic acids by chemical synthesis. Several applications of oligonucleotides labeled with biotin-streptavidin for the study of helicase mechanisms are described.     

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.     

Electricity-free, sequential nucleic acid and protein isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.     

Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA

Electrophoretic mobility shift assays (EMSAs) are commonly used to analyze nucleic acid-protein interactions. When nucleic acid is bound by protein, its mobility during gel electrophoresis is reduced. Similarly, the final position of protein within a complex is shifted when compared to its free state. Here we provide a protocol for a simple approach that uses these mobility differences to identify nucleic acid-binding proteins. Following EMSA, denaturing gel electrophoresis is implemented to provide a second dimension of separation. Protein that binds a specific nucleic acid is identified as a spot(s) whose presence at a particular position(s) is dependent on nucleic acid within the initial binding reaction. The polypeptide in a spot can be subsequently identified by mass spectrometry. As EMSAs can be performed using partially purified or cell extracts, this approach substantially reduces the need for protein purification. It should facilitate the identification of a nucleic acid-binding protein within approximately 4 d.     

Electron spectroscopic imaging of chromatin.

The analytical electron microscope technique called electron spectroscopic imaging (ESI) has a number of applications in the study of DNA:protein complexes. The method offers an intermediate level of spatial resolution for in vitro structural studies of complexes that may be too large or heterogeneous to study by crystallography or magnetic resonance spectroscopy. An advantage of ESI is that the distribution of nucleic acids can be resolved in a nucleoprotein complex by mapping the element phosphorus, present at high levels in nucleic acid compared to protein. Measurements of phosphorus content together with mass determination allows estimates to be made of stoichiometric relationships of protein and nucleic acids in these complexes. ESI is also suited to in situ studies of nuclear structure. Mass-sensitive images combined with nitrogen and phosphorus maps can be used to distinguish nucleic acid components from nuclear structures that are predominantly protein based. Interactions between chromatin on the periphery of interchromatin granule clusters (IGC) with the protein substructure that connects the exterior of the IGC to its core can be studied with this technique. The method also avoids the use of heavy atom stains, agents required in conventional electron microscopy, that preclude the distinguishing of structures on the basis of their biochemical composition. The principles of ESI and technical aspects of the method are discussed.     

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2-oxo group it can form a hydrazone with N4-aminocytosine. Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl2 at pH 7.0 and 37 degrees C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N4-aminocytosines. 35S-labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37 degrees C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA. The co-sedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA-protein complex was prepared from unlabeled 30S subunits. The protein portion was labeled with 125I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.     

Simulation of non-specific protein-mRNA interactions

Protein–nucleic acid interactions exhibit varying degrees of specificity. Relatively high affinity, sequence-specific interactions, can be studied with structure determination, but lower affinity, non-specific interactions are also of biological importance. We report simulations that predict the population of nucleic acid paths around protein surfaces, and give binding constant differences for changes in the protein scaffold. The method is applied to the non-specific component of interactions between eIF4Es and messenger RNAs that are bound tightly at the cap site. Adding a fragment of eIF4G to the system changes both the population of mRNA paths and the protein–mRNA binding affinity, suggesting a potential role for non-specific interactions in modulating translational properties. Generally, the free energy simulation technique could work in harness with characterized tethering points to extend analysis of nucleic acid conformation, and its modulation by protein scaffolds.     

生物条形码检测技术及其研究进展

生物条形码检测技术是利用磁场作用收集"金纳米颗粒-被检物-磁性微球"复合物,再释放其中的条形码链,然后可选用多种方法对条形码链进行下一步检测。生物条形码检测技术在蛋白质及核酸的检测方面显示出极高的灵敏度,在不依赖酶反应的情况下,检测蛋白质可以达到10-18mol水平,同时在核酸检测方面也能达到PCR反应的灵敏度。该技术是一种新型的诊断技术,在临床诊断方面有着广泛的应用前景。     

A spin probe approach for measuring the nucleic acid affinity of gene 32 protein.

A novel and fast procedure for determining by electron spin resonance the affinity of proteins for nucleic acids is described. The assay makes use of nitroxide radicals which are covalently bound to various polynuleotides to the extent of one probe per 75 to 100 nucleotides. As a test example gene 32 protein was used, a protein known to interact with single stranded nucleic acids. Competition experiments with unlabeled nucleic acids made it feasible to directly monitor the gene 32 protein affinity for different nucleic acids. It was observed that DNA single strands are not necessarily favored by this protein for preferential binding. The experimental data also suggest that the difference in the binding constants for most of the complexes is remarkably large; for instance, (dT)_n binds at least 3 to 4 orders of magnitude better to gene 32 protein than (dA)_n.     

Photochemical crosslinking of protein and DNA in chromatin. II. Synthesis and application of psoralen-cystamine-arylazido photocrosslinking reagents

The synthesis and testing of a new type of nucleic acid-protein photocrosslinking reagent is described. The reagents are composed of a psoralen ligand for nucleic acid photoattachment, which is linked to an azidobenzoyl group, for protein photoattachment. The linker contains a disulfide bridge which can be opened by reduction with mercaptans. The reagents were tested in a chromatin system, where it was found that they induced cleavable crosslinks between the histones and the DNA upon irradiation with long-wavelength ultraviolet light (lambda greater than 300 nm).     

Useful Microsoft Word Macros for molecular biologists and protein chemists

Biologists today make extensive use of word processing programs for the production of research reports, literature reviews and grant proposals. Frequently, such programs become the default platform for viewing and the later publication of protein and nucleic acid sequence data. Thus, researchers often switch between their word processor and more specialized programs designed to analyze protein and nucleic acid sequences. It would be more convenient to perform these simple sequence analyses using the word processor without switching to another program. The focus here is on the use of the Visual Basic programming language, which is built into all recent versions of Microsoft Word to generate surprisingly complex and useful macros that can conveniently analyze several important features of protein and nucleic acid sequences. The standard Word interface can also be easily modified to display and run these macros from a pull-down menu. Several examples of this approach are provided.     

Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

Heat capacity changes associated with nucleic acid folding

Whereas heat capacity changes (DeltaCPs) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Recent advances in the understanding of nucleic acid folding and improved technology for measuring the energetics of folding transitions have allowed a greater experimental window for measuring these effects. We present in this review a survey of current literature that confronts the issue of DeltaCPs associated with nucleic acid folding transitions. This work helps to gather the molecular insights that can be gleaned from analysis of DeltaCPs and points toward the challenges that will need to be overcome if the energetic contribution of DeltaCP terms are to be put to use in improving free energy calculations for nucleic acid structure prediction.     

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se‐Met) strategy). This selenium derivatization strategy via MAD (multi‐wavelength anomalous dispersion) phasing has revolutionized protein X‐ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X‐ray crystal‐structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O‐atom at the positions 2′, 4′, 5′, and in nucleobases and non‐bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid‐phase chemical synthesis and enzymatic methods, and the Se‐derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein–nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2′‐position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom‐specific substitution of the nucleotide O‐atom, better stability under X‐ray radiation, and structure isomorphism. Therefore, our Se‐derivatization strategy has great potentials to provide rational solutions for both phase determination and high‐quality crystal growth in nucleic‐acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site‐specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se‐derivatization strategy in nucleic acid and protein research are also described in this review.     

The C-terminal domain of nucleolin accelerates nucleic acid annealing

We report that the abundant nucleolar protein nucleolin accelerates nucleic acid annealing. Nucleolin accelerates annealing of complementary oligonucleotides and of oligonucleotides that contain a limited number of mismatches. The annealing activity of nucleolin can be localized to a C-terminal region consisting of two RNA binding domains (RBD3 and RBD4) and the RGG(9) domain (RBD3-RBD4-RGG(9)). This same region mediates self-association of nucleolin. The RGG(9) domain of nucleolin, believed to mediate interactions between nucleolin and several ribosomal proteins, is neither sufficient for self-association, as determined by small-angle X-ray scattering, nor can it independently accelerate annealing. Acceleration of nucleic acid annealing by nucleolin is likely to depend on self-association of nucleolin molecules bound to nucleic acid.     

马尾松毛虫蛋白质、核酸酶和羧酸酯酶与耐药性的关系

马尾松毛虫(Dendrolimus Punctatus Walder)幼虫蛋白质含量,羧酸酮酶和多酚氧化酶活力与虫龄大小成正相关;氰戊菊酯处理后,兴奋期的蛋白质含量和羧酸酯酶活力上升,到抑制期均降低到正常虫体的水平,而多酚氧化酶的变动不大。正常虫体中脱氧核糖核酸酶(Dnase)和核糖核酸酶(Rnase)的活力存在差异：Dnase从3-5龄幼虫随虫体增大而上升,到6龄时明显降低;Rnase与虫龄大小成负相关;氰戊菊酯处理后,Dnase便开始下降并低于正常虫体的水平,而Rnase在兴奋期上升,到抑制期下降亦低于正常虫体水平。结果说明,除多酚氧化酶外,蛋白质、核酸酶和羧酸酯酶均与耐药性存在一定的相关性,研制酶的抑制剂具有实用意义。根据毒力测定结果,马尾松毛虫幼虫随虫龄增大而耐药力增加,氰戊菊酯对5龄幼虫是3龄的2.8倍,6龄是4龄的2.3倍。因此,掌握在4龄前进行药物防治是合理的。     

Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex.

The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr. Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to [32P]p(dT)16 and 70% of the nonspecific binding energy of the intact protein. However, peptide CN6 does not exhibit the binding specificity of the intact protein. Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.     

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.     

Ferrocene-functionalized SWCNT for electrochemical detection of T4 polynucleotide kinase activity

A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti(4+)) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing 5'-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic 5'-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The 5'-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product can be linked with the signal indicator by Ti(4+). The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4 PNK. This assay can measure activity of T4 PNK down to 0.01 UmL(-1). The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.