Synthesis and Nucleic Acids Binding Properties of Diastereomeric Aminoethylprolyl Peptide Nucleic Acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the “cis-” homothymine aepPNA decamers with (2′R,4′R) and (2′S,4′S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2′R,4′S) and (2′S,4′R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2′R,4′R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

Binding Specificity and Affinity of Acriflavine for Nucleic Acids

A set of conditions was developed for the specific binding of acriflavine to the DNA of intact squamous cells. This was achieved through a series of studies into the relative affinities for dye between DNA and various biopolymers by an agar gel diffusion technique. Specificity was ascertained by DNase and RNase treatment of the cells. The final conditions, based on an estimated DNA-to-dye ratio of 4:1, required a constant cell count of 100,000 and dye at a concentration of 0.0025 μg per ml in 10 ml of phosphate buffer, pH 6.0-7.4. These quantities were dictated by the sensitivity limitations of the analytical apparatus. To make use of standard fluorometric instrumentation, the whole cell population method for determining average values was followed. Free dye was analyzed after cell samples were stained, and the difference between this value and that of an aliquot of working dye was taken as the amount of bound dye. To ensure cell-free residues of dye, cells were removed by centrifugation through Teflon membrane filters. The average amount of dye which was bound to the DNA of normal squamous cells was 1.6 × 10^-7 μg (0.7 × 10^-15 mole) per cell, and the amount bound to HeLa cells was 2.3 × 10^-7 μg (1 × 10^-15 mole). These values were highly replicable, making it possible to use them as an expression of the DNA content of the cells. The mildness of the staining conditions, and the preservation of cellular integrity in this technique may permit quantitative measurements of the DNA content of living cells.     

Effect of LNA Modifications on Small Molecule Binding to Nucleic Acids

Abstract

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2′-O, 4′-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.     

The Cleavage of Nucleic Acids in Reversed Micelles Using Site Specific Endonucleases

Plasmid and λ DNA molecules of between 2.2 and 48.5 kb pairs can be solubilised in n-hexane containing the surfactant sodium dioctyl sulfosuccinate (AOT) and aqueous buffers. Linear λ phage DNA fragments (2.2-23.1 kb pairs) and intact λ bio 1 DNA (48.5 kb pairs) are efficiently cleaved by Bam HI and Em RI in systems containing 100 mM AOT. Under these conditions, λ bio 1 DNA undergoes regioselective restriction by Hind III at only one site but is completely cleaved when the surfactant concentration is lowered to 50 mM. Covalent closed circular plasmid DNA (pUC8, 2.73 kb pairs) is only partially linearised by Eco RI and Bam HI in reversed micelles; Hae II cleavage affords both complete and partial restriction fragments. The results suggest that the tertiary structures adopted by substrate DNA in reversed micelles influence the availability of restriction sites.     

Blind Prediction of Charged Ligand Binding Affinities in a Model Binding Site

Predicting absolute protein–ligand binding affinities remains a frontier challenge in ligand discovery and design. This becomes more difficult when ionic interactions are involved because of the large opposing solvation and electrostatic attraction energies. In a blind test, we examined whether alchemical free-energy calculations could predict binding affinities of 14 charged and 5 neutral compounds previously untested as ligands for a cavity binding site in cytochrome c peroxidase. In this simplified site, polar and cationic ligands compete with solvent to interact with a buried aspartate. Predictions were tested by calorimetry, spectroscopy, and crystallography. Of the 15 compounds predicted to bind, 13 were experimentally confirmed, while 4 compounds were false negative predictions. Predictions had a root-mean-square error of 1.95 kcal/mol to the experimental affinities, and predicted poses had an average RMSD of 1.7 Å to the crystallographic poses. This test serves as a benchmark for these thermodynamically rigorous calculations at predicting binding affinities for charged compounds and gives insights into the existing sources of error, which are primarily electrostatic interactions inside proteins. Our experiments also provide a useful set of ionic binding affinities in a simplified system for testing new affinity prediction methods.     

A Differential Stain for Nucleic Acids

An easily used trichrome stain consisting of orange G, methyl green, and toluidine blue is proposed as a method of differentiating desoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells. Carnoy's acetic-alcohol is the fixative of choice, though cold acetone is also satisfactory. Photomicrographs taken with ultraviolet and visible light show that the structures containing nucleic acid are exactly those which stain with methyl green and toluidine blue. Studies with nucleases and extraction of nucleic acids with cold and hot perchloric acid further indicate a specificityy of the dyes for DNA and RNA. Present experiments are directed toward using the stain for quantitative estimation of the nucleic acids.     

Aryldiazomethane Derivatives as Reagents for Site Specific Labeling of Nucleic Acids at Phosphate

Abstract

An efficient and direct labeling method based on direct alkylation of nucleic acids at phosphates by aryldiazomethane derivatives is described.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

Role of Aromatic Amino-acid Residues in the Binding of Enzymes and Proteins to Nucleic Acids

MANY studies have been made of the specificity of interaction between nucleic acids and polypeptides, proteins and enzymes^1,2. Electrostatic forces between basic amino-acids and phosphate groups contribute to the stability of the complexes, but selective recognition requires more specific interactions which are not yet understood. The recognition of a specific region of a nucleic acid could be explained if this region has some particular conformation or if there are specific interactions between a few amino-acid residues and the bases of this region. We wish to report results which show that the aromatic amino-acids tryptophan and tyrosine can interact with nucleic acid bases in double stranded nucleic acids. They suggest that aromatic amino-acid residues of enzymes and proteins could participate in the binding to nucleic acids by intercalating between the bases and thus constraining the nucleic acid molecule to adopt a definite position with respect to the protein molecule.     

Boranophosphate Nucleic Acids - A Versatile DNA Backbone

Abstract

Important chemical and biochemical properties of boranophosphate DNA and RNA oligonucleotides are reviewed. Stereoregular boranophosphate oligomers can be synthesized enzymatically and form stable duplexes with DNA. Fully boronated, non-stereoregular oligothymidylates, synthesized chemically, form hybrids with poly(A) that have lower melting points than oligothymidylate:poly(A), yet they nevertheless can support the RNase H mediated cleavage of RNA.     

Investigations of Amino Acids in the ATP Binding Site of 5,10-Methenyltetrahydrofolate Synthetase

5-Formyltetrahydrofolate is a compound that is administered as a rescue agent in methotrexate chemotherapy and in 5-fluorouracil chemotherapy for synergistic effects. It has also recently been suggested to play a role in bacterial resistance to antifolate therapy. 5,10-methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme known to catalyze the conversion of this compound to 5,10-methenyltetrahydrofolate along with the hydrolysis of ATP to ADP. To better understand the roles of specific amino acids in the ATP binding pocket of this enzyme, we used site-directed mutagenesis to create 10 modified forms of the Mycoplasma pneumoniae ortholog. The Michaelis constant (K_m) for each substrate and the turnover number (k_cat) was determined for each mutant to help elucidate the role of individual amino acids. Data were compared to crystal structures of human and M. pneumoniae orthologs of MTHFS. Results were largely consistent with a simple coulombic and proximity model; the larger the predicted charges of an interaction and the closer those interactions were to the phosphate transferred between the substrates, the greater the reduction in ATP binding and catalytic activity of the enzyme.     

Nucleic Acids of Entamoeba histolytica

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 178 (0.8 kDa) and 58 rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 258 rRNA: guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 28 RNA relative to 178 RNA. The 258 RNA is “nicked” (apparently during nuclear processing) and dissociates readily into 1 78 (0.7 kDa) and 168 (0.6 kDa) species, and a more rigidly bound 5.88 species. A small amount of “unnicked” 258 RNA was recovered with guanidine. Two DNA-dcpendent RNA polymerases (I and II) with a pronounced preference for denatured DNA as template were eluted from DEAE-Sephadex in reverse order of what occurs in other eukaryotes, except Physarum polycephalum. This conclusion was based on salt optima and alpha-amanitin sensitivity studies. Initial characterization of DNA isolated with a procedure capable of isolating > 100-kbp Leishmania DNA showed that undigested DNA migrates as a broad band between markers 6 and 24 kbp. The persistent recovery of such a “band” by us and Perez-Mutul et al. no larger than ca. 24 kbp (with the exception of >48 kbp DNA isolated by Hernandez et al. using an in situ lysis technique which did not include a proteinase) may be due to nicks introduced during isolation; or, perhaps much of the amebal DNA exists in vivo as gene sized fragments. However, preliminary data generated using orthogonal pulse-field agarose gel electrophoresis do suggest that amebal DNA may be in small chromosomes.     

How to Use Not-Always-Reliable Binding Site Information in Protein-Protein Docking Prediction

In many protein-protein docking algorithms, binding site information is used to help predicting the protein complex structures. Using correct and accurate binding site information can increase protein-protein docking success rate significantly. On the other hand, using wrong binding sites information should lead to a failed prediction, or, at least decrease the success rate. Recently, various successful theoretical methods have been proposed to predict the binding sites of proteins. However, the predicted binding site information is not always reliable, sometimes wrong binding site information could be given. Hence there is a high risk to use the predicted binding site information in current docking algorithms. In this paper, a softly restricting method (SRM) is developed to solve this problem. By utilizing predicted binding site information in a proper way, the SRM algorithm is sensitive to the correct binding site information but insensitive to wrong information, which decreases the risk of using predicted binding site information. This SRM is tested on benchmark 3.0 using purely predicted binding site information. The result shows that when the predicted information is correct, SRM increases the success rate significantly; however, even if the predicted information is completely wrong, SRM only decreases success rate slightly, which indicates that the SRM is suitable for utilizing predicted binding site information.     

A Multi-sample Microdialysis Apparatus for Proteins and Nucleic Acids

Abstract

A multiposition microdialysis system suitable for simultaneous microsample applications (between 10 μL and 500 μL), has been developed. Each sample, contained in a specially designed microfuge dialysis tube (mDT), is dialysed independently from the other samples. Each mDT has its own membrane, and this feature allows the use of different membranes and dialysis times for different samples.

The microdialysis apparatus is kept at constant temperature by an external thermostat, avoiding the use of a cold box. The dialysis release time for small ions, a parameter used for quantitation of microdialysis efficiency, decreases from 22.9 min (for a 200 μL sample) to 7 min (for a 50 μL sample). The sample is efficiently recovered by centrifugation. Quantitative recoveries (90%) of different proteins and DNA were achieved after microdialysis by mDT.     

Structural Pre-organization of Peptide Nucleic Acids

Abstract

Introduction of constraint via chemical bridging in the aegPNA leads to the five or six membered cyclic structures that may contribute towards maintaining the balance between rigidity and flexibility of the PNA backbone. The significant promise of our approach to use the naturally occurring trans-4-hydroxy-L-proline to arrive at different chirally pure cyclic PNA analogs and their DNA binding properties will be presented.     

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer.Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample.