Recovery of Small DNA Fragments from Serum Using Compaction Precipitation

Background: While most nucleic acids are intracellular, trace amounts of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including micro RNAs, can also be found in peripheral blood. Many studies have suggested the potential utility of these circulating nucleic acids in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. However, DNA circulating in blood is usually present at very low concentrations (ng/ml), and is in the form of relatively small fragments (,1,000 bp), making its isolation challenging. Methods: Here we report an improved method for the isolation of small DNA fragments from serum using selective precipitation by quaternary ammonium compaction agents. A 151 bp fragment of double-stranded DNA from the Escherichia coli bacteriophage lambda served as the model DNA in our experiments. DNA was serially diluted in serum until undetectable by conventional polymerase chain reaction (PCR), before being enriched by compaction precipitation. Results: Starting with concentrations two to three orders of magnitude lower than the PCR-detectable level (0.01 ng/ml), we were able to enrich the DNA to a detectable level using a novel compaction precipitation protocol. The isolated DNA product after compaction precipitation was largely free of serum contaminants and was suitable for downstream applications. Conclusions: Using compaction precipitation, we were able to isolate and concentrate small DNA from serum, and increase the sensitivity of detection by more than four orders of magnitude. We were able to recover and detect very low levels (0.01 ng/ml) of a small DNA fragment in serum. In addition to being very sensitive, the method is fast, simple, inexpensive, and avoids the use of toxic chemicals

The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years.Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against <it>S. pneumoniae </it>and other bacterial pathogens.</p> <p>Results</p> <p>Here we report the crystal structure of alanine racemase from <it>S. pneumoniae </it>(Alr_SP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3₁21 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = β = 90° and γ = 120°. Structural comparisons show that Alr_SPshares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway.</p> <p>Conclusions</p> <p>We have solved the structure of Alr_SP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.</p

Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

<p>Abstract</p> <p>Background</p> <p><it>Bacillus anthracis </it>is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of <it>B. anthracis </it>(Ames) alanine racemase (Alr_<it>Bax</it>), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr_{<it>Bax </it>}structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.</p> <p>Results</p> <p><it>B. anthracis </it>has two open reading frames encoding for putative alanine racemases. We show that only one, <it>dal1</it>, is able to complement a D-alanine auxotrophic strain of <it>E. coli</it>. Purified Dal1, which we term Alr_<it>Bax</it>, is shown to be a dimer in solution by dynamic light scattering and has a V_maxfor racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified Alr_{<it>Bax </it>}is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, Alr_{<it>Bax </it>}makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.</p> <p>Conclusion</p> <p>The chloride ion in Alr_{<it>Bax </it>}is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two Alr_{<it>Bax </it>}structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.</p

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.</p> <p>Results</p> <p>Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the <it>Vibrio proteolyticus </it>5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into <it>Escherichia coli</it>, the chimeric RNA (3×<it>pen </it>aRNA) was expressed constitutively from <it>E. coli rrnB </it>P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse.</p> <p>Conclusions</p> <p>The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs <it>in vivo </it>using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.</p

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

Background: Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results: Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3譸en aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions:The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research

Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae

<p>Abstract</p> <p>Background</p> <p>Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen <it>Streptococcus pneumoniae </it>has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.</p> <p>Results</p> <p>The alanine racemases gene from <it>S. pneumoniae </it>(<it>alr</it>_<it>SP</it>) was amplified by PCR and cloned and expressed in <it>Escherichia coli</it>. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an <it>alr </it>auxotroph of <it>E. coli </it>growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg^-1were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.</p> <p>Conclusion</p> <p>We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen <it>S. pneumoniae</it>. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.</p

The hookworm Ancylostoma ceylanicum intestinal transcriptome provides a platform for selecting drug and vaccine candidates

BACKGROUND: The intestine of hookworms contains enzymes and proteins involved in the blood-feeding process of the parasite and is therefore a promising source of possible vaccine antigens. One such antigen, the hemoglobin-digesting intestinal aspartic protease known as Na-APR-1 from the human hookworm Necator americanus, is currently a lead candidate antigen in clinical trials, as is Na-GST-1 a heme-detoxifying glutathione S-transferase. METHODS: In order to discover additional hookworm vaccine antigens, messenger RNA was obtained from the intestine of male hookworms, Ancylostoma ceylanicum, maintained in hamsters. RNA-seq was performed using Illumina high-throughput sequencing technology. The genes expressed in the hookworm intestine were compared with those expressed in the whole worm and those genes overexpressed in the parasite intestine transcriptome were further analyzed. RESULTS: Among the lead transcripts identified were genes encoding for proteolytic enzymes including an A. ceylanicum APR-1, but the most common proteases were cysteine-, serine-, and metallo-proteases. Also in abundance were specific transporters of key breakdown metabolites, including amino acids, glucose, lipids, ions and water; detoxifying and heme-binding glutathione S-transferases; a family of cysteine-rich/antigen 5/pathogenesis-related 1 proteins (CAP) previously found in high abundance in parasitic nematodes; C-type lectins; and heat shock proteins. These candidates will be ranked for downstream antigen target selection based on key criteria including abundance, uniqueness in the parasite versus the vertebrate host, as well as solubility and yield of expression. CONCLUSION: The intestinal transcriptome of A. ceylanicum provides useful information for the identification of proteins involved in the blood-feeding process, representing a first step towards a reverse vaccinology approach to a human hookworm vaccine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1795-8) contains supplementary material, which is available to authorized users

New Classes of Alanine Racemase Inhibitors Identified by High-Throughput Screening Show Antimicrobial Activity against Mycobacterium tuberculosis

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents

Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

We report a novel, modular approach to immuno-detection based on antibody recognition and PCR read-out that employs antibody-conjugated bacteriophage, easily-manipulated nonpathogenic viruses, as affinity agents. Our platform employs phage genetically tagged for in vivo biotinylation during phage maturation that can easily be linked, through avidin, to any biotinylatable affinity agent, including full-length antibodies, peptides, lectins or aptamers. The presence of analyte is reported with high sensitivity through real-time PCR. This approach avoids the need to clone antibody-encoding DNA fragments, allows the use of full-length, high affinity antibodies and, by having DNA reporters naturally encapsulated inside the bacteriophage, greatly reduces nonspecific binding of DNA. We validate the efficacy of this new approach through the detection of VEGF (Vascular Endothelial Growth Factor), a known angiogenic cancer biomarker protein, at attomolar concentrations in bronchoalveolar lavage (BAL) fluid