Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA

Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences.     

Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

Recently, we developed a simple isothermal nucleic acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), to detect specific DNA sequences with great sensitivity and large dynamic range. In this paper, we combined PG-RCA with a three-way junction (3WJ) formation, and detected specific RNA molecules with high sensitivity and specificity in a one-step and isothermal reaction format. In the presence of target RNA, 3WJ probes (primer and template) are designed to form a 3WJ structure, from which multiple signal primers for the following PG-RCA can be generated by repeating primer extension, nicking and signal primer dissociation. Although this signal primer generation is a linear amplification process, the PG-RCA exponentially can amplify these signal primers and thus even a very small amount of RNA specimen can be detected. After optimizing the structures of 3WJ probes, the detection limit of this assay was 15.9 zmol (9.55 × 10(3) molecules) of synthetic RNA or 143 zmol (8.6 × 10(4) molecules) of in vitro transcribed human CD4 mRNA. Further, the applicability of this assay to detect CD4 mRNA in a human mRNA sample was demonstrated.     

Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3′-end

We improved the target RNA-primed RCA technique for direct detection and analysis of RNA in vitro and in situ. Previously we showed that the 3′ → 5′ single-stranded RNA exonucleolytic activity of Phi29 DNA polymerase converts the target RNA into a primer and uses it for RCA initiation. However, in some cases, the single-stranded RNA exoribonucleolytic activity of the polymerase is hindered by strong double-stranded structures at the 3′-end of target RNAs. We demonstrate that in such hampered cases, the double-stranded RNA-specific Escherichia coli RNase III efficiently assists Phi29 DNA polymerase in converting the target RNA into a primer. These observations extend the target RNA-primed RCA possibilities to test RNA sequences distanced far from the 3′-end and customize this technique for the inner RNA sequence analysis.     

A homogeneous nucleic acid hybridization assay based on strand displacement.

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement.     

Sequence-specific detection using a universal probe system based on the formation of a four-way junction structure

We have developed a novel hybridization detection system using a universal probe based on the formation of a four-way junction (4WJ) structure. This methodology employs a combination of two sequence-specific probes and a universal quenching probe, and the same universal probe can be used for any target gene, allowing cost-effective assays. This 4WJ detection is ideal for extensive parallel identification of nucleic acids such as in multiplex polymerase chain reaction (PCR) systems. Compared with gel electrophoresis, this detection procedure is not only sensitive and rapid but also free of hazardous chemicals such as ethidium bromide. In addition, the 4WJ hybridization technology is more specific as an identifier than the size of a band on an agarose gel. We used a model multiplex PCR method that detected eight different virulence genes in Escherichia coli isolates, demonstrating that our 4WJ detection system is rapid, sensitive, and specific.     

Substrate specificities of bacterial and human AlkB proteins

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.     

In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional,ATP-dependent Degradation of DNA Target

Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. In Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid degradation in vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.     

Intracellular inactivation of specific nucleotide sequences: a general approach to the treatment of viral diseases and virally-mediated cancers.

A general approach is proposed for development of anti-viral complexes capable of specific intracellular inactivation of viral genetic sequences. Such complexes would consist of a specially designed bifunctional crosslinking agent bound to a single-stranded segment of virus-specific nucleic acid (the carrier). Pairing this complex with its complementary target sequence would generate covalent interstrand crosslinks between carrier and target, thereby irreversibly inactivating the target sequence. Since cells have natural mechanisms for taking up nucleic acids and pairing the newly-taken-up material with complementary sequences within the cell, it is proposed such mechanisms can be exploited for delivery of nucleic acid-agent complexes to their intracellular viral targets. The feasibility of developing and delivering such antiviral complexes is discussed in light of currently available compounds and techniques.     

Aptamer-based bio-barcode assay for the detection of cytochrome-c released from apoptotic cells

The recently developed bio-barcode (BBC) assay using polymerase chain reaction (PCR) to generate signals has been shown to be an extraordinarily sensitive method to detect protein targets. The BBC assay involves a magnetic microparticle (with antibody to capture the target of interest) and gold nanoparticle (with recognition antibody and thiolated single-stranded barcode DNAs) to form a sandwich around the target. The concentration of target is determined by the amount of barcode DNA released from the nanoparticles. Here we describe a modification using aptamers to substitute the gold nanoparticles for the BBC assay. In this study, we isolated a 76-mer monoclonal aptamer against cytochrome-c (cyto-c) and this single-stranded DNA in defined 3D structure for cyto-c was used in the BBC assay for both recognition and readout reporting. After magnetic separation, the aptamer was amplified by PCR and this aptamer-based barcode (ABC) assay was sensitive enough to detect the cyto-c in culture medium released from the apoptotic cells after drug treatment at the picomolar level. When compared to the conventional cyto-c detection by Western blot analysis, our ABC assay is sensitive, and time for the detection and quantification with ready-made probes was only 3 h.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10⁶-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.