Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis

A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.     

Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres

Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5′-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.     

Experimental and theoretical analysis of the invasive signal amplification reaction

The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling. We have examined the performance of the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction. We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction. We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate. The experimental data are compared with a theoretical model of the invasive signal amplification reaction.     

Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescent adsorbent.

We studied various parameters affecting the sensitivity of assays that use nucleic acid hybridization in solution followed by capture of the hybrid on a solid phase. Sensitivity is limited not only by nonspecific binding of the detection components but also by reannealing of the target or probe to itself. To perform sensitive assays, the probe concentration must be low enough to reduce high nonspecific binding. Under these conditions, however, the strand displacement reaction or the reannealing of the target to itself drastically decreases the hybridization yield, particularly when the target and the probes are different sizes. To improve DNA detection, we propose a sandwich method based on hybridization of oligonucleotides with a single-strand DNA obtained by polymerase chain reaction under asymmetric conditions. The assay can be performed in one step using a bioluminescent detection procedure which does not require any separation step. The specificity of the method is sufficient to perform a rapid detection and quantification of papillomavirus in biological samples.     

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.     

Electric chips for rapid detection and quantification of nucleic acids

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.     

Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification

A novel method for regenerating biosensors has been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems requiring low power consumption such as remote field sensing applications. Gold-sputtered 96-well polystyrene microplates and a fluorescent label were used to explore the detection limits of the surface-associated RCA technique, specificity for different MPP, conditions for regeneration of the biomolecular sensing surface, and reproducibility of measurements on regenerated surfaces. The technique was used to create highly selective biomolecular surfaces capable of discriminating between DNA oligonucleotides with sequences identical to RNA from infectious salmon anemia (ISA) and infectious hematopoietic necrosis (IHN) virus. As little as 0.6 fmol of circularized MPP was detectable with this fluorimetric assay. The sensing layers could be reused for at least four cycles of amplification using thermal denaturation, with less than 33% decrease in RCA response over time. Because the nucleic acid product of the test is attached to a surface during amplification, the technique is directly applicable to a variety of existing sensing platforms, including acoustic wave and optical devices.     

Detection of Nucleic Acids by Cycling Probe Technology on Magnetic Particles: High Sensitivity and Ease of Separation

Abstract

Cycling Probe Technology (CPT) is a signal amplification system that allows detection of nucleic acid target sequences without target amplification. CPT employs a sequence specific chimeric probe, typically DNA-RNA-DNA, which hybridizes to a complementary target DNA sequence and becomes a substrate for RNase H. Cleavage occurs at the RNA internucleotide linkages and results in dissociation of the probe from the target, thereby making it available for the next probe molecule. This communication describes the use of oligonucleotides attached to solid supports for target capture and release followed by solution and solid phase cycling. Through the attachment of chimeric probes to Sera-Mag^TM magnetic particles (SMP) a simple and effective method of separating the cleaved probe from non-cycled probe has been developed. By capturing the target DNA on particles and separating it from the extraneous non-specific DNA we are able to dramatically reduce background and thus discriminate between samples of Methicillin Resistant (MRSA) and Methicillin Sensitive (MSSA) Staphylococcus Aureus. We conjugated oligonucleotide probes to SMPs (～1 um) and Nylon beads (NB) which were coated with ID Biomedical's proprietary coating materials (R, patent pending). The general structure of the constructs is shown below:     

Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.     

DNA and protein microarray printing on silicon nitride waveguide surfaces

Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.     

Real-time determination of telomerase activity in cell extracts using an optical biosensor

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3'-end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS)-modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signal-to-noise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.     

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.     

Magnetic bead-based solid phase for selective extraction of genomic DNA

Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media. This article presents a general platform for the fabrication of a magnetic bead-based selective solid phase that can be used for subtractive hybridization or sequence capture applications. Phosphorus dendrimers are used for the first time as linkers in a magnetic bead-based selective solid phase for capture of genomic DNA. Aside from providing a high loading capacity, they render a stable bond between the capture probe and the surface under the high temperature and salt conditions required for denaturation and capture to proceed in a single step. The thermal stability of the solid phase under these conditions is first demonstrated by hybridizing a Cy3-labeled target. The selective capture of DNA targets in a single step is then demonstrated by subtractive hybridization of fragmented human genomic DNA. The specificity and selectivity of the solid phase are demonstrated by the recovery of adenovirus serotype 4 DNA spiked into the human DNA target. The effect of steric and electrostatic constraints was also investigated by using dendrimers of different generations that vary in their size and the number of branches. The results demonstrate that this platform can be used for single-step subtractive hybridization applications with better performance over the conventional two-step method using streptavidin-coated magnetic beads.     

Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization

Background

State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.

Methodology and Principal Findings

The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.

Conclusions and Significance

The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.     

Liquid-based hybridization assay with real-time detection in miniaturized array platforms

An assay for the fluorescent detection of short oligonucleotide probe hybridization in miniaturized high-density array platforms is presented. It combines hybridization in solution with real-time fluorescent detection, which involves measurement of fluorescence increase by means of an induced fluorescence resonance energy transfer. The feasibility of this approach using DNA or RNA as a target, and short DNA- as well as LNA (locked nucleic acid)-modified oligonucleotides as probes is shown. The presented approach could potentially contribute to a significant increase in the throughput of large-scale genomic applications, such as oligofingerprinting and genotyping, and also reduce material consumption.     

Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore.

A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target. In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer. The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal. The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution. Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole. We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.     

Preparation of radioiodinated peptide nucleic acids with high specific activity

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.     

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA)

We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.     

Invasive cleavage reactions on DNA-modified diamond surfaces

Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions. An important application of these surfaces is for surface invasive cleavage reactions, in which target DNA strands added to the solution may result in specific cleavage of surface-bound probe oligonucleotides, permitting analysis of single nucleotide polymorphisms (SNPs). Our previous work demonstrated the feasibility of performing such cleavage reactions on planar gold surfaces using PCR-amplified human genomic DNA as target. The sensitivity of detection in this earlier work was substantially limited by a lack of stability of the gold surface employed. In the present work, detection sensitivity is improved by a factor of approximately 100 (100 amole of DNA target compared with 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface.