Development of a statistically robust quantification method for microorganisms in mixtures using oligonucleotide microarrays

High-density oligonucleotide arrays can be extremely useful for identifying and quantifying specific targets (i.e., ribosomal RNA of microorganisms) in mixtures. However, current array identification schemes are severely compromised by nonspecific hybridization, resulting in numerous false-positive and false-negative calls, they lack an adequate internal control for assessing the quality of identification, and are dependent on amplification of specific target sequences which introduce biases. We have developed a novel approach for the routine quantification and identification of metabolically active microorganisms in mixed samples. The advantage of our approach over conventional ones is that it avoids designing, optimizing, validating, and selecting oligonucleotide probes for arrays; also, nonspecific hybridization is no longer a problem. The basic principle of the approach is that a fluorescence pattern of a mixed sample is a superposition of the fluorescent patterns for each target. The superposition can be quantitatively deconvoluted in terms of concentrations of each microbe. We demonstrated the utility of our approach by extracting rRNA from three microorganisms, making test mixtures, labeling the rRNA, and hybridizing each test mixture to DNA oligonucleotide (20-mers, n=346,608) arrays. Comparison of known concentrations of individual targets in mixtures to those estimated by the solution revealed highly consistent results. The goodness-of-fit of the solution revealed that about 90% of the variability in the data could be explained. A new analytical approach for microbial identification and quantification has been presented in this report. Our findings demonstrate that including signal intensity values from all duplexes on the array, which are essentially nonspecific to the target organisms, significantly improved predictions of known microbial targets. To our knowledge, this is the first study to report this phenomenon. In addition, we demonstrate that the method is a self-sufficient analytical procedure since it provides statistical confidence of the quantification.     

Affinity capture of specific DNA fragments with short synthetic sequences

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures.     

Protein microarrays: a chance to study microorganisms?

Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein–protein interactions or protein–small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides) are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard to investigations of microorganisms.     

A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics.

We describe a high throughput microtiter-based assay to measure binding of oligodeoxyribonucleotides to nucleic acid targets. The assay utilizes oligodeoxyribonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE). Reaction of AE with sodium sulfite renders it non-chemiluminescent. When an AE-labeled probe hybridizes to a target nucleic acid AE is protected from reaction with sodium sulfite and thus remains chemiluminescent. In contrast, unhybridized probe readily reacts with sodium sulfite and is rendered non-chemiluminescent. Hybridization of an AE-labeled probe to a target nucleic acid can therefore be detected without physical separation of unhybridized probe by treatment of the hybridization reaction with sodium sulfite and measurement of the remaining chemiluminescence. Using this method we measured hybridization rate constants and thermodynamic affinities of oligodeoxyribonucleotide probes binding to simple synthetic targets as well as large complex biological targets. The kinetic and thermodynamic parameters were measured with a high degree of accuracy and were in excellent agreement with values measured by other established techniques.     

Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridizations

A key issue in applications of short oligonucleotide-based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single-stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications.     

Brush effects on DNA chips: thermodynamics, kinetics, and design guidelines

In biology experiments, oligonucleotide microarrays are contacted with a solution of long nucleic acid targets. The hybridized probes thus carry long tails. When the surface density of the oligonucleotide probes is high enough, the progress of hybridization gives rise to a polyelectrolyte brush due to mutual crowding of the nucleic acid tails. The free-energy penalty associated with the brush modifies both the hybridization isotherms and the rate equations: the attainable hybridization is lowered significantly as is the hybridization rate. When the equilibrium hybridization fraction, x(eq), is low, the hybridization follows a Langmuir type isotherm, x(eq)/(1 - x(eq)) = c(t)K where c(t) is the target concentration and K is the equilibrium constant. K is smaller than its bulk value by a factor (n/N)(2/5) due to wall effects where n and N denote the number of bases in the probe and the target. At higher x(eq), when the brush is formed, the leading correction is x(eq)/(1 - x(eq)) = c(t)K exp - const'x(eq)(2/3) - x(B)(2/3) where x(B) corresponds to the onset of the brush regime. The denaturation rate constant in the two regimes is identical. However, the hybridization rate constant in the brush regime is lower, the leading correction being exp -const' x(2/3) - x(B)(2/3).     

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (T_d-w, temperature of maximum specific dissociation rate constant), obtained from probe–target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.     

Dendron-modified surfaces provide an ideal environment for stem-loop DNA probes

Specificity and sensitivity are important factors affecting DNA microarrays. Stem-loop DNA probes (SLPs) can be more specific in their recognition of target sequences than linear DNA probes, but unless they are carefully designed, surface interactions can disrupt the native stem-loop structure. In this study, we show how dendron-modified surfaces with well-defined, uniform spacing of aldehyde chemical functionalities offer an ideal substrate to immobilize SLPs and use them to detect nucleic acid targets. The mesospacing provided by the dendron-modified surfaces produces a solution-like environment that allows the SLPs to detect target nucleic acids at concentrations as low as 1pM in concentration.     

Oligonucleotides form a duplex with non-helical properties on a positively charged surface

The double helix is known to form as a result of hybridization of complementary nucleic acid strands in aqueous solution. In the helix the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and are available for interaction with cationic groups. Cation-coated glass surfaces are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cationic surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Here we describe a simple method to fabricate DNA microarrays based upon adsorptive rather than covalent attachment of oligonucleotides to a positively charged surface. We show that such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base pair-specific hybridization with a solution state DNA target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest, on symmetry grounds, that the target DNA binds to such adsorbed oligonucleotides to form a highly asymmetrical and unwound duplex. Thus, it is suggested that, at least on a charged surface, a non-helical DNA duplex can be the preferred structural isomer under standard biochemical conditions.     

Snap-to-it probes: chelate-constrained nucleobase oligomers with enhanced binding specificity

We describe snap-to-it probes, a novel probe technology to enhance the hybridization specificity of natural and unnatural nucleic acid oligomers using a simple and readily introduced structural motif. Snap-to-it probes were prepared from peptide nucleic acid (PNA) oligomers by modifying each terminus with a coordinating ligand. The two coordinating ligands constrain the probe into a macrocyclic configuration through formation of an intramolecular chelate with a divalent transition metal ion. On hybridization with a DNA target, the intramolecular chelate in the snap-to-it probe dissociates, resulting in the probe ‘snapping-to’ and binding the target nucleic acid. Thermal transition analysis of snap-to-it probes with complementary and single-mismatch DNA targets revealed that the transition between free and target-bound probe conformations was a reversible equilibrium, and the intramolecular chelate provided a thermodynamic barrier to target binding that resulted in a significant increase in mismatch discrimination. A 4–6°C increase in specificity (ΔT_m) was observed from snap-to-it probes bearing either terminal iminodiacetic acid ligands coordinated with Ni²⁺, or terminal dihistidine and nitrilotriacetic acid ligands coordinated with Cu²⁺. The difference in specificity of the PNA oligomer relative to DNA was more than doubled in snap-to-it probes. Snap-to-it probes labeled with a fluorophore-quencher pair exhibited target-dependent fluorescence enhancement upon binding with target DNA.     

Nucleic acid capture assay,a new method for direct quantitation of nucleic acids

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.     

Conjugates of PNA with naphthalene diimide derivatives having a broad range of DNA affinities

Peptide nucleic acids (PNAs) are neutral DNA analogues, which bind single-stranded DNA (ssDNA) strongly and with high sequence specificity. However, binding efficiency is dependent on the purine content of the PNA strand. This property make more difficult application of PNA as hybridization probes in, e.g., PNA chips, since at a set temperature the hybridization of a fraction of the DNA targets to the PNA probes does not obey Watson-Crick binding rules. The polypurine PNAs, for example, bind the mismatch containing DNA targets stronger, than the pyrimidine rich PNAs their fully complementary targets. Herein we show that PNA-DNA binding efficiency can be finely tuned by the conjugation of derivatives of naphthalene diimide (NADI) to the N-terminus of PNA using polyamide linkers of different lengths. This approach can potentially be used for the design of PNA probes, which bind their DNA targets with similar affinity independently of the PNA sequence.     

ChipCheckII - predicting binding curves for multiple analyte strands on small DNA microarrays

Incomplete binding, saturation, and cross-hybridization between partially complementary strands complicate the parallel detection of nucleic acids via DNA microarrays. Treating the competing equilibria governing binding to microarrays requires computational tools. We have developed the web-based program ChipCheckII that calculates total hybridization matrices for target strands interacting with probes on small DNA microarrays. The program can be used to compute the extent of cross-hybridization and other phenomena affecting fidelity of detection based on sequences, quantities of strands, and hybridization conditions as inputs. Enthalpy and entropy of duplex formation are generated locally with UNAfold, including those for complexes that are partially matched. Simulated binding versus temperature curves for portions of a commercial genome chip demonstrate the extent to which cross-hybridization can complicate DNA detection. ChipCheckII is expected to aid nucleic acid chemists in developing high fidelity DNA microarrays.     

ChipcheckII—Predicting Binding Curves for Multiple Analyte Strands on Small DNA Microarrays

Incomplete binding, saturation, and cross-hybridization between partially complementary strands complicate the parallel detection of nucleic acids via DNA microarrays. Treating the competing equilibria governing binding to microarrays requires computational tools. We have developed the web-based program ChipCheckII that calculates total hybridization matrices for target strands interacting with probes on small DNA microarrays. The program can be used to compute the extent of cross-hybridization and other phenomena affecting fidelity of detection based on sequences, quantities of strands, and hybridization conditions as inputs. Enthalpy and entropy of duplex formation are generated locally with UNAfold, including those for complexes that are partially matched. Simulated binding versus temperature curves for portions of a commercial genome chip demonstrate the extent to which cross-hybridization can complicate DNA detection. ChipCheckII is expected to aid nucleic acid chemists in developing high fidelity DNA microarrays.     

PNA and LNA throw light on DNA

In some aspects, homogeneous (all-in-solution) nucleic acid hybridization assays are superior to the traditionally used heterogeneous (solution-to-surface) alternatives. Profluorescent probes, which reveal fluorescence enhancement or fluorescence polarization upon their binding to DNA and RNA targets, are a paradigm for the real-time sequence-specific homogeneous detection of nucleic acids. A variety of such DNA or RNA-derived probes of different constructs has already been developed with numerous applications. However, the recent additions to the field - locked nucleic acids (LNAs) and peptide nucleic acids (PNAs) - significantly increase the potential of profluorescent probes and provide a robust impulse for their new uses.     

Evaluation of gel-pad oligonucleotide microarray technology by using artificial neural networks

Past studies have suggested that thermal dissociation analysis of nucleic acids hybridized to DNA microarrays would improve discrimination among duplex types by scanning through a broad range of stringency conditions. To more fully constrain the utility of this approach using a previously described gel-pad microarray format, artificial neural networks (NNs) were trained to recognize noisy or low-quality data, as might derive from nonspecific fluorescence, poor hybridization, or compromised data collection. The NNs were trained to classify dissociation profiles (melts) into groups based on selected characteristics (e.g., initial signal intensity, area under the curve) using a data set of 21,044 profiles derived from 186 probes hybridized to a study set of RNA extracted from 32 microbes common to the human oral cavity. Three melt profile groups were identified: one group consisted mostly of ideal melt profiles; another group consisted mostly of poor melt profiles; and, the remainder were difficult to classify. Screening of melting profiles of perfect-match hybrids revealed inconsistencies in the form of melting profiles even for identical probes on the same microarray hybridized to same target rRNA. Approximately 18% of perfect-match duplex types were correctly classified as poor. Experimental variability and deviation from ideal melt behavior were shown to be attributable primarily to a method of local background subtraction that was very sensitive to displacement of the grid frames used for image capture (both determined by the image analysis system) and duplexes with low binding constants. Additional results showed that long RNA fragments limit the discriminating power among duplex types.     

一种新的基于适体和酶切保护分析的超灵敏蛋白质检测方法

以凝血酶适体(aptamer)为例,利用适体和核酸外切酶特性,通过定量PCR扩增建立一种高灵敏的蛋白质检测方法.首先合成3段寡核苷酸序列即凝血酶适体探针,上游连接子和下游连接子.将适体探针与凝血酶温育结合后,再加入核酸外切酶I降解未能结合的探针.接着将保护下来的探针与连接子杂交、连接和对连接产物进行定量PCR .分别建立连接产物标准品浓度与Ct 值的标准曲线和凝血酶浓度与连接产物浓度的标准曲线,通过定量PCR对凝血酶进行定量.结果显示,基于适体的外切酶保护凝血酶检测方法灵敏度较高,连接产物标准品浓度的对数值和Ct 值之间的方程为y =- 2 95x + 33 6 5 (R2 =0. 990 ,P <0 .0 1) ;凝血酶浓度和连接产物浓度对数值之间的方程为y =0 94x - 0 . 2 9(R2 =0 . 998,P <0 . 0 1) ,还对可能影响检测的有关参数举行了探讨.     

Conjugated polyelectrolytes for label-free DNA microarrays

Classical strategies for gene microarrays require labeling of probes or target nucleic acids with signaling molecules, a process that is expensive, time consuming and not always reliable. Bazan and colleagues showed that a nucleic acid-binding cationic conjugated polyelectrolyte can be used in label-free DNA microarrays based on surfaces modified with neutral peptide nucleic acid (PNA) probes. This technique provides a simple and sensitive method for DNA detection without the need for covalent labeling of target DNA.