Mismatch formation in solution and on DNA microarrays: how modified nucleosides can overcome shortcomings of imperfect hybridization caused by oligonucleotide composition and base pairing

The DNA microarray technology is a well-established and widely used technology although it has several drawbacks. The accurate molecular recognition of the canonical nucleobases of probe and target is the basis for reliable results obtained from microarray hybridization experiments. However, the great flexibility of base pairs within the DNA molecule allows the formation of various secondary structures incorporating Watson-Crick base pairs as well as non-canonical base pair motifs, thus becoming a source of inaccuracy and inconsistence. The first part of this report provides an overview of unusual base pair motifs formed during molecular DNA interaction in solution highlighting selected secondary structures employing non-Watson-Crick base pairs. The same mispairing phenomena obtained in solution are expected to occur for immobilized probe molecules as well as for target oligonucleotides employed in microarray hybridization experiments the effect of base pairing and oligonucleotide composition on hybridization is considered. The incorporation of nucleoside derivatives as close shape mimics of the four canonical nucleosides into the probe and target oligonucleotides is discussed as a chemical tool to resolve unwanted mispairing. The second part focuses non-Watson-Crick base pairing during hybridization performed on microarrays. This is exemplified for the unusual stable dG.dA base pair.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

DNA hybridization on microparticles: determining capture-probe density and equilibrium dissociation constants

Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-self-complementary sequences that can be used to calculate the equilibrium dissociation constant ( Kd ) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.     

Sequence dependence of fluorescence emission and quenching of doubly thiazole orange labeled DNA: effective design of a hybridization-sensitive probe

We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.     

Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

Facilitating RNA structure prediction with microarrays

Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.     

Formation of the double helix: a mutational study

To investigate the mechanisms by which oligonucleotides hybridize to target molecules, the binding of two oligodeoxynucleotide probes to RNA targets was measured over a broad range of temperatures. Mutations were then scanned across each DNA/RNA hybrid to map, at single base resolution, sequences important for hybridization. Despite being unrelated in sequence, each hybrid formed by a similar mechanism. In the absence of secondary structure, two stretches of bases, termed nucleation regions, cooperated with one another by a looping mechanism to nucleate hybridization. Mutations inside each nucleation region strongly decreased hybridization rates, even at temperatures well below the melting temperature (T_m) of the hybridized duplex. Surprisingly, nucleation regions were detected in a RNA target but not a corresponding DNA target. When either nucleation region was sequestered in secondary structure, the hybridization rate fell and the mechanism of hybridization changed. Single-stranded bases within the nucleation region of the probe and target first collided to form a double helix. If sufficiently G + C rich, the double helix then propagated throughout the oligonucleotide by a strand invasion process. On the basis of these results, general mechanisms for the hybridization of oligonucleotides to complementary and mutant targets are proposed.     

The effect of surface probe density on DNA hybridization

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.     

Preparation and characterization of spin-labeled oligonucleotides for DNA hybridization.

Sequence-specific spin-labeled oligodeoxynucleotides with conformation-sensitive electron paramagnetic resonance (EPR) signals are synthesized and examined as solution-phase nucleic acid hybridization probes. Either a proxyl or tempo ring linked to the C(5) position of deoxyuridine (dU) by a nonrigid two-atom methylamino tether is incorporated within 15-mers by phosphotriester chemistry yielding stable spin-labeled probes with distinctive EPR specific activity (AEPR) values. The AEPR is greater for a proxyl-labeled than for a tempo-labeled probe and is consistent with EPR data of enzymatically labeled 26-mers [Bobst, A. M., Pauly, G. T., Keyes, R. S., and Bobst, E. V. (1988) FEBS Lett. 228, 33-36], after normalizing for percent labeling. The spectral characteristics of the free probes and the probe/target complexes are similar to those of enzymatically spin-labeled nucleic acids containing a different nonrigid two-atom-tethered spin label [Bobst, A. M., Kao, S.-C., Toppin, R. C., Ireland, J. C., and Thomas, I. E. (1984) J. Mol. Biol. 173, 63-70]. The presence of target DNA is detected in solution by EPR spectroscopy and the assay is based on the characteristic line-shape change associated with hybridization. The EPR spectra of free and bound probe reflect little interference from changes in global dynamics of the probe, and the line-shape change upon complexation results primarily from a change in local base dynamics. The presence or absence of hybridization can be detected in a loop-gap resonator with about 1 pmol of spin-labeled 15-mer within minutes.     

Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer

When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate limited.     

An intramolecular DNA triplex is disrupted by point mutations associated with hereditary persistence of fetal hemoglobin.

Several investigators have suggested that secondary structures in DNA may be involved with physiologic gene regulatory processes in higher organisms. This hypothesis has been difficult to prove, however, since naturally occurring mutations that alter secondary DNA structures have not yet been identified. In this report, we describe a secondary DNA structure upstream from the human gamma-globin genes; this structure is formed in a homopyrimidine-homopurine tract and is stabilized by acidic pH and negative supercoiling of plasmid DNA. Since this structure is asymmetrically cleaved by S1 nuclease, it probably contains a single-stranded region and an intramolecular triplex. The single-stranded region is actually accessible for Watson-Crick base pairing with exogenous oligomers, a characteristic that permitted us to directly map the secondary DNA structure without additional chemical modifications of the supercoiled DNA. Five different point mutations just downstream from the single-stranded region are associated with hereditary persistence of fetal hemoglobin; four of these mutations dramatically reduce the stability of the secondary DNA structure, suggesting that these mutations alter formation of the intramolecular triplex by destabilizing critical Hoogsteen (triple-stranded) base pairs. These mutations may therefore represent a novel class of genetic defects that alter gene expression by changing the interaction of a critical regulatory molecule with a secondary DNA structure.     

Computer simulation study of molecular recognition in model DNA microarrays

DNA microarrays have been widely adopted by the scientific community for a variety of applications. To improve the performance of microarrays there is a need for a fundamental understanding of the interplay between the various factors that affect microarray sensitivity and specificity. We use lattice Monte Carlo simulations to study the thermodynamics and kinetics of hybridization of single-stranded target genes in solution with complementary probe DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct and each segment represents a sequence of nucleotides ( approximately 11 nucleotides). Each probe segment interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how the probe length, temperature, or hybridization energy, and the stretch along the target that the probe segments complement, affect the extent of hybridization. For systems containing single probe and single target molecules, we observe that as the probe length increases, the probability of binding all probe segments to the target decreases, implying that the specificity decreases. We observe that probes 12-16 segments ( approximately 132-176 nucleotides) long gave the highest specificity and sensitivity. This agrees with the experimental results obtained by another research group, who found an optimal probe length of 150 nucleotides. As the hybridization energy increases, the longer probes are able to bind all their segments to the target, thus improving their specificity. The hybridization kinetics reveals that the segments at the ends of the probe are most likely to start the hybridization. The segments toward the center of the probe remain bound to the target for a longer time than the segments at the ends of the probe.     

Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization

DNA microarray measurements are susceptible to error caused by non-specific hybridization between a probe and a target (cross-hybridization), or between two targets (bulk-hybridization). Search algorithms such as BLASTN can quickly identify potentially hybridizing sequences. We set out to improve BLASTN accuracy by modifying the substitution matrix and gap penalties. We generated gene expression microarray data for samples in which 1 or 10% of the target mass was an exogenous spike of known sequence. We found that the 10% spike induced 2-fold intensity changes in 3% of the probes, two-third of which were decreases in intensity likely caused by bulk-hybridization. These changes were correlated with similarity between the spike and probe sequences. Interestingly, even very weak similarities tended to induce a change in probe intensity with the 10% spike. Using this data, we optimized the BLASTN substitution matrix to more accurately identify probes susceptible to non-specific hybridization with the spike. Relative to the default substitution matrix, the optimized matrix features a decreased score for A–T base pairs relative to G–C base pairs, resulting in a 5–15% increase in area under the ROC curve for identifying affected probes. This optimized matrix may be useful in the design of microarray probes, and in other BLASTN-based searches for hybridization partners.     

Competitive displacement of DNA during surface hybridization

Using real-time dual-color fluorescence detection, we have experimentally tracked individual target species during competitive DNA surface hybridization in a two-component sample. Our experimental results demonstrate displacement of the lower affinity species by the higher affinity species and corroborate recent theoretical models describing competitive DNA surface hybridization. Competition at probe sites complementary to one of the two DNA species was monitored in separate experiments for two different target pairs. Each pair differs in sequence by a single nucleotide polymorphism, and one pair includes a folding target. We propose a mechanistic interpretation of the differences between hybridization curves of targets in multi-component and single-component experiments.     

Dissecting the hybridization of oligonucleotides to structured complementary sequences

BackgroundWhen oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood.MethodsI used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and > 20 complementary oligonucleotides (‘antisenses’).ResultsHybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem.ConclusionsThe experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation–invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates.General significanceWhile analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process.     

Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres

Hybridization kinetics of DNA sequences with known secondary structures and random sequences designed with similar melting temperatures were studied in solution and when one strand was bound to 5 μm silica microspheres. The rates of hybridization followed second-order kinetics and were measured spectrophotometrically in solution and fluorometrically in the solid phase. In solution, the rate constants for the model sequences varied by almost two orders of magnitude, with a decrease in the rate constant with increasing amounts of secondary structure in the target sequence. The random sequences also showed over an order of magnitude difference in the rate constant. In contrast, the hybridization experiments in the solid phase with the same model sequences showed almost no change in the rate constant. Solid phase rate constants were approximately three orders of magnitude lower compared with the solution phase constants for sequences with little or no single-stranded structure. Sequences with a known secondary structure yielded solution phase rate constants as low as 3 × 10³ M⁻¹ s⁻¹ with solid phase rate constants for the same sequences measured at 2.5 × 10² M⁻¹ s⁻¹. The results from these experiments indicate that (i) solid phase hybridization occurs three orders of magnitude slower than solution phase, (ii) trends observed in structure-dependent kinetics of solution phase hybridization may not be applicable to solid phase hybridization and (iii) model probes with known secondary structure decrease reaction rates; however, even random sequences with no known internal single-stranded structure can yield a broad range of reaction rates.     

A polymerase chain reaction-based ribosomal DNA detection technique using a surface plasmon resonance detector for a red tide causing microalga, Alexandrium affine

A detection technique with a DNA probe was developed for the bloom‐forming alga Alexandrium affine harvested in Japan. The design of this probe was based on the sequence polymorphism within the 28S ribosomal DNA (rDNA) of this strain using the BIAcore? 2000 biosensor, which determines surface plasmon resonance. The specific DNA sequence in 28S rDNA for A. affine was determined by sequence data analysis, and a probe was designed for the detection of A. affine. A fragment of the 28S rDNA from A. affine was amplified by polymerase chain reaction and applied to the BIAcore? sensor system, and the target DNA was selectively recognized by species‐specific hybridization using two DNA probes: a fluorescein isothiocyanate (FITC)‐labeled probe and a biotin‐labeled DNA probe. Using FITC‐labeled anti‐immunogloblin G antibody, enhancement of the response for the target DNA can be detected directly as a resonant unit change. In this detection method, a difference within only 20 base pairs of the target could be detected, and specific detection of A. affine was achieved intraspecifically.     

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.     

Hybridization kinetics and thermodynamics of molecular beacons

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem–loop structure of molecular beacons provides a competing reaction for probe–target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure–function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon–target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.