Enhanced recognition of non-complementary hybridization by single-LNA-modified oligonucleotide probes

Locked nucleic acid (LNA) is a deoxyribonucleotide analogue with an unusual ‘locked’ furanose conformation. LNA-modified oligonucleotide probes have demonstrated an enhanced binding affinity towards their complementary strands; however, their potential to discriminate non-complementary hybridization of mismatches has not been explored. In this study, we investigated the effect of the chemical nature of LNA nucleobases on the hybridization stability and the capability of LNA-modified oligonucleotides to discriminate the LNA:DNA mismatched base pairs. It was observed that LNA modification indeed improves the discrimination capability of oligonucleotides by increasing the melting temperature differences between the complementary duplexes and hybrids containing mismatches. Particularly, LNA purines offer a greater potential to recognize the mismatches than LNA pyrimidines and DNA purines. Real-time PCR experiments further confirmed that LNA modifications at the 3′-end are more effective. The results and conclusions in this study provide useful information for hybridization-based nucleic acid analysis where designing sound oligonucleotide probes is crucial to the success of the analyses.   Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ChipCheck--a program predicting total hybridization equilibria for DNA binding to small oligonucleotide microarrays

Presented here is the program ChipCheck that allows the computation of total hybridization equilibria for hybridization experiments involving small oligonucleotide arrays. The calculation requires the free energies of binding for all pairs of probes and targets as well as total strand concentrations and probe molecule numbers. ChipCheck has been tested computationally on microarrays with up to 100 spots and 42 target strands (4200 binding equilibria). It arrives at solutions through iterations employing the multidimensional Newton method. While currently running in simulation mode only, an extension of the approach to the exhaustive analysis of chip results is being outlined and may be implemented in the future. The output displays the extent of correct and cross hybridization both graphically and numerically. In principle, calculating total hybridization equilibria allows for eliminating noise from DNA chip results and thus an improvement in sensitivity and accuracy.     

Imaging TOF-SIMS analysis of oligonucleotide microarrays

Single strand thiolated oligonucleotide (25-mer) was printed onto chemically modified glass and silicon surfaces. Confirmation of the level of attachment attained in each case was effected through detection by conventional confocal fluorescence microscopy. Both positive-ion and negative ion imaging time-of-flight mass spectra were recorded for the visualization of micro-patterned oligonucleotide arrays. This represents the first report of such detection by this form of mass spectrometry on glass. Ultimately, we are interested in the possibility that imaging time-of-flight secondary ion mass spectrometry can discern the orientation and conformation of DNA strands present on the surface of a substrate.     

Heterotypic cell pair co-culturing on patterned microarrays

We present a pair-wise co-culturing technique that creates large numbers of heterotypic cell pairs in patterned arrays. Lithographic patterning produces arrays with thousands of traps, each designed to accommodate only two cells and confine them at these sites for co-culturing. Two variants are introduced: a random seeding method that sediments a mixture of two cell types onto the array, and an approach that incorporates ferromagnetic thin films into the arrays and attracts cells that have been attached to ferromagnetic nanowires to the array sites through dipole interactions. The array technique includes the utilization of custom image analysis software that extracts data from multi-channel fluorescence images and records information about the cells in every trap, enabling the acquisition of accurate, high-statistics data. The applicability of the technique was demonstrated in experiments examining proliferation rates in pairs of bovine pulmonary artery endothelial and smooth muscle cells. Results demonstrated that heterotypic interactions favored smooth muscle cell proliferation while disfavoring endothelial cell proliferation. This is one example of a variety of cell-cell interactions that could be probed with this method.     

A fluorescent molecular switch for room temperature operation based on oligonucleotide hybridization without labeling of probes or targets

A molecular switch was prepared by self-assembly. Neutravidin served as a template that allowed for a biotinylated probe oligonucleotide to be placed adjacent to a biotinylated long-chain linker that was terminated with thiazole orange (TO). Hybridization of probe oligonucleotide with target to form double-stranded DNA resulted in intercalation of the adjacent TO probe. This was a reversible process that could be tracked by fluorescence intensity changes. Formamide was used as a denaturant for double-stranded DNA, and could be used to depress thermal denaturation temperatures. In this work formamide had a dual function, providing for control of hybridization selectivity at room temperature, while concurrently ameliorating non-specific adsorption to improve signal-to-noise when using thiazole orange as a fluorescence signalling agent to determine oligonucleotide hybridization. Room temperature single nucleotide polymorphism (SNP) discrimination for oligonucleotide targets was achieved both in solution and for molecular switches that were immobilized onto optical fibers. In solution, a concentration of 18.5% formamide provided greater than 40-fold signal difference between single-stranded DNA and double-stranded DNA, in contrast to only a 2-fold difference in the absence of formamide. Selectivity for SNP determination in solution was demonstrated using targets of varying lengths including a 141-base PCR amplicon. The improved signal-to-noise achieved by use of formamide is likely due to preferential displacement of dye molecules that are otherwise electrostatically bound to the polyanionic nucleic acid backbone.     

Hybridization of mismatched or partially matched DNA at surfaces

We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.     

Investigation of matched and mismatched duplex DNA by electrospray ionization-mass spectrometry

In this research, the gas-phase stabilities of matched and mismatched duplex DNA were investigated by electrospray ionization-mass spectrometry (ESI-MS). The wild-type p53 duplex DNA [ds1, perfectly-matched (PM) DNA] was successfully distinguished from its three mutated DNAs [double-base mismatched DNA (DM)]. Moreover, the three DM DNAs were also well discriminated from each other using ESI-MS. Results show that the gas-phase thermodynamic stability of the DM DNAs decreased as the two mismatch spots moved closer. This implies that the dissociation of DM duplexes into two single strands prefers the mode "from middle to terminals".     

Alteration of the selectivity of hybridization of immobilized oligonucleotide probes by co-immobilization with charged oligomers of ethylene glycol

A significant challenge exists in the creation of an environment for immobilized probe oligonucleotides that offer good structural regularity and reproducibility, where nearest neighbour interactions provide for control of selectivity, yet where the degree of hybridization does not alter nearest neighbour interactions. This new work explores whether a “matrix isolation” method will produce the desired environment for the probe molecules. The DNA oligonucleotide probes are polyelectrolytes with charged backbones and significant flexibility. It is possible to isolate the probe molecules by surrounding each, on average, with a sheath of immobilized oligomer that is not based on complementary nucleic acid, yet that is a polyelectrolyte in order to control the surface density and charge within the mixed film. Preliminary work investigates a mixture of dT₂₀ as the probe oligonucleotide, and a 20-mer oligomer primarily containing ethylene glycol phosphate, as a matrix isolation material in a 1:20 mole ratio, respectively. Melt temperature (T_m) measurements indicate that the thermodynamic stability of the probe molecules can be adjusted using the oligomer matrix to achieve lower T_m values by up to 5 °C, with full retention of selectivity for discrimination of single base pair mismatches even under conditions where the probes at a surface are saturated with complementary target.     

Optimization of DNA hybridization analysis on microarrays with colorimetric detection

A method of hybridization analysis on a DNA microarray using colorimetric detection on the basis of horseradish peroxidase has been developed. The effectiveness of the incorporation of biotin as a label in the DNA molecule in the PCR process is estimated and the conditions of hybridization of the biotin-labeled DNA with oligonucleotides immobilized on the surface of the array are optimized. The possibility of using the developed method is shown by the example of genotyping of CTX-M β-lactamases.     

Protein microarrays and quantum dot probes for early cancer detection

We describe here a novel approach for detection of cancer markers using quantum dot protein microarrays. Both relatively new technologies; quantum dots and protein microarrays, offer very unique features that together allow detection of cancer markers in biological specimens (serum, plasma, body fluids) at pg/ml concentration. Quantum dots offer remarkable photostability and brightness. They do not exhibit photobleaching common to organic fluorophores. Moreover, the high emission amplitude for QDs results in a marked improvement in the signal to noise ratio of the final image. Protein microarrays allow highly parallel quantitation of specific proteins in a rapid, low-cost and low sample volume format. Furthermore the multiplexed assay enables detection of many proteins at once in one sample, making it a powerful tool for biomarker analysis and early cancer diagnostics.

In a series of multiplexing experiments we investigated ability of the platform to detect six different cytokines in protein solution. We were able to detect TNF-, IL-8, IL-6, MIP-1β, IL-13 and IL-1β down to picomolar concentration, demonstrating high sensitivity of the investigated detection system.

We have also constructed and investigated two different models of quantum dot probes. One by conjugation of nanocrystals to antibody specific to the selected marker—IL-10, and the second by use of streptavidin coated quantum dots and biotinylated detector antibody. Comparison of those two models showed better performance of streptavidin QD–biotinylated detector antibody model. Data quantitated using custom designed computer program (CDAS) show that proposed methodology allows monitoring of changes in biomarker concentration in physiological range.     

Allosteric control of oligonucleotide hybridization by metal-induced cyclization

A bis(terpyridine) modified single-stranded DNA smoothly forms a stable cycle by Fe2+-assisted ring closure. A novel, complex type of allosteric behavior is observed with Zn2+, which reversibly off-regulates binding of a complementary oligonucleotide only in a narrow concentration range.     

Photochemically-activated probes of protein-protein interactions

The activity of light-activatable ("caged") compounds can be temporally and spatially controlled, thereby providing a means to interrogate intracellular biochemical pathways as a function of time and space. Nearly all caged peptides contain photocleavable groups positioned on the side chains of key residues. We describe an alternative active site targeted strategy that disrupts the interaction between the protein target (SH2 domain, kinase, and proteinase) and a critical amide NH moiety of the peptide probe.     

Chemoselective covalent coupling of oligonucleotide probes to self-assembled monolayers

A chemoselective route to routinely and rapidly attach oligonucleotide probes to well-defined surfaces is presented. Cu(I) tris(benzyltriazolylmethyl)amine-catalyzed coupling of terminal acetylenes to azides on a self-assembled monolayer is used instead of traditional nucleophilic-electrophilic coupling reactions. The reaction proceeds well even in the presence of purposely introduced nucleophilic and electrophilic impurities. The density of oligonucleotide probes can be controlled by controlling the amount of azide functionality. Although most of our work was done on gold surfaces, this technique should be readily applicable to any surface on which an azide-containing monolayer can be assembled as we have preliminarily demonstrated by derivatizing azidotrimethoxysilane-modified glass slides with fluorescein-containing oligonucleotides.     

LNA-modified oligodeoxynucleotide hybridization with DNA microarrays printed on nanoporous membrane slides

We report a robust method for the detection of hybridization events using a microarray-based assay on a nanoporous membrane platform. The technique is characterized by a hybridization time of only 1 hour and uses Cy5-labeled, 7-mer oligodeoxynucleotide probes modified with locked nucleic acid (LNA) nucleotides. We show that the volume of the DNA spotted onto a nanomembrane can be reduced to approximately 4 nL with detectable signal intensity. Moreover, the amount of the DNA target could be reduced to 4 fmol. The described approach could dramatically increase the throughput of techniques based on sequencing by hybridization, such as oligofingerprinting, by decreasing the total number of probes that are needed for analysis of large clone sets and reduction of the sample/reagent consumption. The method is particularly advantageous when numerous hybridization-based assays must be performed for characterization of sample sets of 100,000 or more.     

Drug-induced stabilisation of a mismatched C-T base pair in a DNA hairpin

We have shown that a key feature of drug binding, namely specific G-C base pair recognition at a 5'-TG step, can induce a number of novel structural features when an extrahelical base is inserted in close proximity to the drug binding site; we have clearly demonstrated the formation of a stabilised C-T mismatched base pair at a non-terminal site.     

Design and utility of oligonucleotide gene probes for fungal polyketide synthases

BACKGROUND: Recent advances in the molecular biology of polyketide biosynthesis have allowed the engineering of polyketide synthases and the biological ('combinatorial') synthesis of novel polyketides. Additional structural diversity in these compounds could be expected if more diverse polyketide synthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the structure and function of fungal PKS complexes. Relatively few fungal synthases have been investigated, perhaps because of a lack of suitable genetic techniques available for the isolation and manipulation of gene clusters from diverse hosts. We set out to devise a general method for the detection of specific PKS genes from fungi. RESULTS: We examined sequence data from known fungal and bacterial polyketide synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conservation. Using individual domains such as beta-ketoacylsynthases (KS), beta-ketoreductases (KR) and methyltransferases (MeT) we determined specific short (ca 7 amino acid) sequences showing high conservation for particular functional domains (e.g. fungal KR domains involved in producing partially reduced metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching these regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing species. Products obtained from these reactions were sequenced and shown to be fragments from as-yet undiscovered PKS gene clusters. The fragments could be used in blotting experiments with either homologous or heterologous fungal genomic DNA. CONCLUSIONS: A number of sequences are presented which have high utility for the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, partially reduced or highly reduced KS domains). We have also developed primers suitable for amplifying segments of fungal genes encoding polyketide C-methyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potential as aids for the eventual cloning of new fungal PKS gene clusters.     

Non-contact production of oligonucleotide microarrays using the highly integrated TopSpot nanoliter dispenser

For the first time we report on the production of oligonucleotide microarrays using a highly parallel and highly integrated, pressure driven TopSpot nanoliter dispenser. The system enables non-contact printing of different media like oligonucleotides, DNA or protein solutions. We optimized the printing buffer needed for oligonucleotides microarrays production with respect to two major aspects: microfluidical optimum for droplet dispensing and biochemical coupling efficiency on different commercially available microarray slides. Coefficient of variations (CVs) of generated spot diameters were measured to be smaller than 1% within one single dispensing nozzle and smaller than 1.5% within all 24 parallel nozzles of the printhead for all printing buffers used. No carry-over and no cross-talk was found, in extensive experiments with oligonucleotides. Optimized printing buffer compositions and concentrations for oligonucleotide microarrays were found, as well as optimized coupling protocols. Furthermore, buffers and protocols were adapted to a host of different microarray slides used. With this system, prime critical points of microarray production are solved, leading to high quality high throughput microarray fabrication.     

Fidelity discrimination in DNA polymerase beta: differing closing profiles for a mismatched (G:A) versus matched (G:C) base pair

Understanding fidelity-the faithful replication or repair of DNA by polymerases-requires tracking of the structural and energetic changes involved, including the elusive transient intermediates, for nucleotide incorporation at the template/primer DNA junction. We report, using path sampling simulations and a reaction network model, strikingly different transition states in DNA polymerase beta's conformational closing for correct dCTP versus incorrect dATP incoming nucleotide opposite a template G. The cascade of transition states leads to differing active-site assembly processes toward the "two-metal-ion catalysis" geometry. We demonstrate that these context-specific pathways imply different selection processes: while active-site assembly occurs more rapidly with the correct nucleotide and leads to primer extension, the enzyme remains open longer, has a more transient closed state, and forms product more slowly when an incorrect nucleotide is present. Our results also suggest that the rate-limiting step in pol beta's conformational closing is not identical to that for overall nucleotide insertion and that the rate-limiting step in the overall nucleotide incorporation process for matched as well as mismatched systems occurs after the closing conformational change.     

Multiplexed analyte and oligonucleotide detection on microarrays using several redox enzymes in conjunction with electrochemical detection

We show that multiple enzyme tags may be used in an immunoassay format or for the detection of sequence-specific DNA on microarrays. The assays may be multiplexed and monitored under separate solution and voltage differences. Thus, the detection method relies on an electrochemical detection format, whereby multiple enzymes can be sensed. In our case we utilize horseradish peroxidase, laccase, and glucose dehydrogenase as enzymes attached to specific antibodies or to streptavidin.