Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips.

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.     

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T_m) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T_m. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.     

Massive parallel analysis of DNA-Hoechst 33258 binding specificity with a generic oligodeoxyribonucleotide microchip.

A generic oligodeoxyribonucleotide microchip was used to determine the sequence specificity of Hoechst 33258 binding to double-stranded DNA. The generic microchip contained 4096 oxctadeoxynucleo-tides in which all possible 4(6)= 4096 hexadeoxy-nucleotide sequences are flanked on both the 3'- and 5'-ends with equimolar mixtures of four bases. The microchip was manufactured by chemical immobilization of presynthesized 8mers within polyacrylamide gel pads. A selected set of immobilized 8mers was converted to double-stranded form by hybridization with a mixture of fluorescently labeled complementary 8mers. Massive parallel measurements of melting curves were carried out for the majority of 2080 6mer duplexes, in both the absence and presence of the Hoechst dye. The sequence-specific affinity for Hoechst 33258 was calculated as the increase in melting temperature caused by ligand binding. The dye exhibited specificity for A:T but not G:C base pairs. The affinity is low for two A:T base pairs, increases significantly for three, and reaches a plateau for four A:T base pairs. The relative ligand affinity for all trinucleotide and tetranucleotide sequences (A/T)(3)and (A/T)(4)was estimated. The free energy of dye binding to several duplexes was calculated from the equilibrium melting curves of the duplexes formed on the oligonucleotide microchips. This method can be used as a general approach for massive screening of the sequence specificity of DNA-binding compounds.     

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.     

DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA–8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10–15°C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA–8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.     

Comparison of Complex DNA Mixtures with Generic Oligonucleotide Microchips

Abstract

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to VD-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.     

Comparison of complex DNA mixtures with generic oligonucleotide microchips.

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to V-D-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.     

Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded oligonucleotides containing a true abasic site.

A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11 degrees C. Schiff base related fluorescence and beta-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial beta-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction.     

Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements

In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed.     

A method for DNA sequencing by hybridization with oligonucleotide matrix.

A new technique of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) which could also be applied for DNA mapping and fingerprinting, mutant diagnostics, etc., has been tested in model experiments. A dot matrix was prepared which contained 9 overlapping octanucleotides (8-mers) complementary to a common 17-mer. Each of the 8-mers was immobilized as individual dot in thin layer of polyacrylamide gel fixed on a glass plate. The matrix was hybridized with the 32P-labeled 17-mer and three other 17-mers differing from the first one by a single base change. The hybridization enabled us to distinguish perfect duplexes from those containing mismatches in 32 out of 35 cases. These results are discussed with respect to the applicability of the approach for sequencing. It was shown that hybridization of DNA with an immobilized 8-mer in the presence of a labeled 5-mer led to the formation of a stable duplex with the 5-mer only if the 5- and the 8-mers were in continuous stacking making a perfect nicked duplex 13 (5+8) base pairs long. These experiments and computer simulations suggest that continuous stacking hybridization may increase the efficiency of sequencing so that random or natural coding DNA fragments about 1000 bases long could be sequenced in more than 97% of cases. Miniaturized matrices or sequencing chips were designed, where oligonucleotides were immobilized within 100 x 100 micron dots disposed at 100 micron intervals. Hybridization of fluorescently labeled DNA fragments with microchips may simplify sequencing and ensure sensitivity of at least 10 attomoles per dot. The perspectives and limitations of SHOM are discussed.     

An RNA microchip containing immobilized oligoribonucleotides with protective groups at 2'-O-positions

To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.     

Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization.

Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 x 0.1 x 0.02 mm or 1 x 1 x 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of 'microtest tube' of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.     

Oligonucleotide analogues bearing an acyclonucleoside linked by an internucleotide amide bond

Oligonucleotide analogues bearing an acyclocytidine linked to thymidine with an amide (3′-O-CH₂-CO-N-5′) bond were synthesized. Melting curves of duplexes formed by modified oligonucleotides and complementary natural oligomers were obtained and thermodynamic parameters of their formation were measured. Replacement of dCpT by a modified dinucleotide only moderately decreased the melting temperature of these modified duplexes in comparison with unmodified duplexes containing complementary natural bases. CD spectra of modified duplexes were studied, and the duplex spatial structures are discussed.     

Biological microchips with hydrogel-immobilized nucleic acids,proteins, and other compounds: properties and applications in genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain the gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes proceeding on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, the protein microchips have been created, containing the immobilized antibodies, antigens, enzymes, and many other substances, as well as the microchips with the gel-immobilized live cells.     

Analysis of Perfect and Mismatched DNA Duplexes with a Generic Hexanucleotide Microchip

Thermodynamic analysis was performed for the duplexes formed by fluorescently labeled oligonucleotide targets on a generic hexanucleotide microchip. All 4096 different hexanucleotide chains were immobilized as probes in individual gel pads of the microchip. To strengthen the hybridization, each hexamer was extended at both ends by one nucleotide from the equimolar mixture of all four nucleotides to serve as nonselective linkers. It has been shown that the melting curves for oligonucleotide duplexes formed on the microchip and in a solution are quite similar. The influence of ionic surrounding has been studied in terms of the hybridization efficiency and discrimination between the mismatched and perfect duplexes. Different approaches have been tested to compensate the dependence of duplex stability on the GC content. It has been demonstrated that the use of chaotropic agents, addition of nonlabeled GC-rich competitor oligonucleotides, as well as creation of a temperature gradient along the microchip reproducing the distribution of melting temperatures, efficiently level out the AT/GC differences. The use of tetramethylammonium chloride for the same purpose was accompanied by weakening to some extent the discrimination between the mismatched duplexes and the perfect ones.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480–530 nm and fluorescence maxima at 500–550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled oligonucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

Analysis of perfect and mismatched DNA duplexes by a generic hexanucleotide microchip

The thermodynamic analysis was done for the duplexes formed by fluorescently labeled oligonucleotide targets on a genetic hexanucleotide microchip. All 4096 different hexanucleotide chains were immobilized as probes in individual gel pads of the microchip. To strengthen the hybridization, each hexamer was extended at both ends by one nucleotide from the equimolar mixture of all four nucleotides to serve as nonselective linkers. It has been shown that the melting curves for oligonucleotide duplexes formed on the microchip and in a solution are quite similar. The influence of ionic surrounding has been studied in terms of the hybridization efficiency and discrimination between the mismatched and perfect duplexes. Different approaches have been tested to compensate the dependence of duplex stability on the GC content. It has been demonstrated that the use of chaotropic agents, addition of nonlabeled GC-rich competitor oligonucleotides, as well as creation of a temperature gradient along the microchip reproducing the distribution of melting temperatures, efficiently level out the AT/GC differences. The use of tetramethylammonium chloride for the same purpose was accompanied by weakening to some extent the discrimination between the mismatched duplexes and the perfect ones.     

Oligonucleotide analogues bearing an acyclonucleoside linked by an internucleotide amide bond

Oligonucleotide analogues bearing an acyclocytidine linked to thymidine by an amide (3'-O-CH2-CO-N-5') bond were synthesized. Melting curves of duplexes formed by modified oligonucleotides and complementary natural oligomers were obtained and thermodynamic parameters of their formation were measured. Replacement of dCpT by a modified dinucleotide only moderately decreased the melting temperature of these modified duplexes in comparison with unmodified duplexes containing complementary natural bases. CD spectra of modified duplexes were studied, and the duplex spatial structures are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Biological Microchips with Hydrogel-Immobilized Nucleic Acids,Proteins, and Other Compounds: Properties and Applications in Genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single-nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, protein microchips have been created, containing immobilized antibodies, antigens, enzymes, and many other substances, as well as microchips with gel-immobilized live cells.