Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays.

We have investigated the use of spacer molecules to reduce steric interference of the support on the hybridisation behaviour of immobilised oligonucleotides. These spacers are built up from a variety of monomeric units, using phosphoramidite chemistry, by condensation onto an amine-functionalised polypropylene support. The optimal spacer length was determined to be at least 40 atoms in length, giving up to 150-fold increase in the yield of hybridisation. The effects of different charged groups in the spacer were also examined, and it was shown that both positively and negatively charged groups in the spacer diminish the yield of hybridisation. Steric hindrance in hybridisation can also be a problem if the oligonucleotides attached to the support are too close to each other. Surface coverage was varied using a combination of cleavable and stable linkers, giving the highest hybridisation yields for surfaces containing approximately 50% of the maximum concentration of oligonucleotides.     

Reactive oligonucleotide derivatives and sequence-specific modification of nucleic acids

Selective modification of nucleic acid base sequences can be achieved by complementary oligonucleotides carrying reactive groups. Different types of reactive groups are briefly presented.     

Disulfide-linked oligonucleotide phosphorothioates: Novel analogues of nucleic acids

Summary The synthesis of phosphorothioate analogues of oligonucleotides by the oxidation of deoxyadenosine 3,5-bisphosphorothioate (3) was attempted. Cyclization of3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5-terminal phosphorothioate group undergoes efficient chain-extension when oxidized in the presence of3.     

Real-time genotyping with oligonucleotide probes containing locked nucleic acids

Oligonucleotide probes containing locked nucleic acid (LNA) hybridize to complementary single-stranded target DNA sequences with an increased affinity compared to oligonucleotide DNA probes. As a consequence of the incorporation of LNA residues into the oligonucleotide sequence, the melting temperature of the oligonucleotide increases considerably, thus allowing the successful use of shorter LNA probes as allele-specific tools in genotyping assays. In this article, we report the use of probes containing LNA residues for the development of qualitative fluorescent multiplex assays for the detection of single nucleotide polymorphisms (SNPs) in real-time polymerase chain reaction using the 5'-nuclease detection assay. We developed two applications that show the improved specificity of LNA probes in assays for allelic discrimination. The first application is a four-color 5'-nuclease assay for the detection of SNPs for two of the most common genetic factors involved in thrombotic risk, factor V Leiden and prothrombin G20210A. The second application is a two-color assay for the specific detection of the A-to-T tranversion in codon 6 of the beta-globin gene, responsible for sickle cell anemia. Both real-time genotyping assays were evaluated by comparing the performance of our method to that of a reference method and in both cases, we found a 100% concordance. This approach will be useful for research and molecular diagnostic laboratories in situations in which the specificity provided by oligonucleotide DNA probes is insufficient to discriminate between two DNA sequences that differ by only one nucleotide.     

Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly

This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5' hexanethiol anchoring group with that of unfunctionalized oligonucleotides for lengths from 8 to 48 bases. Qualitatively, the results indicate that the thiol anchoring group strongly enhances oligonucleotide immobilization, but that the enhancement is reduced for longer strand lengths. Interestingly, examination of the probe coverage as a function of strand length suggests that adsorbed thiol-ssDNA oligonucleotides shorter than 24 bases tend to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. For strands longer than 24 bases, the surface coverage begins to decrease notably with probe length. The decrease is consistent with a less ordered arrangement of the DNA chains, presumably reflecting increasingly polymeric behavior.     

Evaluation of locked nucleic acids for signal enhancement of oligonucleotide probes for microalgae immobilised on solid surfaces

Biosensors and microarrays are powerful tools for species detection and monitoring of microorganisms. A reliable identification of microorganisms with probe-based methods requires highly specific and sensitive probes. The introduction of locked nucleic acid (LNA) promises an enhancement of specificity and sensitivity of molecular probes. In this study, we compared specificity and sensitivity of conventional probes and LNA modified probes in two different solid phase hybridisation methods: sandwich hybridisation on biosensors and on DNA microarrays. In combination with DNA-microarrays, the LNA probes displayed an enhancement of sensitivity, but also gave more false-positive signals. With the biosensor, the LNA probes showed neither signal enhancement nor discrimination of a single mismatch. In all cases, conventional DNA probes showed equal or better results than LNA probes. In conclusion, LNA technology may have great potential in methods that use probes in suspension and in gene expressions studies, but under certain solid surface-hybridisation applications, they do not improve signal intensity.     

Contrast normalization of oligonucleotide arrays.

Affymetrix high-density oligonucleotide array is a tool that has the capacity to simultaneously measure the abundance of thousands of mRNA sequences in biological samples. In order to allow direct array-to-array comparisons, normalization is a necessity. When deciding on an appropriate normalization procedure there are a couple questions that need to be addressed, e.g., on which level should the normalization be performed: On the level of feature intensities or on the level of expression indexes? Should all features/expression indexes be used or can we choose a subset of features likely to be unregulated? Another question is how to actually perform the normalization: normalize using the overall mean intensity or use a smooth normalization curve? Most of the currently used normalization methods are linear; e.g., the normalization method implemented in the Affymetrix software GeneChip is based on the overall mean intensity. However, along with alternative methods of summarizing feature intensities into an expression index, nonlinear methods have recently started to appear. For many of these alternative methods, the natural choice is to normalize on the level of feature intensities, either using all feature intensities or only perfect match intensities. In this report, a nonlinear normalization procedure aimed for normalizing feature intensities is proposed.     

Effective complementarily-addressed photomodification of nucleic acids by oligonucleotide derivatives, containing aromatic azido groups]

Highly effective site-specific photomodification of a DNA-target was carried out with oligonucleotide reagents carrying aromatic azido groups. Oligonucleotide derivatives with a photoactive function R on the 5'-terminal phosphate and at C-5 atom of deoxyuridine were synthesized: R1NH(CH2)3NHpd(TCCACTT) and d(ULNHRCCACTT), where R1 is p-azidotetrafluorobenzoyl, R2 is 2-nitro, 5-azidobenzoyl, R3 is p-azidobenzoyl; LNH = -CH2NH-, -CH2OCH2CH2NH- or -CH2NHCOCH2CH2NH-. The prepared compounds form stable complementary complexes and effect site-specific photomodification of the target DNA. The modification of pentadecanucleotide d(TAAGTGGAGTTTGGC) with the reagents was investigated. Maximum extent of modification strongly depended on the reagent's type, the photoreagent with R1 being the most effective. Whatever the binding site was, this agent provided a 65-70% modification in all cases except LNH = -CH2NH-, when the yield was twice lower. For the reagents bearing R1 the modification sites were identified. Selective modification at the G9 residue was detected in the case of LNH = -CH2OCH2CH2NH- and when a photoactive group was linked to the terminal phosphate.     

Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization.

We described a solid-supported desulfurization procedure allowing easy access to the sequence analysis of oligonucleotide phosphorothioates. The described method is based upon selective removal of the 2-cyanoethyl phosphate protecting groups, followed by iodine-promoted desulfurization of the resulting phosphorothioate diesters. Automatic oxidation of oligonucleotide phosphorothioates, anchored via an ester linkage to a standard solid support (LCAA/CPG), is combined with Maxam-Gilbert solid-support sequencing. The overall procedure allows rapid simultaneous sequence analysis of several oligonucleotide analogs.     

Photolithographic synthesis of high-density oligonucleotide probe arrays

High-density DNA probe arrays provide a massively parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research and diagnostics. Light-directed combinatorial oligonucleotide synthesis has enabled the large-scale production of GeneChip probe arrays which contain several hundred of thousand oligonucleotide sequences on glass "chips" about one cm2 in size. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms ("genotyping"), and in a wide range of gene expression studies. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques adapted from the microelectronics industry. The present-generation methodology employs MeNPOC photo-activatable nucleoside monomers with proximity photolithography, and is currently capable of printing individual 10 microns 2 probe features at a density of 10(6) probes/cm2.     

Activation by laser irradiation of a "switched on" oligonucleotide reagent for an addressed modification of nucleic acids

Properties of oligonucleotide reagents containing an alkylating group of regulated reactivity (nitrogen mustard residue activatable upon mild borohydride reduction of the aromatic formyl group) have been studied. It was shown that these reagents can also be activated by irradiation with nitrogen laser light (lambda 337 nm). Activation of the reagent in complex with a target polydeoxyribonucleotide resulted in the addressed chemical modification of the target. The positional direction of the modification depended on the way of the activation (borohydride reduction or laser irradiation).     

Effects of different fixatives on detection of nucleic acids from paraffin-embedded tissues by in situ hybridization using oligonucleotide probes

Detection of nucleic acids from paraffin-embedded material by in situ hybridization with oligonucleotide probes is increasingly being used. To determine the effect of fixation on the preservation of DNA and mRNA, we studied 18 lymphoid tissues fixed in B5, formalin, OmniFix, ethanol, and Bouin's fixatives and embedded in paraffin by in situ hybridization, using biotinylated oligonucleotide poly d(T) probes and immunoglobulin light chain probes. Detection of DNA using the poly d(T) probe was most consistent and most intense in tissue fixed in formalin, followed by OmniFix and ethanol, with B5 and Bouin's fixatives yielding unsatisfactory results. Detection of mRNA, using the light chain probes, was most consistent and most intense with tissue fixed in formalin and Bouin's solution, followed by B5 fixative, with OmniFix and ethanol fixatives yielding unsatisfactory results. The results of mRNA detection using the poly d(T) probe were found not to correlate with mRNA content as determined by the light chain probes for several fixatives, possibly owing to selective degradation of portions of the mRNA molecule.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

Predicting oligonucleotide affinity to nucleic acid targets

A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.     

Modeling nucleic acids

Nucleic acids are an important class of biological macromolecules that carry out a variety of cellular roles. For many functions, naturally occurring DNA and RNA molecules need to fold into precise three-dimensional structures. Due to their self-assembling characteristics, nucleic acids have also been widely studied in the field of nanotechnology, and a diverse range of intricate three-dimensional nanostructures have been designed and synthesized. Different physical terms such as base-pairing and stacking interactions, tertiary contacts, electrostatic interactions and entropy all affect nucleic acid folding and structure. Here we review general computational approaches developed to model nucleic acid systems. We focus on four key areas of nucleic acid modeling: molecular representation, potential energy function, degrees of freedom and sampling algorithm. Appropriate choices in each of these key areas in nucleic acid modeling can effectively combine to aid interpretation of experimental data and facilitate prediction of nucleic acid structure.     

Extracellular nucleic acids

Extracellular nucleic acids are found in different biological fluids in the organism and in the environment: DNA is a ubiquitous component of the organic matter pool in the soil and in all marine and freshwater habitats. Data from recent studies strongly suggest that extracellular DNA and RNA play important biological roles in microbial communities and in higher organisms. DNA is an important component of bacterial biofilms and is involved in horizontal gene transfer. In recent years, the circulating extracellular nucleic acids were shown to be associated with some diseases. Attempts are being made to develop noninvasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Recent observations demonstrated the possibility of nucleic acids exchange between eukaryotic cells and extracellular space suggesting their participation in so far unidentified biological processes.