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.     

Stabilization of duplex DNA and RNA by dangling ends studied by free energy simulations

Single unpaired nucleotides at the end of double‐stranded nucleic acids, termed dangling ends, can contribute to duplex stability. Umbrella sampling free energy simulations of dangling cytosine and guanine nucleotides at the end of duplex and single stranded RNA and DNA molecules have been used to investigate the molecular origin of dangling end effects. In unrestraint simulations, the dangling end nucleotides stayed close to placements observed in experimental structures. Calculated free energy contributions associated with the presence of dangling nucleotides were in reasonable agreement with experiment predicting the general trend of a more stabilizing effect of purine vs. pyrimidine dangling ends. In addition, the calculations indicate a more significant stabilizing effect of dangling ends at the 5′‐end vs. 3′‐end in case of DNA and the opposite trend in case of RNA. Both electrostatic and van der Waals interactions contribute to the duplex stabilizing effect of dangling end nucleotides. The free energy simulation scheme could also be used to design dangling end nucleotides that result in enhanced duplex stabilization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 418–427, 2014.     

Influence of dangling ends and surface-proximal tails of targets on probe-target duplex formation in 16S rRNA gene-based diagnostic arrays

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.     

Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability

Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8kcal/mol and increased T(m) up to 4.3 degrees C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ approximately Black Hole 2>Cy5 approximately Cy3>Black Hole 1>QSY7 approximately Iowa Black FQ>Texas Red approximately TAMRA>FAM approximately HEX approximately Dabcyl>TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.     

Determining the influence of structure on hybridization using oligonucleotide arrays.

We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies.     

Parallel analysis of oligodeoxyribonucleotide (oligonucleotide) interactions. I. Analysis of factors influencing oligonucleotide duplex formation.

A novel method for the analysis of oligonucleotide-oligonucleotide interactions is described. Oligonucleotides of different sequence are synthesised in situ as stripes on the surface of a glass slide (see accompanying paper). Multiple hybridizations are then carried out on each oligonucleotide simultaneously to determine the dependence of oligonucleotide duplex formation on duplex length, base composition, hybridisation solvent and sequence complexity.     

Effect of dangling ends on the stability of a DNA double helix.

The effect of 3' and 5' dangling ends has been studied on the stability of a self-complementary double-helix of d(ATGCGCAT) in 1 mol dm-3 NaCl-phosphate buffer. It was shown that the effect on the DNA was smaller than that on r(AUGCAU).     

Studies on the base pairing properties of deoxyinosine by solid phase hybridisation to oligonucleotides.

Extensive analyses of the base-pairing properties of deoxyinosine to A, C, G, and T were carried out by measuring the hybridisation of oligonucleotides with deoxyinosine in various positions to complementary sets of oligonucleotides made as an array on the surface of a glass microscope slide. With deoxyinosine in internal positions, results are consistent with previous studies, showing a preferential order for pairing of I-C > I-A > I-G approximately I-T. With two adjacent deoxyinosines in the centre of the oligonucleotide, the order in duplex yield is CC > CA > AA > AC > GC > GA > CG > TA > TC > CT = AG > AT > GT > TT. With deoxyinosine at the ends of the oligonucleotide, we find that at the 3' end there is the same order in duplex yield as for the deoxyinosine in internal positions, though with lower discrimination between the bases. When hybridisation is carried out in TMACI there is little base pairing discrimination with deoxyinosine, or indeed any of the four natural bases at the 5' end. Changing the cation to Na+ increased discrimination slightly.     

Oligonucleotide lipoplexes: the influence of oligonucleotide composition on complexation.

Despite extensive investigations into oligonucleotide lipoplexes, virtually no work has addressed whether the physicochemical properties of these assemblies vary as a function of the constituent oligonucleotide (ODN) sequence and/or composition. The present study was aimed at answering this question. To this end, we complexed N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) liposomes, in dispersion, with either 18-mer phosphorothiote homo-oligonucleotides composed of either adenine, thymidine or cytosine; or one of three structurally related 18-mer phosphorothioate oligonucleotides (S-ODNs) (G3139, its reverse sequence and its two-base mismatch). After ODN addition to vesicles at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin while particle size distributions were analyzed by static-light scattering. The results indicate that each homo-oligonucleotide does indeed exhibit different complexation behavior. In particular, the maximal level of DOTAP neutralization by the polyadenine S-ODN is much lower than that for the two other homo-oligonucleotides and hence its lipoplex is much more positively charged. Much smaller electrostatic differences are also apparent between lipoplexes formed from each of the G3139-related ODNs. This paper identifies nucleotide base selection and sequence as a variable that can affect the physicochemical properties of oligonucleotide lipoplexes and hence probably their transfection competency.     

Stability of 3' dangling ends on the core helix of AUGCAU at various Na+ concentrations.

Stability increments of 3' dangling ends on the core helices AUGCAU at various Na+ concentrations are reported. The results show that all 3' dangling ends except 3'U dangling at low Na+ concentrations can stabilize the helix and this stabilization is very sequence dependent.     

Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.

A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.     

Studies of DNA dumbbells VII: evaluation of the next-nearest-neighbor sequence-dependent interactions in duplex DNA

Melting experiments were conducted on 22 DNA dumbbells as a function of solvent ionic strength from 25-115 mM Na(+). The dumbbell molecules have short duplex regions comprised of 16-20 base pairs linked on both ends by T(4) single-strand loops. Only the 4-8 central base pairs of the dumbbell stems differ for different molecules, and the six base pairs on both sides of the central sequence and adjoining loops on both ends are the same in every molecule. Results of melting analysis on the 22 new DNA dumbbells are combined with our previous results on 17 other DNA dumbbells, with stem lengths containing from 14-18 base pairs, reported in the first article of this series (Doktycz, Goldstein, Paner, Gallo, and Benight, Biopoly 32, 1992, 849-864). The combination of results comprises a database of optical melting parameters for 39 DNA dumbbells in ionic strengths from 25-115 mM Na(+). This database is employed to evaluate the thermodynamics of singlet, doublet, and triplet sequence-dependent interactions in duplex DNA. Analysis of the 25 mM Na(+) data reveals the existence of significant sequence-dependent triplet or next-nearest-neighbor interactions. The enthalpy of these interactions is evaluated for all possible triplets. Some of the triplet enthalpy values are less than the uncertainty in their evaluation, indicating no measurable interaction for that particular sequence. This finding suggests that the thermodynamic stability of duplex DNA depends on solvent ionic strength in a sequence-dependent manner. As a part of the analysis, the nearest-neighbor (base pair doublet) interactions in 55, 85, and 115 mM Na(+) are also reevaluated from the larger database.     

DNA-surfactant interactions: coupled cooperativity in ligand binding leads to duplex stabilization.

The cooperative nature of interaction of cationic surfactants with short oligonucleotides leading to eventual stabilization of DNA duplexes is demonstrated. At submicellar concentrations and DNA:surfactant charge ratios of 0.2 to 0.8, the association of single chain (CTAB) and double chain (DOTAP) surfactants to oligonucleotides is initiated by electrostatic interaction of cationic ligands with polyanionic DNA that aligns the surfactant molecules on the DNA template. This is followed by binding of new surfactant ligands to the initial complex, driven cooperatively by the hydrophobic forces, leading to in situ formation of surfactant-bound and bare duplexes as separate species. These exhibit independent melting behaviour characterised by double transition in thermal UV profiles, with a higher T(m) for surfactant-DNA complexes. Understanding the cooperative binding of the cationic surfactants to the DNA described here may have implications for rational design of DNA binding drugs and DNA delivery systems.     

Oligonucleotide lipoplexes: the influence of oligonucleotide composition on complexation

Despite extensive investigations into oligonucleotide lipoplexes, virtually no work has addressed whether the physicochemical properties of these assemblies vary as a function of the constituent oligonucleotide (ODN) sequence and/or composition. The present study was aimed at answering this question. To this end, we complexed N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) liposomes, in dispersion, with either 18-mer phosphorothiote homo-oligonucleotides composed of either adenine, thymidine or cytosine; or one of three structurally related 18-mer phosphorothioate oligonucleotides (S-ODNs) (G3139, its reverse sequence and its two-base mismatch). After ODN addition to vesicles at different mole ratios, changes in pH and electrical surface potential at the lipid–water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin while particle size distributions were analyzed by static-light scattering. The results indicate that each homo-oligonucleotide does indeed exhibit different complexation behavior. In particular, the maximal level of DOTAP neutralization by the polyadenine S-ODN is much lower than that for the two other homo-oligonucleotides and hence its lipoplex is much more positively charged. Much smaller electrostatic differences are also apparent between lipoplexes formed from each of the G3139-related ODNs. This paper identifies nucleotide base selection and sequence as a variable that can affect the physicochemical properties of oligonucleotide lipoplexes and hence probably their transfection competency.     

Normalization of oligonucleotide arrays based on the least-variant set of genes

Background  

It is well known that the normalization step of microarray data makes a difference in the downstream analysis. All normalization methods rely on certain assumptions, so differences in results can be traced to different sensitivities to violation of the assumptions. Illustrating the lack of robustness, in a striking spike-in experiment all existing normalization methods fail because of an imbalance between up- and down-regulated genes. This means it is still important to develop a normalization method that is robust against violation of the standard assumptions     

Kinetics of oligonucleotide hybridization to DNA probe arrays on high-capacity porous silica substrates

We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of approximately 23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-microm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-microm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22 degrees C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (k(a), k(d), and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22 degrees C is described well by the predictive equations for morphology, whereas the behavior at 45 degrees C deviates from expectations and suggests that more complex phenomena may be occurring in that temperature regime.     

Evolution by hybridisation. The influence of reticulate evolution on biosymmetrical patterns and processes in plants

Natural hybridisation in plants is one of the most striking modes of evolution, when looking at the effects reticulation may have on patterns and processes of the evolutionary history of the affected system. Among other functional constraints, symmetry is regarded as a major factor of or for evolution. A loss of symmetry, that means origin of asymmetrical phenomena can in some cases be linked to environmental or genetic stresses, such as hybridisation. It has been shown that the discussion on reticulation and symmetrical evolution is strongly influenced by a zoocentric evolutionary view, but more focus is needed on the differences between the organisms subject to the study. Plant hybridogenic evolution with the subsequent possibility of polyploidisation and the consequent reduced homology is a completely different phenomenon compared to animal evolution. This article examines the role of symmetry for plant evolution by concentration on two topics: (a) spatial asymmetry on multiple levels caused by hybridisation, and (b) temporal asymmetry (changes of rhythms inherent to specific systems) as an effect of reticulation.     

Nucleic acid duplex stability: influence of base composition on cation effects.

The effects of counter ion on a nucleic acid duplex stability were investigated. Since a linear free energy relationship for the thermostability of oligonucleotide duplexes between those in 1 M and in 100 mM NaCl-phosphate buffer were observed regardless of whether they are DNA-DNA, RNA-RNA or RNA-DNA duplexes, simple prediction systems for [Delta] G degrees 37as well as T mvalues in 100 mM NaCl-phosphate buffer were established. These predictions were successful with an average error of only 2.4 degrees C for T mand 5. 7% for G degrees 37values. The number of Na+newly bound to a duplex when the duplex forms (-[Delta] n) was significantly influenced by the base composition, and -[Delta] n for d(GCCAGTTAA)/d(TTAACTGGC) was different for MgCl2, CaCl2, BaCl2and MnCl2(from 0.70 to 0.76 with the same order of the duplex stability). Almost no additive effects on the duplex stability was observed for NaCl and MgCl2, suggesting a competitive binding for these cations. The sequence-dependent manner of [Delta] n suggests the presence of preferential base pairs or nearest-neighbor base pairs for the cation binding, which would affect nearest-neighbor parameters.