Thermodynamic Behavior of Short Oligonucleotides in Microarray Hybridizations Can Be Described Using Gibbs Free Energy in a Nearest-Neighbor Model

Abstract

While designing oligonucleotide-based microarrays, cross-hybridization between surface-bound oligos and non-intended labeled targets is probably the most difficult parameter to predict. Although literature describes rules-of-thumb concerning oligo length, overall similarity, and continuous stretches, the final behavior is difficult to predict. The aim of this study was to investigate the effect of well-defined mismatches on hybridization specificity using CodeLink Activated Slides, and to study quantitatively the relation between hybridization intensity and Gibbs free energy (∆G), taking the mismatches into account. Our data clearly showed a correlation between the hybridization intensity and ∆G of the oligos over three orders of magnitude for the hybridization intensity, which could be described by the Langmuir model. As ∆G was calculated according to the nearest-neighbor model, using values related to DNA hybridizations in solution, this study clearly shows that target-probe hybridizations on microarrays with a three-dimensional coating are in quantitative agreement with the corresponding reaction in solution. These results can be interesting for some practical applications. The correlation between intensity and ∆G can be used in quality control of microarray hybridizations by designing probes and corresponding RNA spikes with a range of ∆G values. Furthermore, this correlation might be of use to fine-tune oligonucleotide design algorithms in a way to improve the prediction of the influence of mismatching targets on microarray hybridizations. KEYWORDSMicroarray oligonucleotide design, nearest-neighbor model, Langmuir model, microarray quality control 3

Nonequilibrium Effects in DNA Microarrays: A Multiplatform Study

“…[2][3][4][5][6][7][8][9][10][11][12] The tacit assumption behind the use of these models is that hybridization in typical microarray experiments is sufficiently long (usually 15 − 17h), so that thermal equilibrium can be considered to be attained.…”

“…There, the logarithm of the fraction of hybridized probes varies linearly with ∆G with a slope equal to γ/RT , where the parameter γ is defined in Eq. (9). We note that a regime characterized by a slope smaller than expected from the equilibrium isotherm, is equivalent to introducing an effective temperature T eff = T exp /γ higher than the experimental one.…”

“…Last, we present another set of experiments on custom Codelink activated slides by Weckx et al 9 In these experiments the target solution contained 8 different sequences. Note that only four were with a fluorophore.…”

Development and Validation of a Species-Independent Functional Gene Microarray That Targets Lactic Acid Bacteria

“…An algorithm able to design 30-mer oligonucleotides with limited and controlled cross-hybridization capabilities for species for a given gene was implemented by applying parameters for the amount and position of mismatches in hybridization processes (19,21,22,30,52,55,61,62). Based on data obtained from a comparative analysis of all coding sequences for LAB strains that were present in the public domain (71 LAB species and 4 non-LAB species), a set of 4,851 oligonucleotides was designed, and 2,269 30-mer oligonucleotides for 46 LAB species and 4 non-LAB species were selected for synthesis.…”

“…The validation hybridizations also provided insight into the specificity of the microarray, although it remains difficult to fully understand this specificity in terms of an ecosystem approach. The fact that 21.0% of the species-specific oligonucleotides tested had a hybridization intensity below the background intensity could be explained not only by experimental performance, such as random labeling and differences between hybridization conditions and thermodynamic parameters for the oligonucleotides (55), but also by sequence variations between the strains tested and the public sequences that were used for oligonucleotide design, which is well illustrated by the four L. plantarum strains that were hybridized. When the cross-hybridizing oligonucleotides were focused on, only 41.9% of the perfectly matching cross-hybridizing oligonucleotides resulted in a signal whose intensity was above the background level when DNA was hybridized.…”