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Probing hybridization parameters from microarray experiments: nearest-neighbor model and beyond.

ABSTRACT: In this article, it is shown how optimized and dedicated microarray experiments can be used to study the thermodynamics of DNA hybridization for a large number of different conformations in a highly parallel fashion. In particular, free energy penalties for mismatches are obtained in two independent ways and are shown to be correlated with values from melting experiments in solution reported in the literature. The additivity principle, which is at the basis of the nearest-neighbor model, and according to which the penalty for two isolated mismatches is equal to the sum of the independent penalties, is thoroughly tested. Additivity is shown to break down for a mismatch distance below 5 nt. The behavior of mismatches in the vicinity of the helix edges, and the behavior of tandem mismatches are also investigated. Finally, some thermodynamic outlying sequences are observed and highlighted. These sequences contain combinations of GA mismatches. The analysis of the microarray data reported in this article provides new insights on the DNA hybridization parameters and can help to increase the accuracy of hybridization-based technologies.

SUBMITTER: Hadiwikarta WW

PROVIDER: S-EPMC3467032 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Probing hybridization parameters from microarray experiments: nearest-neighbor model and beyond.

Hadiwikarta W W WW Walter J-C JC Hooyberghs J J Carlon E E

Nucleic acids research 20120531 18

In this article, it is shown how optimized and dedicated microarray experiments can be used to study the thermodynamics of DNA hybridization for a large number of different conformations in a highly parallel fashion. In particular, free energy penalties for mismatches are obtained in two independent ways and are shown to be correlated with values from melting experiments in solution reported in the literature. The additivity principle, which is at the basis of the nearest-neighbor model, and acc ...[more]

PMID: 22661582

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Project description:Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest-neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching complementary sequence and other spots with one or two mismatches: in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays. Keywords: experiment to assess technical aspects of hybridization in microarrays The study consists of four experiments. Each experiments consists of one slide with eight identical microarrays which were hybridized with an identical solution but at a different concentration. Per experiment a single target sequence in solution was used for hybridization: target1 to target4 (Table 1 main manuscript or description of samples below). Each target oligo was bought in duplicate, referred to as oligos a and b. In experiment 1 and 2 a fragmentation step (f) was applied. More details can be found in the main manuscript especially Table 3.

Project description:BackgroundThe enumeration of tetrameric and other sequence motifs that are positively or negatively correlated with in vivo antisense DNA effects has been a useful addition to the arsenal of information needed to predict effective targets for antisense DNA control of gene expression. Such retrospective information derived from in vivo cellular experiments characterizes aspects of the sequence dependence of antisense inhibition that are not predicted by nearest-neighbor (NN) thermodynamic parameters derived from in vitro experiments. However, quantitation of the antisense contributions of motifs is problematic, since individual motifs are not isolated from the effects of neighboring nucleotides, and motifs may be overlapping. These problems are circumvented by a next-nearest-neighbor (NNN) analysis of antisense DNA effects in which the overlapping nature of nearest-neighbors is taken into account.ResultsNext-nearest-neighbor triplet combinations of nucleotides are the simplest that include overlapping sequence effects and therefore can encompass interactions beyond those of nearest neighbors. We used singular value decomposition (SVD) to fit experimental data from our laboratory in which phosphorothioate-modified antisense DNAs (S-DNAs) 20 nucleotides long were used to inhibit cellular protein expression in 112 experiments involving four gene targets and two cell lines. Data were fitted using a NNN model, neglecting end effects, to derive NNN inhibition parameters that could be combined to give parameters for a set of 49 sequences that represents the inhibitory effects of all possible overlapping triplet interactions in the cellular targets of these antisense S-DNAs. We also show that parameters to describe subsets of the data, such as the mRNAs being targeted and the cell lines used, can be included in such a derivation. While NNN triplet parameters provided an adequate model to fit our data, NN doublet parameters did not.ConclusionsThe methodology presented illustrates how NNN antisense inhibitory information can be derived from in vivo cellular experiments. Subsequent calculations of the antisense inhibitory parameters for any mRNA target sequence automatically take into account the effects of all possible overlapping combinations of nearest-neighbors in the sequence. This procedure is more robust than the tallying of tetrameric motifs that have positive or negative antisense effects. The specific parameters derived in this work are limited in their applicability by the relatively small database of experiments that was used in their derivation.

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Probing hybridization parameters from microarray experiments: nearest-neighbor model and beyond.

Publications

Probing hybridization parameters from microarray experiments: nearest-neighbor model and beyond.

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