Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted

Abstract

Hybridization of rRNAs to microarrays is a promising approach for prokaryotic and eukaryotic species identification. Typically, the amount of bound target is measured by fluorescent intensity and it is assumed that the signal intensity is directly related to the target concentration. Using thirteen different eukaryotic LSU rRNA target sequences and 7693 short perfect match oligonucleotide probes, we have assessed current approaches for predicting signal intensities by comparing Gibbs free energy (ΔG°) calculations to experimental results. Our evaluation revealed a poor statistical relationship between predicted and actual intensities. Although signal intensities for a given target varied up to 70-fold, none of the predictors were able to fully explain this variation. Also, no combination of different free energy terms, as assessed by principal component and neural network analyses, provided a reliable predictor of hybridization efficiency. We also examined the effects of single-base pair mismatch (MM) (all possible types and positions) on signal intensities of duplexes. We found that the MM effects differ from those that were predicted from solution-based hybridizations. These results recommend against the application of probe design software tools that use thermodynamic parameters to assess probe quality for species identification. Our results imply that the thermodynamic properties of oligonucleotide hybridization are by far not yet understood.

Absolute quantification of microRNAs by using a universal reference

“…Again, in contrast to Goff et al (2005), we did not find a uniform hybridization for oligo variants that had been adjusted with respect to their melting temperature but rather a severe loss in sensitivity, and most notably, a variable influence of individual miRNA sequences. Therefore, as also described by Pozhitkov et al (2006) and Dimitrov and Zuker (2004), we doubt that there is, at least in complex assays, a simple consistency between Gibbs free energy and target sequence.…”

Introduction to Microarray‐Based Detection Methods

“…In silico approaches allow for partial prediction of the hybridization behavior of designed probes. However, it has become clear that the simple notion that short oligonucleotides with a mismatch (MM) should hybridize less efficiently than perfect-match (PM) probes is not always applicable [137]. It has been demonstrated that the hybridization intensity of MM probes can depend on the nucleotide type (i.e.…”

“…More generally, there is a lack of a simple relationship between hybridizations of probe-target duplexes as inferred from signal intensity values and in silico predictions based on Gibbs free energies [137]. This does not apply as strictly for highdensity microarrays where the high level of redundancy accounts for the specificity of the signals nor for long oligonucleotide microarrays where inter-allele distinction is not required.…”

“…The intensity of this signal provides a measure of the amount of bound nucleic acid from a sample [137]. Schematically, we have divided this section into spotted and in situ synthesized arrays.…”

Influence of Dangling Ends and Surface-Proximal Tails of Targets on Probe-Target Duplex Formation in 16S rRNA Gene-Based Diagnostic Arrays

“…Low reproducibility and false-positive and false-negative signals with 16S rRNA gene-based arrays are partly accredited to bias caused by the characteristics of target molecules (8,17), and target-background interaction may be one of the main reasons for these phenomena. Poor statistical relationships between experimental and predicted signal intensities (from Gibbs free energy calculations) suggest that thermodynamic parameters between target-and-probe duplexes are not fully understood (26). Thus, an evaluation of the influence of dangling ends and surface-proximal tails on signal intensity and target-background interaction is needed in order to interpret the signal intensities from 16S rRNA gene-based diagnostic and microbial community analysis arrays.…”