Secondary structure in the target as a confounding factor in synthetic oligomer microarray design

Abstract

BackgroundSecondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment.ResultsDespite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. For example, at 65°C, 28 ± 7% of the average cDNA target sequence is predicted to be inaccessible to hybridization. We also analyzed the specific binding sites of a set of 70mer probes previously designed for Brucella using a freely available oligo design software package. 21 ± 13% of the nucleotides in each probe binding site are within a double-stranded structure in over half of the folds predicted for the cDNA target at 65°C. The intramolecular structures formed are more stable and extensive when an RNA target is modeled rather than cDNA. When random shearing of the target is modeled for fragments of 200, 100 and 50 nt, an overall destabilization of secondary structure is predicted, but shearing does not eliminate secondary structure.ConclusionSecondary structure in the target is pervasive, and a significant fraction of the target is found in double stranded conformations even at high temperature. Stable structure in the target has the potential to interfere with hybridization and should be a factor in interpretation of microarray results, as well as an explicit criterion in array design. Inclusion of this property in an oligonucleotide design procedure would change the definition of an optimal oligonucleotide significantly.

Optimization and clinical validation of a pathogen detection microarray

“…The random primer used in our experiments was a 26-mer composed of a random nonamer (3') tagged with a fixed 17-mer sequence (5'-GTTTC-CCAGTCACGATA) [4,9,22]. Intra-primer secondary structure formation, such as dimer and hairpin formation between the 17-mer tag and nonamer, and probe melting temperature are known to influence binding efficiency [23,24]. To explore our hypothesis, we designed an algorithm to model the RT-PCR process using experimental data (see Additional data file 1 for details).…”

Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens

“…In general, DNA-DNA hybridization is a complex process that is difficult to predict (Mueckstein et al 2010;Pozhitkov et al 2007). Possible secondary structures of the amplicon represent a critical factor by blocking the probe binding site and therefore should be reduced by minimizing the amplicon length (Ratushna et al 2005). In this study, the amplicon length was reduced to mainly polymorphic regions, resulting in lengths between 250 and 500 bp.…”

Typing of Enteroviruses by Use of Microwell Oligonucleotide Arrays

“…Although this effect can be anticipated to some extent by secondary-structure prediction algorithms (9,20), the random occurrence of point mutations in the vicinity of the conserved probe binding sites makes this difficult. It will still be safe to include some redundancy in the probe sets, as was done here.…”