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

Abstract

Background: Secondary 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.

Results: Despite 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 degrees 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 degrees 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.

Conclusion: Secondary 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.

Figure 1

Secondary structure in a sample transcript. Circular diagrams of structure in a sample transcript (moeB homolog designated BR0004) from Brucella suis. Circular diagrams show hydrogen bonds between individual nucleotides, color-coded according to single-strandedness – the fraction of structures in which that bond is not present. Black bonds indicate 0% single-strandedness; red bonds indicate 100% single-strandedness.

Figure 2

Stability of transcript secondary structure in Brucella suis. Average free energy change on global secondary structure formation for Brucella suis targets, modeled as DNA or RNA. ΔG values are normalized to global mean target length.

Figure 3

Fractional accessibility of nucleotides in the target. Fraction of the complete transcript classified as inaccessible due to the presence of stable structure in >50% of predicted conformations. Data shown are for 37, 42, 52 and 65°C simulations in Brucella suis.

Figure 4

Stability of secondary structure in sheared fragments. Free energy change on secondary structure formation for the ureG-1 RNA transcript from Brucella suis. The transcript is modeled as sheared into fragments of length 200 nt, 100 nt or 50 nt; fragments are chosen starting at every 10th residue.

Figure 5

Accessibility of the probe binding site. Fraction of the average probe binding site in the Brucella genomic array that is found to be inaccessible at 37°, 42°, 52° and 65°C, for DNA or RNA target. Inaccessible sites are defined here using three different cutoffs for the fraction of structures in which the site is base-paired: 25%, 50%, and 75%.

Figure 6

Structure in a binding site – full length target and sheared fragments. The position of a 70mer oligonucleotide probe (green) binding site (red dots) within a full-length optimal transcript structure, as well as examples of stable structure in 200mer and 100mer fragments which overlap the probe binding site. Corresponding ΔG values for these fragments modeled at 42° and 52°C are shown in Table 1.

Figure 7

Accessibility prediction using three common methods. Pairing probabilities computed using RNAFold (top), MFold (middle) and SFold (bottom) for the human ICAM-1 transcript. Extendable sites detected by Allawi et al [21]