Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays

Abstract

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.

FIG. 1.

Metal and sulfate reducer 16S rRNA target region and detection strategy. (A) 16S rRNA secondary structure model for the target region based on the secondary structure models of Gutell (13) for D. desulfuricans. Numbering is based on the G. chapellei sequence (GenBank accession number U41561). (B) Conceptual representation of the proximal chaperone detector hybridization strategy. (C) Alignment of Geobacter targets showing the sequences and locations of capture and detector probes: +0, 0-nt separation between the 3′ end of the capture probe and the 5′ end of the detector probe; +10, 10-nt separation; +14, 14-nt separation.

FIG. 2.

Increased hybridization specificity with increased separation between the capture probe and the detector probe. Hybridizations were performed as described in Materials and Methods with 2 μg of intact G. chapellei RNA (A) or intact G. sulfurreducens RNA (B). Numbers on the y axes are mean fluorescence intensity (in arbitrary units). White columns indicate the perfectly matched probe for the target RNA. Error bars indicate standard deviations.

FIG. 3.

Effect of nucleotide mismatches between the detector probe and target rRNA. Detector probe sequences and perfect matches are listed in Table 1. Probes1 to 3 are specific for Geobacter species; probes 4 to 8 are specific for Desulfovibrio species; probe 9 is specific for S. putrefaciens; and probe 10 is identical to probe 1 and is specific for Geobacter species, except that it is shortened by 4 nt to achieve a +14 hybridization condition. Signals represent the average hybridization intensity from at least three replicate arrays (across two slide lots), with each array containing three replicate spots. Hybridizations were performed as described in Materials and Methods with 2 μg of fragmented G. chapellei target RNA (A) or fragmented D. desulfuricans target RNA (B). Similar results were obtained with intact RNA targets (not shown). The graphing software did not permit error bars to be plotted in the three-dimensional graphing format, and so they are not shown.

FIG. 4.

Multigenus microarray. (A) Print pattern for oligonucleotide (Oligo) probes. QC, quality control. (B) Hybridization results for 2 μg of intact RNA. Target RNA species are shown beneath the hybridization results.