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, 41 (9), e103

The Effect of Hybridization-Induced Secondary Structure Alterations on RNA Detection Using Backscattering Interferometry

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The Effect of Hybridization-Induced Secondary Structure Alterations on RNA Detection Using Backscattering Interferometry

Nicholas M Adams et al. Nucleic Acids Res.

Abstract

Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼10(5) target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

Figures

Figure 1.
Figure 1.
Depiction of the optical train and mechanism of signal generation for RNA detection using BSI. (A) Schematic of BSI optical train. (B) Digital representation of interference fringes. (C) Representation of signal generation as DNA probes bind complementary RNA targets.
Figure 2.
Figure 2.
Comparison of the BSI binding response and net hybridization upon adding the 15-mer, 22-mer, 44-mer, 88-mer or four consecutive 22-mer DNA probes to the RNA target. (A) The probe length that produces optimal BSI signal is 22 nt. Four short 22-mer DNA probes have improved signal over one 88-mer spanning the same target sequence. A scrambled negative control sequence produced negligible signal. (B) Net hybridization of the four consecutive 22-mers is significantly greater than any of the four probe lengths.
Figure 3.
Figure 3.
Comparison of the BSI binding response and net hybridization of various numbers and distributions of probes incubated with the RNA target. (A) Increasing the number and distribution of distinct probes improves sensitivity. (B) Illustration of the relative positions of the DNA probes along the RNA target. (C) Hybridization studies confirm that increased number of probes bound correlates with increased binding signal.
Figure 4.
Figure 4.
Saturation curves of target RNA incubated with increasing concentrations of either a single 22-mer probe or a mixture of nine distributed probes. The mixture of nine probes saturates at a higher level than the single probe.
Figure 5.
Figure 5.
Evaluations of BSI specificity for (A) mismatched targets or (B) RNA targets in complex samples using a single 22-mer probe. (A) BSI signal drops off moderately when probing for RNA targets with increasing numbers of mismatched nucleotides. (B) BSI signal is consistent when probing for the ∼1300 nt RNA biomarker in a sample of total RNA extracted from HEp-2 cell lysates of increasing concentrations (open circles), whereas BSI signal diminishes in unextracted samples of increasing background concentration (closed circles). (C) qRT-PCR cycle threshold values for the extracted samples correlate with the BSI fringe shift values (R2 = 0.92).
Figure 6.
Figure 6.
DNA probes designed to bind different regions of the RNA target generate a range of BSI binding responses. With the exception of two probes, binding response correlates positively with the number of nucleotides predicted to be unpaired in the RNA target (R2 = 0.86). x-axis values are averages of predicted unpaired nucleotides in the five lowest energy folding structures of mfold ± standard error.
Figure 7.
Figure 7.
Comparison of the BSI binding response and net hybridization of LNA and DNA probes of the same sequence and length incubated with target RNA. (A) LNA probes improve the BSI signal. (B) DNA:RNA hybrids and LNA:RNA hybrids produce virtually the same net hybridization.
Figure 8.
Figure 8.
Relative degree of A-form nucleic acid character of the DNA:DNA, DNA:RNA and LNA:RNA hybrids corresponds with increased BSI signal. (A) The CD spectrum of the DNA:DNA duplex (green) corresponds to B-form secondary helical structure with a maximum near 280 nm, a deep minimum near 250 nm. LNA:RNA hybrid (red) produces a spectra corresponding to A-form secondary structure with a maximum near 270 nm and a shallow minimum near 245 nm. The DNA:RNA hybrid produces a spectra that is intermediate of A-form and B-form. (B) BSI binding curves of LNA:RNA, DNA:RNA and DNA:DNA.
Figure 9.
Figure 9.
Relative degree of A-form character corresponds to increased BSI signal. (A) CD spectra of the DNA duplex demonstrate a shift from A-form to B-form structure with decreasing concentrations of TFE. Inset: A-form to B-form transition monitored at 270 nm. (B) Ellipticity at 270 nm correlates positively with the shift in the RI as detected by BSI.

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References

    1. Bordelon H, Adams NM, Klemm AS, Russ PK, Williams JV, Talbot HK, Wright DW, Haselton FR. Development of a low-resource RNA extraction cassette based on surface tension valves. ACS Appl. Mater. Interfaces. 2011;3:2161–2168. - PMC - PubMed
    1. Bae H-G, Nitsche A, Teichmann A, Biel SS, Niedrig M. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. J. Virol. Methods. 2003;110:185–191. - PubMed
    1. Jayagopal A, Halfpenny KC, Perez JW, Wright DW. Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells. J. Am. Chem. Soc. 2010;132:9789–9796. - PMC - PubMed
    1. Mehlmann M, Dawson ED, Townsend MB, Smagala JA, Moore CL, Smith CB, Cox NJ, Kuchta RD, Rowlen KL. Robust sequence selection method used to develop the FluChip diagnostic microarray for influenza virus. J. Clin. Microbiol. 2006;44:2857–2862. - PMC - PubMed
    1. Marras SAE, Tyagi S, Kramer FR. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes. Clin. Chim. Acta. 2006;363:48–60. - PubMed

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