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The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.

Adams NM, Olmsted IR, Haselton FR, Bornhop DJ, Wright DW - Nucleic Acids Res. (2013)

Bottom Line: Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.

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.

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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.
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gkt165-F8: 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.

Mentions: It has been established that LNA:RNA hybrids primarily form A-form secondary helical structures, whereas DNA:RNA hybrids consist of a mixture of A-form and B-form character (20). Since the net hybridization measured for DNA versus LNA probes binding the RNA target was approximately equivalent but the BSI signal of the LNA probes was significantly greater than that of the DNA probes (Figure 7A and B, respectively), we hypothesized that the induced A-form helical character of the LNA:RNA hybrid was responsible for the greater RI change upon formation compared to the DNA:RNA hybridization. Accordingly, we evaluated nucleic acid hybrids of the same length and sequence that exhibit a range of secondary structures using CD and BSI. To reduce the background noise from the unbound regions of the ∼1300 RNA target, the RNA and DNA complements used in these studies are the same length as the LNA and DNA probes (22 nt). Based on the CD spectra, we verified that the LNA:RNA hybrid resulted in a characteristic A-form secondary structure, the DNA:RNA hybrid resulted in a secondary structure consisting of a mixture of A- and B-form, and the DNA:DNA duplex resulted in a characteristic B-form secondary structure (Figure 8A). These same hybrids were then evaluated using BSI. The LNA:RNA interaction produced the largest RI shift (1.53 × 10−2 rad/nM), followed by the DNA:RNA interaction (5.48 × 10−3 rad/nM), and the DNA:DNA interaction produced the smallest RI shift (3.75 × 10−3 rad/nM) (Figure 8B). These data indicate that the BSI signal or slope reflects the extent to which the hybridized product displays A-form secondary structure. These observations are consistent with the hypothesis that BSI signal can be maximized using oligonucleotide probes that induce the greatest net change in the nucleic acid secondary structure.Figure 8.


The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.

Adams NM, Olmsted IR, Haselton FR, Bornhop DJ, Wright DW - Nucleic Acids Res. (2013)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkt165-F8: 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.
Mentions: It has been established that LNA:RNA hybrids primarily form A-form secondary helical structures, whereas DNA:RNA hybrids consist of a mixture of A-form and B-form character (20). Since the net hybridization measured for DNA versus LNA probes binding the RNA target was approximately equivalent but the BSI signal of the LNA probes was significantly greater than that of the DNA probes (Figure 7A and B, respectively), we hypothesized that the induced A-form helical character of the LNA:RNA hybrid was responsible for the greater RI change upon formation compared to the DNA:RNA hybridization. Accordingly, we evaluated nucleic acid hybrids of the same length and sequence that exhibit a range of secondary structures using CD and BSI. To reduce the background noise from the unbound regions of the ∼1300 RNA target, the RNA and DNA complements used in these studies are the same length as the LNA and DNA probes (22 nt). Based on the CD spectra, we verified that the LNA:RNA hybrid resulted in a characteristic A-form secondary structure, the DNA:RNA hybrid resulted in a secondary structure consisting of a mixture of A- and B-form, and the DNA:DNA duplex resulted in a characteristic B-form secondary structure (Figure 8A). These same hybrids were then evaluated using BSI. The LNA:RNA interaction produced the largest RI shift (1.53 × 10−2 rad/nM), followed by the DNA:RNA interaction (5.48 × 10−3 rad/nM), and the DNA:DNA interaction produced the smallest RI shift (3.75 × 10−3 rad/nM) (Figure 8B). These data indicate that the BSI signal or slope reflects the extent to which the hybridized product displays A-form secondary structure. These observations are consistent with the hypothesis that BSI signal can be maximized using oligonucleotide probes that induce the greatest net change in the nucleic acid secondary structure.Figure 8.

Bottom Line: Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.

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.

Show MeSH
Related in: MedlinePlus