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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 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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gkt165-F9: 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.

Mentions: To further validate that the formation of A-form secondary structure is responsible for the observed increase in BSI signal, as opposed to the differences in the primary structures of the nucleotide subunits, we performed BSI measurements on a DNA:DNA duplex matching the sequence of the RSVN(242–263) probe at various stages of a TFE-induced B-form to A-form transition. Incubation with high concentrations of TFE is a well-established method for converting B-form secondary structure in DNA:DNA duplexes to A-form (21,22). TFE was titrated into a solution containing a DNA:DNA duplex and the secondary structure transition was confirmed by CD analysis (Figure 9A). BSI signal magnitude increased as the DNA:DNA duplex adopted a more A-form character as monitored by the ellipticity at 270 nm. These data validate the BSI signal enhancing effect of induced alterations to the helical geometry of the nucleic acid hybrid (Figure 9B).Figure 9.


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

License
Show All Figures
getmorefigures.php?uid=PMC3643578&req=5

gkt165-F9: 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.
Mentions: To further validate that the formation of A-form secondary structure is responsible for the observed increase in BSI signal, as opposed to the differences in the primary structures of the nucleotide subunits, we performed BSI measurements on a DNA:DNA duplex matching the sequence of the RSVN(242–263) probe at various stages of a TFE-induced B-form to A-form transition. Incubation with high concentrations of TFE is a well-established method for converting B-form secondary structure in DNA:DNA duplexes to A-form (21,22). TFE was titrated into a solution containing a DNA:DNA duplex and the secondary structure transition was confirmed by CD analysis (Figure 9A). BSI signal magnitude increased as the DNA:DNA duplex adopted a more A-form character as monitored by the ellipticity at 270 nm. These data validate the BSI signal enhancing effect of induced alterations to the helical geometry of the nucleic acid hybrid (Figure 9B).Figure 9.

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