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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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Related in: MedlinePlus

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.
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gkt165-F1: 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.

Mentions: BSI has been used to successfully monitor binding interactions of a variety of biological molecules with high sensitivity (9–15). The design of this unique interferometer is very simple. A He–Ne laser is used to illuminate a semicircular microfluidic channel containing <1 μl of analyte, creating a set of high contrast interference fringes of reflected and refracted light (Figure 1A). When a specific binding event occurs, the refractive index (RI) of the solution in the channel changes, causing these fringes to shift in a manner that is proportional to the concentration of the analyte (Figure 1B–C). Though BSI has been used to quantify protein biomarkers via antibody–antigen interactions (10), the work presented here represents the first report of its use for detecting and quantifying RNA biomarkers.Figure 1.


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)

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

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

gkt165-F1: 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.
Mentions: BSI has been used to successfully monitor binding interactions of a variety of biological molecules with high sensitivity (9–15). The design of this unique interferometer is very simple. A He–Ne laser is used to illuminate a semicircular microfluidic channel containing <1 μl of analyte, creating a set of high contrast interference fringes of reflected and refracted light (Figure 1A). When a specific binding event occurs, the refractive index (RI) of the solution in the channel changes, causing these fringes to shift in a manner that is proportional to the concentration of the analyte (Figure 1B–C). Though BSI has been used to quantify protein biomarkers via antibody–antigen interactions (10), the work presented here represents the first report of its use for detecting and quantifying RNA biomarkers.Figure 1.

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