Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC3643578&req=5

gkt165-F4: 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.

Mentions: To further demonstrate that the increased signal from the nine-probe cocktail was a result of an increase in the number of available binding sites, as opposed to the effect of having a higher concentration of probes present, a saturation binding isotherm was constructed for both the single probe and the nine-probe cocktail. For this assay, the concentration of the target RNA was held constant while the probe concentration was varied from 0–100 nM. The signal at saturation (Bmax) for the single probe was 0.087 radians, whereas the signal for the nine distributed probes was 0.25 radians, a 2.9-fold overall increase in signal (Figure 4). This result indicates that the sensitivity improvement observed in the nine-probe system is the result of an increased number of available binding sites; therefore, a greater number of binding events can occur before target saturation is reached. This may not be surprising as BSI signal magnitude is directly related to the number of binding events (9). These data are consistent with these previous observations and indicate that the greatest BSI sensitivity over a large dynamic range is achieved by maximizing the number of available target RNA binding sites.Figure 4.


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)

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

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

gkt165-F4: 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.
Mentions: To further demonstrate that the increased signal from the nine-probe cocktail was a result of an increase in the number of available binding sites, as opposed to the effect of having a higher concentration of probes present, a saturation binding isotherm was constructed for both the single probe and the nine-probe cocktail. For this assay, the concentration of the target RNA was held constant while the probe concentration was varied from 0–100 nM. The signal at saturation (Bmax) for the single probe was 0.087 radians, whereas the signal for the nine distributed probes was 0.25 radians, a 2.9-fold overall increase in signal (Figure 4). This result indicates that the sensitivity improvement observed in the nine-probe system is the result of an increased number of available binding sites; therefore, a greater number of binding events can occur before target saturation is reached. This may not be surprising as BSI signal magnitude is directly related to the number of binding events (9). These data are consistent with these previous observations and indicate that the greatest BSI sensitivity over a large dynamic range is achieved by maximizing the number of available target RNA binding sites.Figure 4.

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