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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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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.
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gkt165-F3: 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.

Mentions: Next we investigated the influence on assay sensitivity of distributing the probes along the ∼1300 nt RNA target sequence. Because the four consecutive probes were designed to bind contiguous sequences of the target RNA, it was hypothesized that the conformation of the target RNA secondary structure prevented the probes from fully hybridizing and that distributed sequences would improve signal. By distributing the four probes along the length of the target RNA, the slope of response was improved more than 3-fold over the four contiguous sequences (Figure 3A and Table 1). Furthermore, increasing the total number of probes to nine further improved sensitivity, providing a LOD of 624 pM, or 1.5 × 105 molecules target RNA. Accordingly, studies evaluating the net hybridization of the single probe versus the four and nine probe combinations revealed increased hybridization with the increased number of probes (Figure 3C), further validating that BSI signal is at least partially the result of a net change in hybridization.Figure 3.


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)

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

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

gkt165-F3: 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.
Mentions: Next we investigated the influence on assay sensitivity of distributing the probes along the ∼1300 nt RNA target sequence. Because the four consecutive probes were designed to bind contiguous sequences of the target RNA, it was hypothesized that the conformation of the target RNA secondary structure prevented the probes from fully hybridizing and that distributed sequences would improve signal. By distributing the four probes along the length of the target RNA, the slope of response was improved more than 3-fold over the four contiguous sequences (Figure 3A and Table 1). Furthermore, increasing the total number of probes to nine further improved sensitivity, providing a LOD of 624 pM, or 1.5 × 105 molecules target RNA. Accordingly, studies evaluating the net hybridization of the single probe versus the four and nine probe combinations revealed increased hybridization with the increased number of probes (Figure 3C), further validating that BSI signal is at least partially the result of a net change in hybridization.Figure 3.

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