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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 LNA and DNA probes of the same sequence and length incubated with target RNA. (A) LNA probes improve the BSI signal. (B) DNA:RNA hybrids and LNA:RNA hybrids produce virtually the same net hybridization.
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gkt165-F7: Comparison of the BSI binding response and net hybridization of LNA and DNA probes of the same sequence and length incubated with target RNA. (A) LNA probes improve the BSI signal. (B) DNA:RNA hybrids and LNA:RNA hybrids produce virtually the same net hybridization.

Mentions: With some knowledge of the probe length and spacing parameters that yield good signal in BSI for optimized hybridization, we explored LNAs, a category of oligonucleotides with unique structure and binding characteristics. LNA oligonucleotides have much greater binding affinities for their targets when compared to DNA or RNA of similar length and sequence (19). The first LNA probe we used, RSV(242–263)L, was the same sequence and length as the 22-mer DNA probe used in our initial experiments, except that every third nucleotide in the sequence contains a methylene group bridging the 2′ oxygen and the 4′ carbon of the ribose ring, ‘locking’ the sugar into the 3′-endo conformation. With these simple structural modifications, a 4-fold improvement in sensitivity was achieved over the DNA probes, resulting in a LOD of 2.15 nM of target RNA (Figure 7A and Table 1). Using a mixture of four distributed LNA probes, identical in sequence and length to the four distributed DNA probes, a LOD of 1.05 nM target RNA was achieved. These results compare favorably to the 1.5-fold improvement in LOD observed when increasing the number of DNA probes from one to four (Table 1). Interestingly, this improvement in signal and sensitivity was not attributed to an increase in the net hybridization of the probe to the RNA target. Although the increased affinity of LNA for the RNA target would generally shift the binding equilibrium toward the bound state, both LNA and DNA probes hybridize to approximately the same number of RNA targets (Figure 7B). This result is likely because the LNA and DNA probes are added to the RNA target in such excess that, despite the increased affinity of LNA for the target RNA, the total number of LNA and DNA probes bound to the RNA target was nearly equivalent. Because there is not a significant increase in net hybridization when using LNA probes, we concluded that hybridization alone did not account for the 4-fold improvement in BSI signal using the LNA probe. Therefore, we explored the possibility that the improvement in sensitivity was the result of the unique structural characteristics of LNA:RNA hybrid.Figure 7.


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 LNA and DNA probes of the same sequence and length incubated with target RNA. (A) LNA probes improve the BSI signal. (B) DNA:RNA hybrids and LNA:RNA hybrids produce virtually the same net hybridization.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643578&req=5

gkt165-F7: Comparison of the BSI binding response and net hybridization of LNA and DNA probes of the same sequence and length incubated with target RNA. (A) LNA probes improve the BSI signal. (B) DNA:RNA hybrids and LNA:RNA hybrids produce virtually the same net hybridization.
Mentions: With some knowledge of the probe length and spacing parameters that yield good signal in BSI for optimized hybridization, we explored LNAs, a category of oligonucleotides with unique structure and binding characteristics. LNA oligonucleotides have much greater binding affinities for their targets when compared to DNA or RNA of similar length and sequence (19). The first LNA probe we used, RSV(242–263)L, was the same sequence and length as the 22-mer DNA probe used in our initial experiments, except that every third nucleotide in the sequence contains a methylene group bridging the 2′ oxygen and the 4′ carbon of the ribose ring, ‘locking’ the sugar into the 3′-endo conformation. With these simple structural modifications, a 4-fold improvement in sensitivity was achieved over the DNA probes, resulting in a LOD of 2.15 nM of target RNA (Figure 7A and Table 1). Using a mixture of four distributed LNA probes, identical in sequence and length to the four distributed DNA probes, a LOD of 1.05 nM target RNA was achieved. These results compare favorably to the 1.5-fold improvement in LOD observed when increasing the number of DNA probes from one to four (Table 1). Interestingly, this improvement in signal and sensitivity was not attributed to an increase in the net hybridization of the probe to the RNA target. Although the increased affinity of LNA for the RNA target would generally shift the binding equilibrium toward the bound state, both LNA and DNA probes hybridize to approximately the same number of RNA targets (Figure 7B). This result is likely because the LNA and DNA probes are added to the RNA target in such excess that, despite the increased affinity of LNA for the target RNA, the total number of LNA and DNA probes bound to the RNA target was nearly equivalent. Because there is not a significant increase in net hybridization when using LNA probes, we concluded that hybridization alone did not account for the 4-fold improvement in BSI signal using the LNA probe. Therefore, we explored the possibility that the improvement in sensitivity was the result of the unique structural characteristics of LNA:RNA hybrid.Figure 7.

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