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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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DNA probes designed to bind different regions of the RNA target generate a range of BSI binding responses. With the exception of two probes, binding response correlates positively with the number of nucleotides predicted to be unpaired in the RNA target (R2 = 0.86). x-axis values are averages of predicted unpaired nucleotides in the five lowest energy folding structures of mfold ± standard error.
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gkt165-F6: DNA probes designed to bind different regions of the RNA target generate a range of BSI binding responses. With the exception of two probes, binding response correlates positively with the number of nucleotides predicted to be unpaired in the RNA target (R2 = 0.86). x-axis values are averages of predicted unpaired nucleotides in the five lowest energy folding structures of mfold ± standard error.

Mentions: During the process of testing a variety of oligonucleotide probe sequences, we observed that probes of similar length (i.e. 20–22 nt) and nucleotide content, but composed of different nucleotide sequences, yielded significantly disparate BSI binding responses. Because BSI sensitivity is produced in part by changes in conformation (8), we surmised that the probes were not only interacting at the primary sequence level of the target RNA (i.e. base pairing), but that probe binding signal was also impacted by the complex folding state of the RNA target. To investigate the effects of RNA target folding on BSI response, mfold software was used to predict secondary structure motifs in the regions complementary to the probes that would account for the variation in probe binding. Specifically, the software was used to identify regions of the RNA target that are predicated to be open loops, or sequences that would be available to bind a complementary oligonucleotide probe. Although mfold cannot predict RNA folding with absolute certainty, with the exception of two probes tested, we found a positive correlation between the number of unpaired nucleotides in the open loop regions of the predicted structure of the RNA target and the BSI signal produced by the probe complementary to that sequence (Figure 6). In line with probe design software for microarray oligonucleotide sequences (4), probes designed to bind RNA target sequences predicted to be single-stranded would result in the greatest net change in hybridization and produce the greatest change in BSI signal. Probes RSVN(264–285) and RSVN(308–329), however, produced a much higher signal that does not appear to fit this model. One possible explanation for the large signal of these probes, compared to the other probes, is that tertiary structure rearrangements or allosteric changes in the RNA target may be occurring upon probe binding. We have observed similar binding-order related signal enhancements in other systems, particularly with thrombin binding aptamers (12). These observations cannot be fully explained due to limitations in the current model, but are under investigation.Figure 6.


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)

DNA probes designed to bind different regions of the RNA target generate a range of BSI binding responses. With the exception of two probes, binding response correlates positively with the number of nucleotides predicted to be unpaired in the RNA target (R2 = 0.86). x-axis values are averages of predicted unpaired nucleotides in the five lowest energy folding structures of mfold ± standard error.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt165-F6: DNA probes designed to bind different regions of the RNA target generate a range of BSI binding responses. With the exception of two probes, binding response correlates positively with the number of nucleotides predicted to be unpaired in the RNA target (R2 = 0.86). x-axis values are averages of predicted unpaired nucleotides in the five lowest energy folding structures of mfold ± standard error.
Mentions: During the process of testing a variety of oligonucleotide probe sequences, we observed that probes of similar length (i.e. 20–22 nt) and nucleotide content, but composed of different nucleotide sequences, yielded significantly disparate BSI binding responses. Because BSI sensitivity is produced in part by changes in conformation (8), we surmised that the probes were not only interacting at the primary sequence level of the target RNA (i.e. base pairing), but that probe binding signal was also impacted by the complex folding state of the RNA target. To investigate the effects of RNA target folding on BSI response, mfold software was used to predict secondary structure motifs in the regions complementary to the probes that would account for the variation in probe binding. Specifically, the software was used to identify regions of the RNA target that are predicated to be open loops, or sequences that would be available to bind a complementary oligonucleotide probe. Although mfold cannot predict RNA folding with absolute certainty, with the exception of two probes tested, we found a positive correlation between the number of unpaired nucleotides in the open loop regions of the predicted structure of the RNA target and the BSI signal produced by the probe complementary to that sequence (Figure 6). In line with probe design software for microarray oligonucleotide sequences (4), probes designed to bind RNA target sequences predicted to be single-stranded would result in the greatest net change in hybridization and produce the greatest change in BSI signal. Probes RSVN(264–285) and RSVN(308–329), however, produced a much higher signal that does not appear to fit this model. One possible explanation for the large signal of these probes, compared to the other probes, is that tertiary structure rearrangements or allosteric changes in the RNA target may be occurring upon probe binding. We have observed similar binding-order related signal enhancements in other systems, particularly with thrombin binding aptamers (12). These observations cannot be fully explained due to limitations in the current model, but are under investigation.Figure 6.

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