The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Adams NM,Olmsted IR,Haselton FR,Bornhop DJ,Wright DW

doi

10.1093/nar/gkt165

subject

Has Abstract

pub_date

2013-05-01 00:00:00

pages

e103

issue

9

eissn

0305-1048

issn

1362-4962

pii

gkt165

journal_volume

41

pub_type

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