Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer

Abstract

A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.

Lanthanide‐FRET Molecular Beacons for microRNA Biosensing, Logic Operations, and Physical Unclonable Functions

“…It should be noted that the molar LODs in the actual sample (the assay volume of 100 μL contains 25 μL of sample and 75 μL of assay reagents) are four times higher. Whereas comparison of sensitivities and LODs is not necessarily meaningful if assays were performed and analyzed under different conditions and used different targets, the assay performance parameters of our TG‐FRET MBs correspond well to those found for the same MBs and miRNA targets [48] and for other lanthanide‐based MBs but with different targets, [42–47] all of which were also able to quantify targets in the low to sub nanomolar concentration range.…”

“…[40,41] Lanthanides, including Tb, Eu, Dy, and Sm have also been applied as FRET donors in MBs (or similar structures) for the detection of single-stranded (ss)DNA targets. [42][43][44][45][46][47] Although the application potential of such lanthanide-based TG-FRET MBs is in principle much broader than sensitive quantification of ssDNA, multiplexing with different lanthanides, quantification of RNA biomarkers, Boolean logic operations, or security encryption have not been realized so far.…”

“…This relatively simple but very smart biosensing approach was developed more than 25 years ago and has been used in a myriad of different biosensing applications in research and molecular diagnostics [40,41] . Lanthanides, including Tb, Eu, Dy, and Sm have also been applied as FRET donors in MBs (or similar structures) for the detection of single‐stranded (ss)DNA targets [42–47] . Although the application potential of such lanthanide‐based TG‐FRET MBs is in principle much broader than sensitive quantification of ssDNA, multiplexing with different lanthanides, quantification of RNA biomarkers, Boolean logic operations, or security encryption have not been realized so far.…”

Binary Probes for Nucleic Acid Analysis

“…Another approach to elevate the problem of high background fluorescence employed intramolecular quenching of either the donor or both the donor and acceptor fluorophores. For example, Santangelo et al used a pair of MB probes, one with a donor fluorophore and another with an acceptor.…”