A Biofunctional Molecular Beacon for Detecting Single Base Mutations in Cancer Cells

Abstract

The development of a convenient and sensitive biosensing system to detect specific DNA sequences is an important issue in the field of genetic disease therapy. As a classic DNA detection technique, molecular beacon (MB) is often used in the biosensing system. However, it has intrinsic drawbacks, including high assay cost, complicated chemical modification, and operational complexity. In this study, we developed a simple and cost-effective label-free multifunctional MB (LMMB) by integrating elements of polymerization primer, template, target recognition, and G-quadruplex into one entity to detect target DNA. The core technique was accomplished by introducing a G-hairpin that features fragments of both G-quadruplex and target DNA recognition in the G-hairpin stem. Hybridization between LMMB and target DNA triggered conformational change between the G-hairpin and the common C-hairpin, resulting in significant SYBR-green signal amplification. The hybridization continues to the isothermal circular strand-displacement polymerization and accumulation of the double-stranded fragments, causing the uninterrupted extension of the LMMB without a need of chemical modification and other assistant DNA sequences. The novel and programmable LMMB could detect target DNA with sensitivity at 250 pmol/l with a linear range from 2 to 100 nmol/l and the relative standard deviation of 7.98%. The LMMB could sense a single base mutation from the normal DNA, and polymerase chain reaction (PCR) amplicons of the mutant-type cell line from the wild-type one. The total time required for preparation and assaying was only 25 minutes. Apparently, the LMMB shows great potential for detecting DNA and its mutations in biosamples, and therefore it opens up a new prospect for genetic disease therapy.

Biostable Aptamer Rings Conjugated for Targeting Two Biomarkers on Circulating Tumor Cells in Vivo with Great Precision

“…After phosphorylation at the 5′-end, ligase was added to catalyze the closure reaction between the 3′-and the 5′-ends of the ss to form C1 and CAP1. 35 The latter two hybridized each other to form the ds circular CAP1 (cCAP1), which possesses a unique capture arm (Region 1 that contains AP1 for targeting EpCAM; Figure S3b, Supporting Information) and a circular body (Region 2 as the steric hindrances to degradation; Figure S3b, Supporting Information). Figure 1f showed the structure of cCAP2, which is similar to that of cCAP1 except for the capture arm (Region 1; Figure S3c, Supporting Information) containing HB5 that targets biomarker Her2.…”

Quantum Dot-PNA Conjugates for Target-Catalyzed RNA Detection

“…[89] The achieved LOD is also lower compared to the ones provided by molecular beacons labeled with organic dyes (LOD = 500 pM), [145] or label-free multifunctional molecular beacons (LOD = 250 pM). [146] 5 Polymer-encapsulated QDs were chosen to act as a FRET donor due to their exceptional brightness and good colloidal stability, and subsequently used as a carrier of label acceptor PNA probes. Highly hydrophilic and bright asymmetric tri-sulfo-Cy5 was selected for the role of FRET acceptor and subsequently used for the synthesis of label donor PNA probes.…”

G-Quadruplexes as An Alternative Recognition Element in Disease-Related Target Sensing

“…The split G4 design can also be applied to the molecular beacon (MB) concept devoid of the fluorophore-quencher complex whilst maintaining their specific catalytic activity. MB was first reported in 1996 and has since been one of the most widely used systems for DNA biosensors [66,67]. Originally, the MB system alone relies on the concept of a closed to opened conformational change in the presence of a target DNA.…”