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Bead-Immobilized Multimodal Molecular Beacon-Equipped DNA Machinery for Specific RNA Target Detection: A Prototypical Molecular Nanobiosensor.

ABSTRACT: A variety of nanostructured diagnostic tools have been developed for the precise detection of known genetic variants. Molecular beacon systems are very promising tools due to their specific selectivity coupled with relatively lower cost and time requirements than existing molecular detection tools such as next generation sequencing or real-time PCR (polymerase chain reaction). However, they are prone to errors induced by secondary structure responses to environmental fluctuations, such as temperature and pH. Herein, we report a temperature-insensitive, bead-immobilized, molecular beacon-equipped novel DNA nanostructure for detection of cancer miRNA variants with the consideration of thermodynamics. This system consists of three parts: a molecular beacon for cancer-specific RNA capture, a stem body as a core template, and a single bead for solid-support. This DNA system was selectively bound to nanosized beads using avidin-biotin chemistry. Synthetic DNA nanostructures, designed based on the principle of fluorescence-resonance enhanced transfer, were effectively applied for in vitro cancer-specific RNA detection. Several parameters were optimized for higher performance, with a focus on thermodynamic stability. Theoretical issues regarding the secondary structure of a single molecular beacon and its combinatory forms were also studied. This study provides design guidelines for new sensing systems of miRNA variation for next-generation biotechnological applications.

SUBMITTER: Kim J

PROVIDER: S-EPMC8235652 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Despite the substantial burden posed by osteoarthritis (OA) globally, difficult challenges remain in achieving early OA diagnosis and adopting effective disease-modifying treatments. In this study, we use a biomolecular approach to address these limitations by creating an inherently theranostic molecular beacon whose imaging and therapeutic capabilities are activated by early pathological changes in OA. This platform comprised (1) a peptide linker substrate for metalloproteinase-13 (MMP-13), a pathological protease upregulated in OA, which was conjugated to (2) a porphyrin moiety with inherent multimodal imaging, photodynamic therapy, and drug delivery capabilities, and (3) a quencher that silences the porphyrin's endogenous fluorescence and photoreactivity when the beacon is intact. In diseased OA tissue with upregulated MMP-13 expression, this porphyrin molecular beacon (PPMMP13B) was expected to undergo sequence-specific cleavage, yielding porphyrin fragments with restored fluorescence and photoreactivity that could, respectively, be used as a readout of MMP-13 activity within the joint for early OA imaging and disease-targeted photodynamic therapy. This study focused on the synthesis and characterization of PPMMP13B, followed by a proof-of-concept evaluation of its OA imaging and drug delivery potential. In solution, PPMMP13B demonstrated 90% photoactivity quenching in its intact form and robust MMP-13 activation, yielding a 13-fold increase in fluorescence post-cleavage. In vitro, PPMMP13B was readily uptaken and activated in an MMP-13 cell expression-dependent manner in primary OA synoviocytes without exuding significant cytotoxicity. This translated into effective intra-articular cartilage (to a 50 μm depth) and synovial uptake and activation of PPMMP13B in a destabilization of the medial meniscus OA mouse model, yielding strong fluorescence contrast (7-fold higher signal than background) at the diseased joint site. These results provide the foundation for further exploration of porphyrin molecular beacons for image-guided OA disease stratification, effective articular delivery of disease-modify agents, and OA photodynamic therapy.

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Bead-Immobilized Multimodal Molecular Beacon-Equipped DNA Machinery for Specific RNA Target Detection: A Prototypical Molecular Nanobiosensor.

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