Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs.

Detecting multiple targets in living cells is important in cell biology. However, multiplexed fluorescence imaging beyond two-to-three targets remains a technical challenge. Herein, we introduce a multiplexed imaging strategy, 'sequential Fluorogenic RNA Imaging-Enabled Sensor' (seqFRIES), which enables live-cell target detection via sequential rounds of imaging-and-stripping. In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study four fluorogenic RNA aptamer/dye pairs that can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of critical signalling molecules and mRNA targets was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for multiplexed and dynamic live-cell imaging and cell biology studies.

Binary detection of protein and nucleic acid enabled cancer diagnosis through branched hybridization chain reaction.

Developing isothermal bio-analyzers for amplified detection of multi-factor diseases like cancer biomarkers (nucleic acid and protein) has facilitated the early diagnosis and clinical theranostics. In light of that, a sensitive detection system was developed assisted by the recognition capability of a functional aptamer followed by cyclic self-assembly of three auxiliary hairpins via branched hybridization chain reaction (b-HCR) performance. In the downstream process, in the presence of hemin, split sequences of a DNAzyme brought in close proximity to facilitate the color alteration of the solution to a green appearance. By ingenious exerting multi-level amplification, the assay empowered sensitive detection of miR-21 and PDGF-BB related cancer biomarkers with LOD values of 10 pM and 40 nM, respectively. Taken together, simplicity, enzyme-free nature, excellent generality, affordable cost without any washing steps and immobilization makes the presented system a promising analytical tool in synthetic biology, designing nanomachines and point-of-care detection in resource-constrained settings.

Developing a colorimetric nucleic acid-responsive DNA hydrogel using DNA proximity circuit and catalytic hairpin assembly.

The development of powerful techniques for sensitive detection of nucleic acids has attracted much attention for fabricating accurate biosensors in various fields, such as genomics, clinical diagnostics, and forensic sciences. Up to now, different systems have been introduced, the majority of which are expensive, time-consuming, and relatively low selectivity/limit of detection. These limitations caught our attention to fabricate a nucleic acid responsive system by combining three layers of signal amplification strategy, namely a split proximity circuit (SPC), a catalytic hairpin assembly (CHA), and a DNA hydrogel. Herein, by SPC operation, two initiators and a target strand were assembled and activated the CHA reaction in the presence of three 5'-cytosine (C)-rich hairpins. Then, produced C-rich embedded three-way junction structures could form i-motif structures under acidic environment followed by a transition from sol to gel state. To acquire a quantitative and colorimetric measurement, gold nanoparticles (GNPs) were used that encapsulated and sediment by the gel formation. The resulting platform detected the target with a limit of detection of 1 pM and considerable selectivity.

DNA hydrogel-empowered biosensing.

DNA hydrogels as special members in the DNA nanotechnology have provided crucial prerequisites to create innovative gels owing to their sufficient stability, biocompatibility, biodegradability, and tunable multifunctionality. These properties have tailored DNA hydrogels for various applications in drug delivery, tissue engineering, sensors, and cancer therapy. Recently, DNA-based materials have attracted substantial consideration for the exploration of smart hydrogels, in which their properties can change in response to chemical or physical stimuli. In other words, these gels can undergo switchable gel-to-sol or sol-to-gel transitions upon application of different triggers. Moreover, various functional motifs like i-motif structures, antisense DNAs, DNAzymes, and aptamers can be inserted into the polymer network to offer a molecular recognition capability to the complex. In this manuscript, a comprehensive discussion will be endowed with the recognition capability of different kinds of DNA hydrogels and the alternation in physicochemical behaviors upon target introducing. Finally, we offer a vision into the future landscape of DNA based hydrogels in sensing applications.