In situ generation spatial confinement fluorescence RNA for sensitive and stable imaging of telomerase RNA in cells

2021 
Abstract A critical challenge for cell imaging is that the reduced stability and intensity of signal probe suffering from the complex cellular environment. Here, we present a spatial confinement fluorescence RNA with stable structure and strong signal output for cell imaging, which was generated in situ by a cascaded rolling circle transcription (RCT) and DNA fixation. In this assay, two functional padlock probes (P1, P2), one hairpin probe (HP) and ssDNA were designed as the building blocks, human telomerase RNA (hTR) was chosen as a model. Under the action of hTR, P1 was circularized and initiated the first RCT, producing RNA nanowires for hybridization with HP. The unfolded HP subsequently hybridized with P2 to initiate the second RCT reaction, producing large amounts of spinach RNA aptamer strung to long RNAs. Cycles of the above RCT reactions were repeated in both vertical directions resulting in a high confinement spinach RNA aptamer in a 2D matrix. Finally, spinach RNA aptamer was hybridized with ssDNA and displayed strong fluorescence upon binding the non-fluorescent fluorophore (DFHBI), thus realizing in situ amplified label-free imaging. In our system, the proposed spinach RNA matrix carries amounts of signal reporting sites in a limited space, which facilitates the transporting and binding of DFHBI, thereby providing a high-contrast signal response and a low detection limit (LOD). Importantly, the fluorescence intensity in serum exhibited no significant decrease after 4 h (97.7 %). Its superior biological stability is attributed to the large steric hindrances of the tight frame that prevent intracellular degradation, and the DNA fixation strategy that solidifying the structure of spinach RNA while preventing DFHBI leakage. Moreover, the expression of hTR in different cells (A549, HepG2 and HL-7702) were also performed successfully, providing a powerful platform for assaying biomarkers in cells.
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