Since the level of human telomerase RNA (hTR) in tumor cells is higher than that in normal somatic cells, the quantitative assay of hTR is of significant importance in tumor diagnosis. Herein, graphene oxide (GO) was simultaneously exploited as a fluorescence quencher and a carrier of nucleic acid to successfully deliver two hairpin DNA probes of hybridization chain reaction (HCR) into the cancer cell for detecting telomerase RNA based on DNA nanoassembly of HCR. The sticky end of HCR probes could tightly absorb on the surface of GO, resulting in fluorescence quenching of the dye which was tagged at the sticky end of two hairpin probes. When faced with hTR, the fluorescence of DNA probes is subsequently recovered because hTR could trigger HCR to autonomous assembly of a DNA polymer which released from the GO and led to fluorescence recovery. Taking advantage of nucleic acid nanoassembly of HCR, this intracellular HCR strategy creates enormous signal amplification, and enables ultra-sensitive fluorescence imaging of hTR expression. By monitoring fluorescence change, human telomerase RNA could be specifically studied and this method can also be used for detecting single-base mutation. The GO-aided HCR strategy allowed us to sensitively detect hTR in a living cell, which holds great potential for analyzing other low-abundance biomolecules in living cells via HCR.
Several fluorescence methods have been developed for sensitive detection of PNK activity based on signal amplification techniques, but they need fluorescently labeled DNA probes and superabundant assistant enzymes. We have addressed these limitations and report here a label-free and enzyme-free amplification strategy for sensitively and specifically studying PNK activity and inhibition via hybridization chain reaction (HCR). First, the phosphorylation of hairpin DNA H1 by T4 PNK makes it be specifically digested by lambda exonuclease (λ exo) from 5' to 3' direction to generate a single-stranded initiator which can successively open hairpins H2 and H3 to trigger an autonomous assembly of long DNA nanowires. Meanwhile, an intermolecular G-quadruplex is formed between H2 and H3, thereby providing fluorescence enhancement of N-methyl mesoporphyrin IX (NMM) which is a highly quadruplex-selective fluorophore. So, the PNK activity can be facilely and sensitively detected by using NMM as a signal probe which provides a low background signal to improve the overall sensitivity, resulting in the detection limit of 3.37 × 10(-4) U mL(-1). More importantly, its successful application for detecting PNK activity in a complex biological matrix and studying the inhibition effects of PNK inhibitors demonstrated that it provides a promising platform for screening PNK inhibitors as well as detecting PNK activity. Therefore, it is a highly sensitive, specific, reliable and cost-effective strategy which shows great potential for biological process research, drug discovery, and clinical diagnostics.
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