I-Motif-Based in Situ Bipedal Hybridization Chain Reaction for Specific Activatable Imaging and Enhanced Delivery of Antisense Oligonucleotides

Abstract

The efficient and precise delivery of antisense oligonucleotides (ASOs) to target cells is of great value in gene silencing. However, the specificity and packaging capacity of delivery system still remains challenging. Here, we designed an i-motif forming-initiated in situ bipedal hybridization chain reaction (pH-Apt-BiHCR) amplification strategy for specific target cells imaging and enhanced gene delivery of ASOs. As a proof of concept, an 8-nt ASO modified with locked nucleic acid (LNA) which is complementary to the seed region of microRNA21 (miR-21) was used for gene silencing studies. Benefiting from the design of hairpin-contained i-motif, the stimuli-responsive assembly of pH-Apt-BiHCR was successfully achieved on MCF-7 cells surface based on the specific recognition of aptamer. Using this strategy, the pH-Apt-BiHCR not only contains repeated fluorescence resonance energy transfer (FRET) units for activatable tumor imaging with high contrast but also arrays with plenty of LNA ASOs as interference molecules for cancer cells inhibition. An in vitro assay showed that this strategy presented an excellent response ability in buffer within a narrow pH range (6.0–7.0) with a transition midpoint (pHT) of 6.44 ± 0.06. Moreover, live cell studies revealed that it realized a specific activatable imaging of target cells, while the ASOs arrayed pH-Apt-BiHCR exhibited improved internalization via an endocytosis pathway and enhanced gene silencing to MCF-7 cells compared to single ASO alone. We believe that this design will inspire the development of novel probes for early diagnosis and therapy of cancer cells

Prospects and challenges of dynamic DNA nanostructures in biomedical applications

“… 38 Ma et al developed the pH-Apt-BiHCR technique for specific cell imaging and enhanced gene delivery by separation of the complementary chain followed by formation of the i-motif at low pH and stimulated the downstream cascade reaction. 80 …”

Endogenous miRNA-Activated DNA Nanomachine for Intracellular miRNA Imaging and Gene Silencing

“…According to the principle of operation, there are two main types of DNA nanomachines. One is designed in response to external environmental factors, such as light-, acid-, and temperature-driven DNA nanomachines, which have been employed in bioimaging and drug delivery. − The other type is based on the toehold-mediated strand displacement reaction (TSDR), which is a promising strategy for building self-powered DNA nanomachines that achieve autonomous operation, reliable motion, and improved controllability. In this context, a large number of DNA nanomachines, including DNA walkers and entropy-driven DNA amplifiers, have been established for disease diagnosis, logic computing, and regulation of cell function. − Especially, several linear DNA nanomachines have received extensive attention due to their outstanding performance, such as improved cell internalization efficiency and accelerated reaction rates. − For example, Ren et al designed a responsive DNA “nanostring light” for fast and highly efficient mRNA imaging in living cells based on accelerated DNA cascade reaction along a DNA nanowire .…”

The Recent Development of Hybridization Chain Reaction Strategies in Biosensors

“…This strategy achieved ultrasensitive and specific detection of individual mRNA for discriminating single-nucleotide variation. Ma et al 70 combined pH-sensitive i-motif and HCR to achieve in situ bipedal HCR for highcontrast fluorescence imaging and efficient gene silencing of cancer cells. Once encountering a target cancer cell, the aptamer of DNA hairpins recognized its receptor on the cell surface and i-motif folded into the DNA tetraplex structure, which caused the liberation of initiators and then triggered bipedal HCR.…”