Recombinant Fusion Streptavidin as a Scaffold for DNA Nanotetrads for Nucleic Acid Delivery and Telomerase Activity Imaging in Living Cells.
Zhi-Mei HuangMei-Ya LinChong-Hua ZhangZhen-Kun WuRu-Qin YuJian-Hui JiangPublished in: Analytical chemistry (2019)
Efficient platforms for intracellular delivery of nucleic acids are essential for biomedical imaging and gene regulation. We develop a recombinant fusion streptavidin as a novel protein scaffold for DNA nanotetrads for highly efficient nucleic acid delivery and telomerase activity imaging in living cells via cross-linking hybridization chain reaction (cHCR). The recombinant streptavidin protein is designed to fuse with multiple SV40 NLS (nuclear localization signal) and NES (nuclear export signal) domains and prepared through Escherichia coli expression. The recombinant NLS-SA protein allows facile assembly with four biotinylated DNA probes via high-affinity noncovalent interactions, forming a well-defined DNA tetrad nanostructure. The DNA nanotetrads are demonstrated to confer efficient cytosolic delivery of nucleic acid via a caveolar mediated endocytosis pathway, allowing efficient escape from lysosomal degradation. Moreover, the nanotetrads enable efficient cHCR assembly in response to telomerase in vitro and in cellulo, affording ultrasensitive detection and spatially resolved imaging for telomerase with a detection limit as low as 90 HeLa cells/mL. The fluorescence brightness obtained in live cell imaging is found to be dynamically correlated to telomerase activity and the inhibitor concentrations. Therefore, the proposed strategy may provide a highly efficient platform for nucleic acid delivery and imaging of biomarkers in living cells.
Keyphrases
- nucleic acid
- living cells
- single molecule
- highly efficient
- high resolution
- fluorescent probe
- cell free
- escherichia coli
- fluorescence imaging
- pseudomonas aeruginosa
- signaling pathway
- gold nanoparticles
- binding protein
- high throughput
- cell death
- induced apoptosis
- cell proliferation
- amino acid
- cystic fibrosis
- mass spectrometry
- cell cycle arrest
- loop mediated isothermal amplification
- long non coding rna