scholarly journals Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins

2020 ◽  
Author(s):  
Georgina C. Gavins ◽  
Katharina Gröger ◽  
Michael D. Bartoschek ◽  
Philipp Wolf ◽  
Annette G. Beck-Sickinger ◽  
...  

AbstractDNA nanotechnology is an emerging field, which promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress in the area is the ability to create the nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction, which installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled coil peptide tag. Once installed the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness, and achieving reversible labelling by way of toehold mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled coil systems, with EGFR and ETBR, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of EGFR on CHO cells.

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Guan A. Wang ◽  
Xiaoyu Xie ◽  
Hayam Mansour ◽  
Fangfang Chen ◽  
Gabriela Matamoros ◽  
...  

Abstract Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.


2019 ◽  
Author(s):  
Leo Y.T. Chou ◽  
William M. Shih

AbstractCells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in cell-free gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic-acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDNA domains, and auxiliary nucleic-acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic-acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic-acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bio-analysis, and the design of adaptive materials.


2021 ◽  
Author(s):  
zhangwei lu ◽  
zhe li ◽  
Peng Zheng ◽  
bin jia ◽  
yutong liu ◽  
...  

Methods to efficiently and site-specifically conjugate proteins to nucleic acids could enable exciting application in bioanalytics and biotechnology. Here, we report the use of the strict protein ligase to covalently ligate a protein to a peptide nucleic acid (PNA). The rapid ligation requires only a short N-terminal GL dipeptide in target protein and a C-terminal NGL tripeptide in PNA. We demonstrate the versatility of this approach by conjugating three different types of proteins with a PNA strand. The biostable PNA strand then serves as a generic landing platform for nucleic acid hybridization. Lastly, we show the erasable imaging of EGFR on HEK293 cell membrane through toehold-mediated strand displacement. This work provides a controlled tool for precise conjugation of proteins with nucleic acids through an extremely small peptide linker and facilitates further study of membrane proteins.


Langmuir ◽  
2008 ◽  
Vol 24 (21) ◽  
pp. 12483-12488 ◽  
Author(s):  
Wui Siew Tan ◽  
Christina L. Lewis ◽  
Nicholas E. Horelik ◽  
Daniel C. Pregibon ◽  
Patrick S. Doyle ◽  
...  

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