| Cationic conjugated polymers (CCP) have attracted a great deal of interest because of their excellent optical property and solubility. Recent reported papers have shown that the molecular wire approach of CCP provides a universal method by which to obtain signal amplification relative to single molecule systems. Positively charged CCP can stay close to negatively charged biological macromolecules by electrostatic attraction. For the emission spectra of CCP has a good overlap with the absorption spectrum of fluorescein and green fluorescent protein, we can design fluorescence biosensor based on the fluorescence resonance energy transfer (FRET) between CCP and fluorescein or CCP and green fluorescent protein.(1) A sensitive fluorescence turn-on biosensing platform for protein kinases activities assay has been developed based on the FRET between a fluorophore labeled peptide and CCP. The CCP-based assay is based on electrostatic interaction between peptide and CCP. The FRET efficiency will change with the changing charges around the peptide after phosphorylation. The feasibility of this method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.3 mU μL-1). Based on its simple mechanism, this assay is also sensitive and robust enough to be applied to the evaluation of PKA inhibitor H-89. The IC50 value, the half maximal inhibitory concentration, is 40 nM. Furthermore, our method has excellent selectivity. CCP-based assay is sensitive, versatile, cost-effective and easy to operate, so, this method presents a promising candidate for kinase activity assay and inhibitor screening.(2) Poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in numerous biological processes, such as the DNA damage response and cell cycle regulation. Herein, a simple and convenient fluorescence strategy for PARP-1 activity assay has been developed based on the FRET between supercharged green fluorescent protein (scGFP) and CCP. After PARP-1 is activated by active DNA, PARP-1 itself will be modified with poly(ADP-ribose), possessing lots of electronegative phosphate groups. Then, poly(ADP-ribosyl)ated PARP-1 is mixed with the mixture of electropositive scGFP and CCP, scGFP/poly(ADP-ribosyl)ated PARP-1/CCP complexes would be formed quickly by electrostatic interaction, which would generate efficient FRET from CCP to scGFP. Using this approach, we detected PARP-1 activity with a low detection limit (1 nM). Furthermore, this assay was also used to the evaluation of PARP-1 inhibitors benzamide and AG14361 successfully with the ICso values of 51 μM and 42 nM, respectively. Moreover, our method is robust enough to be applied to detect PARP-1 activity in Hella cells lysates, blood serum, and human urine.(3) At present, many fluorescence methods are designed to study the interaction between protein and DNA, however, most of them need the fluorescent label of probes, which is fussy and costly. Herein, we have developed an unlabelled platform based on the FRET between CCP and scGFP to study the interaction between protein and DNA. When DNA is added to the mixture of CCP and scGFP, CCP/DNA/scGFP complex will be formed, efficient FRET will be generated from CCP to scGFP. However, when single stranded DNA binding protein is present, the generative process of the complex will be prevented, leading to the FRET ratio being decreased. This method has many advantages, such as unlabeled, sensitive, simple.(4) The recent studies of eukaryotic homologs of RecA suggest that the RecA-like homologous DNA pairing reaction is ubiquitous from bacteria to human. So, it is very important to study RecA/triplex DNA complex in the presence of the nearly non-hydrolyzable ATP analog ATPγS. Two kinds of unlabeled methods were developed based on CCP. The first method was based on the FRET between CCP and SYBR Green I, the second method was based on the FRET between CCP and scGFP. We can detect RecA protein with a low detection limit (2 nM). |
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