Protein binding protection in combination with DNA masking for sensitive and reliable transcription factor detection.

Sensitive and reliable detection of transcription factors (TFs) is crucial for disease diagnosis and drug discovery. Herein, a protein binding protection in combination with DNA masking-based strategy is developed for sensitive and reliable detection of NF-κB p50. First, NF-κB p50 binds specifically to a hairpin probe to form a protein-DNA complex. Then, the formed complex protects the hairpin probe from hybridization with the masking strand and subsequently triggers the cascade signal amplification of strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Excess hairpin probes that do not bind NF-κB p50 are masked through hybridization with masking strands into stable duplexes, prohibiting the non-specific amplification and avoiding the risk of false positive signals. The signal results from the presence of NF-κB p50, ensuring the detection reliability. Through the cascade amplification specifically triggered by the target, the method can detect the purified recombinant NF-κB p50 down to 1.0 × 10-13 M. It is further employed for the NF-κB p50 inhibitor screening and analysis, which shows the potential in anti-NF-κB p50 drug discovery. Moreover, the method is applied in detection NF-κB p50 in cell nuclear extracts with a detection limit of 0.1 ng µL-1. The proposed strategy will provide a promising tool for sensitive and reliable assaying NF-κB p50 activity in biomedical study and disease diagnosis.

Colocalization recognition-activated cascade signal amplification strategy for ultrasensitive detection of transcription factors.

Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10-13M. Moreover, NF-κB p50 can be detected in as low as 0.21ngµL-1 HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases.

Label-free and enzyme-free detection of transcription factors with graphene oxide fluorescence switch-based multifunctional G-quadruplex-hairpin probe.

Transcription factors (TFs) play pivotal roles in the regulation of a variety of essential cellular processes and some of them have been recognized as potential diagnostic markers and therapeutic targets of some diseases. Sensitive and accurate detection of TFs is of great importance to better understanding their roles in gene regulation and evaluation of disease state. Here, we developed a simple, label-free and enzyme-free new fluorescent strategy for the detection of TFs by graphene oxide (GO) fluorescence switch-based multifunctional G-quadruplex-hairpin probe (MGHP). The MGHP possessed of three functions simultaneously, adsorbing onto GO with the loop part, binding to target with the stem part and serving as signal carrier with the terminal G-quadruplex. First, the MGHP was adsorbed quickly to GO. Next, the TF bound to the stem part of MGHP to form a huge target-MGHP complex, which led to desorption of the complex from GO. Finally, NMM was inserted into G-quadruplex in the complex to yield an enhanced fluorescence response. The GO used here, as a fluorescence switch, could quickly and efficiently quench the fluorescence of NMM inserted into the MGHP absorbed on the GO, guaranteeing a high signal-to-noise ratio. Sensitive detection of purified NF-κB p50 and HeLa cell nuclear extracts were achieved with detection limits of 0.2nM and 7.8ng/µL, respectively. Moreover, this proposed strategy could be used to screen inhibitors of NF-κB p50 activity. The strategy proposed here might offer a new potential approach for reliable quantification of TFs in clinical diagnostics and treatment research of some diseases.

Fok I cleavage-inhibition strategy for the specific and accurate detection of transcription factors.

Specific and accurate detection of transcription factors is critical for disease diagnosis and drug development. Here, we developed a novel and versatile fluorescent sensing strategy for specific and accurate detection of transcription factors based on the inhibition of endonuclease Fok I-catalyzed DNA cleavage reaction. A FAM-labeled double-stranded DNA probe (dsDNA probe) with transcription factor binding site and Fok I recognition site was designed for target recognition and signal transduction. With the binding of transcription factors, the dsDNA probes are protected from cleavage by Fok I. These protected dsDNA probes then cannot hybridize with the added BHQ-DNA, keeping the fluorescence of FAM in an on state. However, in the absence of targets, the dsDNA probes were cleaved to release the FAM-DNA, which subsequently hybridized with BHQ-DNA, resulting in the fluorescence of the FAM being quenched. With the Fok I-DNA interaction that can specifically recognize the transcription factor-DNA binding and accurately convert the detection of transcription factors to the detection of DNA, the proposed strategy realized the reliable detection of model target NF-κB p50 with a nanomolar detection limit. This strategy was also employed to detect the inhibition effect of oridonin, a known inhibitor of NF-κB. Furthermore, perfect recoveries were obtained when detecting the targets in HeLa cells lysate, demonstrating the feasibility of this strategy for transcription factor detection in biological samples.

Sensitive detection of transcription factors using an Ag(+)-stabilized self-assembly triplex DNA molecular switch.

Based on a Ag(+)-stabilized self-assembly triplex DNA molecular switch (Ag(+)-STDMS), a simple, enzyme-free and sensitive new fluorescent strategy for detection of transcription factors was developed, achieving high sensitivity towards purified targets and real biological samples.