Hypersensitive detection of transcription factors by multiple amplification strategy based on molecular beacon

Highlights

• A fluorescence amplification strategy for the detection of NF-κB p65 was established.

• Multiple amplification was realized by combining molecular beacon and exonuclease.

• Detection of NF-κB p65 in nuclear extract.

• This method can change the specific sequence to detect other transcription factors.

Abstract

Detection and quantification of specific targets at ultralow concentrations are crucial in biotechnological applications and biomedical diagnostics. However, traditional detection methods are time-consuming and complex. In this study, a simple, sensitive, and specific fluorescence-based strategy to detect transcription factors (TFs) was developed by combining molecular beacon (MB) with exonuclease III (Exo III). The strategy was validated using nuclear factor-kappa B (NF-κB) p65 as a model case. In this assay, the TF concentration is determined by estimating the concentration of the nucleic acid released through the reactions of TFs with DNA-1/DNA-2 duplex probe and Exo III; thus, this method can overcome the limitations of the traditional methods. The nucleic acid is then cyclically amplified in the presence of MB and Exo III, and the sequence fragments containing the fluorescent group are released. This method is sensitive, expandable, and suitable for the direct detection of TFs in crude nuclear extracts of cancer cells. Furthermore, it can provide key test data for studies of coronavirus disease 2019-induced acute respiratory distress syndrome and related research.

Introduction

Transcription factors (TFs) contain one or more DNA-binding domains that bind to specific DNA sequences to regulate gene transcription. TFs can function as natural switches to convert physical and chemical signals, such as temperature change, light, drug concentration, and redox state, into transcriptional changes. Therefore, TFs play a central role in the pathway and network of gene expression regulation. The imbalance of TF signals is related to cancer, developmental disorders, inflammation, and autoimmunity [1]. Nuclear factor-kappa B (NF-κB) is an important inducible TF that exists in the cytoplasm together with its inhibitor IκB [2]. Various stimuli induce the phosphorylation, ubiquitination, and proteasome degradation of IκB in cells, thereby leading to the release and transfer of NF-κB into the nucleus. In the nucleus, NF-κB promotes the transcription of pro-inflammatory cytokines and chemokines, stress-response proteins, and anti-apoptotic proteins. Thus, NF-κB has an important role in inflammation-related diseases, such as acute respiratory distress syndrome (ARDS) [3], [4], [5], [6]. However, the anatomical pathological features of patients with coronavirus disease 2019 (COVID-19) suggest the occurrence of ARDS, which has been verified in the clinic [7], [8], [9]. The rapid and sensitive detection of NF-κB in the nucleus is necessary for the diagnosis of COVID-19. NF-κB has gradually become a key target for medical diagnosis and drug development [1].

The role of TFs has spurred interest in the technology of TF detection. Current methods for detecting TFs include electrochemical determination, radioactive electrophoresis mobility shift assay (EMSA), and ELISA [10], [11], [12], [13]. However, these methods have certain drawbacks. Electrochemical strategies consume a large amount of sample, and the complex electrode modification process is usually time-consuming and arduous. Although radioactive EMSA is simple and sensible, the application of radioisotopes poses safety risks to researchers and the surrounding environment. The scope of application of ELISA is too narrow because of the requirement of specific antibodies against TFs. In addition, TFs in solution can be directly detected through typical fluorescence amplification strategies [14], [15], [16], [17]. TFs can promote the formation of DNA double strands, resulting in strong fluorescence resonance energy transfer (FRET). However, the binding of proteins to DNA may produce steric hindrance, resulting in a low FRET signal [18]. Therefore, new detection methods for TFs need to be developed.

Molecular beacon (MB) is a new type of neck-ring-structured probe. An MB is a fluorescence-labelled oligonucleotide chain generally composed of an annular region, stem region, fluorescent group, and quencher. Owing to their high sensitivity, non-toxicity, and high specificity, MBs have been widely used for various purposes, including RNA and DNA detection, biosensing, and protein detection [19], [20], [21], [22], [23]. When MB exists alone, the fluorescent group and quencher on the stem are close to each other, and the fluorescence of the fluorescent group is quenched [24], [25]. In presence of the target nucleic acid in the solution, a conformational change is induced in MB during hybridisation [26], [27], and the fluorescent group and quencher separate from each other, thereby restoring fluorescence. Therefore, an increase in fluorescence intensity indicates the presence of a target nucleic acid in the solution [28], [29]. For protein detection, MBs can be used to detect nucleic acids released through the reaction of target proteins with DNA probes and tool enzymes; thus, the positive detection of nucleic acids indicates the presence of the target protein [27], [30]. Several isothermal signal amplification strategies based on MBs have been applied for protein detection [28], [31]. Unfortunately, these methods are disadvantageous owing to their low sensitivity, time-consuming nature, and complicated design. Therefore, an economical, sensitive, rapid, and highly specific method for the detection of TFs is required.

In this study, we developed a simple and sensitive method for the detection of TFs. This method combines DNA–protein binding, exonuclease III (Exo III), and isothermal exponential amplification. The MB-dependent amplification fluorescence analysis technique can successfully achieve multiple signal amplification. Compared with other methods, the strategy features higher specificity and lower detection limit. Furthermore, the method can be directly applied to detect TFs in nuclear extracts of cancer cells.