Sensitive and Label-Free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-Assisted Signal Amplification

Abstract

Transcription factors (TFs) regulate gene expression by binding to regulatory regions, and their dysregulation is involved in numerous diseases. Thus, they are therapeutic targets and potential diagnostic markers. However, widely used methods for TFs detection are either cumbersome or costly. Herein, we first applied DNA-Ag nanoclusters molecular beacons (AgMBs) in TFs analysis and designed an assay based on the switchable fluorescence of AgMBs. In the absence of TFs, a single-stranded DNA functioned as a reporter is released from a double-stranded DNA probe (referred as dsTFs probe) under exonuclease III (Exo III) digestion. Then, the reporter triggers downstream Exo III-assisted signal amplification by continuously consuming the guanine-rich enhancer sequences in AgMBs, resulting in significant fluorescent decrease eventually. Conversely, the presence of TFs protects the dsTFs probe from digestion and blocks the downstream reaction to keep a highly fluorescent state. To testify this rationale, we utilized nuclear factor-kappa B p50 (NF-κB p50) as a model TFs. Owing to the amplification strategy, this method exhibited high sensitivity toward NF-κB p50 with a limit of detection of 10 pM, and a broad linear range from 30 pM to 1.5 nM. Furthermore, this method could detect multiple TFs in human colon cancer DLD-1 cells and reflect the variation in their cellular levels after stimulation. Finally, by conducting an inhibition assay we revealed the potential of this method for screening TFs-targeted drugs and calculating the IC of corresponding inhibitors.

Tuning DNA–nanoparticle conjugate properties allows modulation of nuclease activity

“…Exonuclease III (ExoIII) was chosen due to its widespread use in biological sensing applications, and in particular, systems co-employing nanomaterials and enzymes. [32][33][34][35] ExoIII is a nonprocessive enzyme that acts on double-stranded DNA (dsDNA), where it cleaves mononucleotides from the 3′-hydroxyl terminus of one strand, leaving the other strand intact. 36,37 The preferred substrates are blunt or recessed 3′-termini of dsDNA, but the enzyme can also act at nicks or abasic sites.…”

Luminescent metal nanoclusters: Biosensing strategies and bioimaging applications

“…[129] Based on this interesting finding, some sensing platforms have been developed to detect various analytes. [130–132] The sensing mechanism is mainly based on the analyte‐induced approaching/separation of the guanine‐rich DNA to/from the Ag NCs. For example, a FL “turn‐on” method for thrombin detection was developed according to the above‐mentioned sensing mechanism.…”

Signal-Amplified Detection of the Tumor Biomarker FEN1 Based on Cleavage-Induced Ligation of a Dumbbell DNA Probe and Rolling Circle Amplification

“…The application of functional nucleic acids and DNA nanotechnology is currently an attractive topic in chemistry and life sciences. − Based on the sequence programmability of DNA, its conformation and function can be rationally designed for recognition, , catalysis, and computing. − Rationally designed DNA probes can be used as recognition elements to recognize analytes ranging from metal ions to microorganisms. − In particular, through their capacity to produce large quantities of nucleic acid fragments under facile conditions, isothermal amplifications such as rolling circle amplification (RCA), , exponential isothermal amplification (EXPAR), and recombinase polymerase amplification (RPA) have significantly improved the performance of nucleic acid-involved biosensors. To the best of our knowledge, biosensors targeting FEN1 that integrate rationally designed DNA probes and signal amplification have not been reported.…”