Yan Jin

Homogeneous Selecting of a Quadruplex-Binding Ligand-Based Gold Nanoparticle Fluorescence Resonance Energy Transfer Assay

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. There is considerable interest in the design of ligands that target G-quadruplex DNA because of their potential anticancer activity. We designed a fluorescence resonance energy transfer (FRET) system to identify molecules that stabilize G-quadruplexes in a homogeneous medium using unmodified gold nanoparticles (GNPs) as a fluorescence quencher. The assay exploits the different adsorption abilities of GNPs for single-stranded DNA and double-stranded DNA. Fluorescein-tagged probe DNA adsorbed onto the surface of GNPs can quench the fluorescence of a DNA probe. Intramolecular folding of an oligonucleotide of the human telomeric sequence into a G-quadruplex structure led to fluorescence enhancement in the presence of quadruplex-binding ligands. G-quadruplex formation, induced by specific binding of GDNA ligands, was investigated by CD measurements. Melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the absorbance at 295 nm. Two series of natural drugs were studied, and flavonoids were shown to increase the melting temperature of the G-quadruplex. This increase in the Tm value was well-correlated with an increase in FRET efficiency. The combined data from fluorescence measurements and melting experiments indicate that the FRET approach offers a simple, sensitive, and effective method to identify ligands with potential anticancer activity.

Label-free fluorescent detection of thrombin using G-quadruplex-based DNAzyme as sensing platform

We report herein a label-free and sensitive fluorescent method for detection of thrombin using a G-quadruplex-based DNAzyme as the sensing platform. The thrombin-binding aptamer (TBA) is able to bind hemin to form the G-quadruplex-based DNAzyme, and thrombin can significantly enhance the activity of the G-quadruplex-based DNAzyme. The G-quadruplex-based DNAzyme is found to effectively catalyze the H(2)O(2)-mediated oxidation of thiamine, giving rise to fluorescence emission. This allows us to utilize the H(2)O(2)-thiamine fluorescent system for the quantitative analysis of thrombin. The assay shows a linear toward thrombin concentration in the range of 0.01-0.12 nM. The present limit of detection for thrombin is 1 pM, and the sensitivity for analyzing thrombin is improved by about 10,000-fold as compared with the reported colorimetric counterpart. The work also demonstrates that thiamine is an excellent substrate for the fluorescence assay using the G-quadruplex-based DNAzyme as the sensing platform.

Label-free protein recognition using aptamer-based fluorescence assay

Monitoring proteins in real time and in homogeneous solution without using external labels has always been a difficult task. In this paper, we have developed a label-free method for the ultrasensitive detection of thrombin in homogeneous solutions. High-affinity thrombin-binding aptamer (TBA) used as molecular recognition probe, and fluorophore, crystal violet (CV), was chose as fluorescence signal probe. The fluorescence of CV enhanced significantly when the free CV solution was mixed with single-stranded TBA. In the presence of human thrombin, the fluorescence of CV decreased after the specific interaction between TBA and thrombin. Using the fluorescence change, we are able to selectively detect the thrombin in homogeneous solutions. The conformation transformation was investigated by circular dichroism (CD) spectra measurements. Our method has been shown to be simple and effective without any labelling of the probe or of the target, and this procedure poses minimum effects on the binding properties of the proteins. This assay is highly selective and ultrasensitive. Under the optimum conditions, the method exhibits a dynamic response range from 2 x 10(-11) to 2 x 10(-9) M with a detection limit of 8 x 10(-12) M.

Highly specific fluorescence detection of T4 polynucleotide kinase activity via photo-induced electron transfer

Sensitive and reliable study of the activity of polynucleotide kinase (PNK) and its potential inhibitors is of great importance for biochemical interaction related to DNA phosphorylation as well as development of kinase-targeted drug discovery. To achieve facile and reliable detection of PNK activity, we report here a novel fluorescence method for PNK assay based on a combination of exonuclease cleavage reaction and photo-induced electron transfer (PIET) by using T4 PNK as a model target. The fluorescence of 3-carboxyfluorescein-labeled DNA probe (FDNA) is effectively quenched by deoxyguanosines at the 5 end of its complementary DNA (cDNA) due to an effective PIET between deoxyguanosines and fluorophore. Whereas FDNA/cDNA hybrid is phosphorylated by PNK and then immediately cleaved by lambda exonuclease (λ exo), fluorescence is greatly restored due to the break of PIET. This homogeneous PNK activity assay does not require a complex design by taking advantage of the quenching ability of deoxyguanosines, making the proposed strategy facile and cost-effective. The activity of PNK can be sensitively detected in the range of 0.005 to 10 U mL(-1) with a detection limit of 2.1×10(-3) U mL(-1). Research on inhibition efficiency of different inhibitors demonstrated that it can be explored to evaluate inhibition capacity of inhibitors. The application for detection of PNK activity in complex matrix achieved satisfactory results. Therefore, this PIET strategy opens a promising avenue for studying T4 PNK activity as well as evaluating PNK inhibitors, which is of great importance for discovering kinase-targeted drugs.

Highly sensitive fluorescence assay of T4 polynucleotide kinase activity and inhibition via enzyme-assisted signal amplification

DNA phosphorylation catalyzed by polynucleotide kinase (PNK) is an indispensable process in the repair, replication, and recombination of nucleic acids. Here, an enzyme-assisted amplification strategy was developed for the ultrasensitive monitoring activity and inhibition of T4 PNK. A hairpin oligonucleotide (hpDNA) was designed as a probe whose stem can be degraded from the 5 to 3 direction by lambda exonuclease (λ exo) when its 5 end is phosphorylated by PNK. So, the 3 stem and loop part of hpDNA was released as an initiator strand to open a molecular beacon (MB) that was designed as a fluorescence reporter, leading to a fluorescence restoration. Then, the initiator strand was released again by the nicking endonuclease (Nt.BbvCI) to hybridize with another MB, resulting in a cyclic reaction and accumulation of fluorescence signal. Based on enzyme-assisted amplification, PNK activity can be sensitively and rapidly detected with a detection limit of 1.0×10(-4)U/ml, which is superior to those of most existing approaches. Furthermore, the application of the proposed strategy for screening PNK inhibitors also demonstrated satisfactory results. Therefore, it provided a promising platform for monitoring activity and inhibition of PNK as well as for studying the activity of other nucleases.

Homogeneous and ultrasensitive detection of telomerase activity via gold nanorod-based fluorescence resonance energy transfer

As a universal biomarker, telomerase is one of the promising targets for cancer diagnosis and therapy. Therefore, it is meaningful to develop facile, robust and sensitive methods for evaluation of telomerase activity. Herein, combined with fluorescence resonance energy transfer (FRET), we creatively designed a gold nanorod (GNR)-based FRET method to detect telomerase activity from cell extracts. As the signal probe, carboxyfluorescein-modified DNA probes (F-DNA) hold negative electricity. The electrostatic interaction between F-DNA and positively charged GNRs makes F-DNA close to GNRs, which leads to weak FRET between the F-DNA and GNRs. In the presence of telomerase, a telomerase substrate (TS) primer was elongated to form a long single-stranded DNA, which could hybridize with numerous F-DNA to form long dsDNAs. The strengthened electrostatic interaction leads to a more efficient FRET between GNRs and dsDNA. Therefore, the amplified fluorescence quenching efficiency can greatly improve the sensitivity. The telomerase activity in the HeLa extracts equivalent to 1 cell was detected sensitively without the polymerase chain reaction (PCR) amplification and enzyme auxiliary signal amplification. Moreover, this facile protocol can be used to distinguish tumor cells from normal cells and study the inhibition effect of telomerase inhibitors, which shows its potential application value in clinical diagnosis and drug screening.