Changbei Ma

Aptamer-based analysis of angiogenin by fluorescence anisotropy

Recognition and monitoring proteins in real time and in homogeneous solution has always been a difficult task. Here, we introduce a signal transduction strategy for quick protein recognition and real-time quantitative analysis in homogeneous solutions based on a high-affinity aptamer for protein angiogenin (Ang). The method takes advantage of the sensitive anisotropy signal change of fluorophore-labelled aptamer upon protein/aptamer binding. When the labelled aptamer is bound with its target protein Ang, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower. Therefore, increasing the amount of Ang results in a raised anisotropy value of the Ang/aptamer. By monitoring the anisotropy change, we are able to detect the binding events between the aptamer and Ang, and measure Ang concentration quantitatively in homogeneous solutions. This assay is highly selective, with a detection limit of 1 nM of Ang. The dissociation constant of the Ang/aptamer binding is determined in the nanomolar range and changes with increasing salt concentration. One can also use our assay to compare the binding affinities of different ligands for the target molecule. Ang in serum samples of malignant lung cancer was also detected. Efficient protein detection using aptamer-based fluorescence anisotropy measurements is expected to find wide applications in protein monitoring, cancer diagnosis, drug screening and other fields.

Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase.

We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.

Real‐Time Monitoring of Nucleic Acid Dephosphorylation by Using Molecular Beacons

Dephosphorylation of the 3’ termini of nucleic acids is important for cellular events, such as DNA replication, recombination, and repair of DNA damage induced by a variety of genotoxic agents, which include ionizing radiation, certain antineoplastic alkylating agents, bleomycin, and topoisomerase inhibitors. Dephosphorylation of the 3’ phosphate is usually catalyzed by various repair enzymes, which include the commonly used T4 polynucleotide kinase (T4 PNK). Since the identification of T4 PNK over 40 years ago, T4 PNK has exemplified a family of bifunctional enzymes with 5’-kinase and 3’phosphatase activities, and has become a molecular biology workhorse as well as an invaluable research tool in biology and bioengineering. Its 3’-phosphatase activity allows T4 PNK to dephosphorylate the 3’ termini of DNA molecules. This converts the DNA to a 3’hydroxyl substrate that is required for DNA polymerases and ligases, which play important roles in RNA and DNA repair. Traditionally, P radiolabeling, PAGE, and autoradiography are used to assay the dephosphorylation of nucleic acids. These methods are discontinuous, time consuming, laborious, require radiolabeled substrates, and cannot be used to analyze the rapid continuous dephosphorylation processes. Recently, Dobson and Allinson used fluorescent probes in combination with denaturing PAGE to study human PNK, but this method also suffers from being discontinuous. There are many advantages to using real-time assays instead of discontinuous ones, particularly in dissecting the molecular mechanisms of enzymes and their regulation. Continuous assays are relatively less laborious than discontinuous assays and they save time. Furthermore, they can be easily adjusted and optimized for high-throughput systems. Therefore, to investigate the dephosphorylation of nucleic acids in real time it is necessary to develop simple and sensitive methods that do not require isotopic labeling. In this paper, a novel method is described for real-time monitoring of the dephosphorylation process by using molecular beacon (MB) DNA probes. Since they were first reported in 1996, MBs have become a class of DNA probes that are widely used in chemistry, biology, biotechnology, and medical sciences for biomolecular recognition, due in part to their high sensitivity and excellent specificity. Previously, we developed techniques for real-time monitoring of nucleic-acid ligation and phosphorylation using MBs; these provided powerful systems for exploring the interactions between nucleic acids and proteins (enzymes). However, these methods are complex. Here, we describe the development of a new technique based on a polymerase-extension reaction to monitor nucleic acid dephosphorylation with MBs in real time. This approach is very simple, rapid, cost effective, and requires only one oligonucleotide. This method is schematically represented in Scheme 1.

Real-time monitoring of double-stranded DNA cleavage using molecular beacons.

Traditional methods to assay enzymatic cleavage of DNA are discontinuous, time-consuming and laborious. Here, we report a new approach for real-time monitoring of double-stranded DNA cleavage by restriction endonuclease based on nucleic acid ligation using molecular beacon. Upon cleavage of DNA, the cleavage product can be ligated by DNA ligase, which results in a fluorescence enhancement of the molecular beacon. This method permits real-time monitoring of DNA cleavage and makes it easy to characterize the activity of restriction endonuclease and to study the cleavage reaction kinetics.

Gold Nanoparticle Loaded Split-DNAzyme Probe for Amplified miRNA Detection in Living Cells

A new class of intracellular nanoprobe, termed AuNP loaded split-DNAzyme probe, was developed to sense miRNA in living cells. Briefly, it consists of an AuNP and substrates hybridized with two half of split DNAzymes. In the absence of target miRNA, the split DNAzymes form an inactive DNAzyme motif with their substrate through partial paring at the end of each strand, and the fluorescence is quenched. Inside the cells, the target miRNA binds with both of the two half of split DNAzymes, forming the active secondary structure in the catalytic cores, which can cleave the substrates, resulting in the rupture of the substrate and recovery of the fluorescence. Meanwhile, the target is released and binds to another inactive DNAzyme motif to drive another cycle of activation. During the cyclic process, a very small number of target miRNAs can initiate the cleavage of many fluorophore-labeled substrate strands from AuNP surface, providing an amplified fluorescent signal of the target miRNA and, thus, offering high detection sensitivity.

Using force spectroscopy analysis to improve the properties of the hairpin probe

The sensitivity of hairpin-probe-based fluorescence resonance energy transfer (FRET) analysis was sequence-dependent in detecting single base mismatches with different positions and identities. In this paper, the relationship between the sequence-dependent effect and the discrimination sensitivity of a single base mismatch was systematically investigated by fluorescence analysis and force spectroscopy analysis. The same hairpin probe was used. The uneven fluorescence analysis sensitivity was obviously influenced by the guanine-cytosine (GC) contents as well as the location of the mismatched base. However, we found that force spectroscopy analysis distinguished itself, displaying a high and even sensitivity in detecting differently mismatched targets. This could therefore be an alternative and novel way to minimize the sequence-dependent effect of the hairpin probe. The advantage offered by force spectroscopy analysis could mainly be attributed to the percentage of rupture force reduction, which could be directly and dramatically influenced by the percentage of secondary structure disruption contributed by each mismatched base pair, regardless of its location and identity. This yes-or-no detection mechanism should both contribute to a comprehensive understanding of the sensitivity source of different mutation analyses and extend the application range of hairpin probes.