Razvan Nutiu

Signaling Aptamers for Monitoring Enzymatic Activity and for Inhibitor Screening

We examined fluorescence-signaling aptamers as real-time reporters for quantifying enzyme activities and identifying enzyme inhibitors. The conversion of adenosine 5’-monophosphate (AMP) into adenosine by alkaline phosphatase (ALP) was used as a model reaction, and a previously described structure-switching signaling DNA aptamer was employed as a model reporter. The signaling aptamer, which has a higher affinity for adenosine than for AMP, is able to generate a twoleg signaling profile. The first leg of the signal is produced upon addition of AMP and indicates the formation of a reactant±aptamer complex. The second leg is produced upon addition of ALP and reports the enzymatic conversion of the reactant into the product. Aptamers are single-stranded nucleic acids with ligand-binding capabilities that can be isolated from random-sequence nucleic acid pools. Several reports have been published that describe the use of fluorescence-based signaling aptamers or signaling ribozymes and deoxyribozymes to detect small molecules and proteins in solution. We recently described a strategy for preparing signaling DNA aptamers that function by a mechanism involving coupled structure switching and fluorescence dequenching. Signaling aptamers usually display signals of great magnitude (more than tenfold fluorescence increase upon target binding) and are capable of real-time reporting at low temperatures (15±37 8C). These properties led us to speculate that structure-switching signaling aptamers might be used as sensitive probes to report enzymemediated reactions in real time. Take a simple chemical reaction A!B as an example. For a signaling aptamer to be useful for reporting this chemical transformation in real time it must exhibit a different level of fluorescence in the presence of A than with B. If the signaling aptamer has a higher level of fluorescence in the presence of B, the A-to-B transformation can be monitored conveniently by following the increase in the fluorescence intensity of the signaling aptamer. If an enzyme mediates the chemical reaction, the presence of the fluorescent aptamer reporter should permit real-time monitoring of the activity of the enzyme, as well as screening for small-molecule inhibitors. We found previously that the structure-switching ATP reporter shown in Figure 1A is able to generate signals with different fluorescence intensities depending on whether adenosine or one of its 5’-phosphorylated analogues is used as the target.

Tau protein binds single-stranded DNA sequence specifically – the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures

Tau is a microtubule‐associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double‐stranded DNA (dsDNA) but not to bind single‐stranded DNA (ssDNA) [Cell. Mol. Life Sci. 2003, 60, 413–421]. Here, we prove that tau binds not only dsDNA but also ssDNA. This finding was facilitated by using two kinetic capillary electrophoresis methods: (i) non‐equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM); (ii) affinity‐mediated NECEEM. Using the new approach, we observed, for the first time, that tau could induce dissociation of strands in dsDNA by binding one of them in a sequence‐specific fashion. Moreover, we determined the equilibrium dissociation constants for all tau–DNA complexes studied.

Engineering DNA aptamers and DNA enzymes with fluorescence-signaling properties

Single-stranded DNA molecules with ligand-binding ability and catalytic function, referred to as DNA aptamers and DNA enzymes, respectively, are special DNA sequences isolated from random-sequence DNA libraries by “in vitro selection”. These two new classes of artificial DNA molecules have the potential of being used as molecular tools in a variety of innovative applications ranging from biosensing to gene regulation. Our laboratory is interested in engineering fluorescence-signaling DNA aptamers and DNA enzymes that can be widely exploited for detection-directed applications. In this article, we will first discuss our recent efforts on the rational design of a new class of signaling aptamers denoted “structure- switching signaling aptamers”, which report target binding by switching structures from DNA/DNA duplex to DNA/target complex. We will then describe the in vitro selection of fluorescence-signaling DNA enzymes that exhibit a synchronized catalysis-signaling capability by cleaving a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by closely spaced fluorophore-quencher pair. Potential utilities of these signaling DNA molecules will also be discussed.

Fluorescence-Signaling Nucleic Acid-Based Sensors

It is widely known that two single-stranded nucleic acids with complementary sequences have the inherent ability to form Watson-Crick duplex structures. The simplicity and sequence-specificity of duplex structure formation, the high chemical stability of a duplex, and the convenience of automated synthesis have made DNA oligonucleotides an ideal choice as probes for the detection of nucleic acids. Recently developed in vitro selection techniques permit creation of DNA and RNA “aptamers” that are capable of binding a wide variety of nonnucleic acid targets with high affinity and specificity. Aptamers have considerably broadened the utility of nucleic acids as probes for detection of biological and nonbiological targets. In vitro selection also allows generation of artificial ribozymes (catalytic RNAs) and deoxyribozymes (catalytic DNAs) with desirable functions. Aptamers, ribozymes, and deoxyribozymes have become increasingly valuable molecular tools in the form of switches and sensors. Unfortunately, binding or catalytic actions by these switches and sensors do not usually lead to an easily detectable signal, and the lack of a facile reporting method could substantially reduce their value. To facilitate the exploitation of nucleic acid switches and sensors for detection-related applications, many recent studies have explored fluorescence signaling as a convenient approach for the reporting of binding and catalytic events. This chapter is devoted to the discussion of these efforts. The reporter molecules to be described include molecular beacons, signaling aptamers, and signaling ribozymes and deoxyribozymes.

Immobilized DNA biosensor based on evanescent wave long-period fiber gratings

A DNA sensor using a long-period grating (LPG) is proposed for sensitive detection of interaction between the single strand DNA (ssDNA) and its target. The ssDNA can be immobilized on the surface of the LPG, and detection of the specific interaction between the ssDNA and its target is monitored optically. This sensor operates on the evanescent field interacting with the ssDNA immobilized over the grating region. The reaction between the ssDNA and its target alter the effective refractive index of the cladding mode and thus change the LPG transmission spectrum, which can be monitored in real time. This technology should permit a label-free detection of DNA/DNA, DNA/proteins or DNA/small molecules interactions. In this paper, for the first time, dependence of the LPG transmission spectrum on the characteristic of the LPG and the property of the immobilized ssDNA are studied both theoretically and experimentally. Experimental demonstration of the immobilization of ssDNA on LPG and its real-time effect on the transmission spectrum are reported.

Characterization of the conformational changes of a tripartite molecular beacon

We have investigated the kinetics of the conformational changes of a new type of molecular beacons called tripartite molecular beacons. The rate constants corresponding to the opening and closing of the beacons have been obtained from fluorescence correlation spectroscopy experiments. We found that both rate constants are larger for the tripartite molecular beacon than for the corresponding molecular beacon. This paper outlines the importance of using very low excitation intensities for this type of measurements, and of considering the fact that the beacon still emits a residual background fluorescence in its closed form. We also report on the exploration of several strategies to improve the precision of the measurements by increasing the characteristic time associated with the diffusion of the beacons so that it would not be confused with the relaxation time associated with the conformational changes.

The Use of Functional Nucleic Acids in Solid-Phase Fluorimetric Assays

The past 15 years have seen a revolution in the area of functional nucleic acid (FNA) research since the demonstration that single-stranded RNA and DNA species can be used for both ligand binding and catalysis. An emerging area of application for such species is in the development of solid-phase fluorimetric assays for biosensing, proteomics, and drug screening purposes. In this chapter, the methods for immobilization of functional nucleic acids are briefly reviewed, with emphasis on emerging technologies such as sol-gel encapsulation. Methods for generating fluorescence signals from aptamers and nucleic acid enzymes are then described, and the use of such species in solid-phase fluorimetric assays is then discussed. Unique features of sol-gel based materials for the development of solid-phase assays are highlighted, and some emerging applications of immobilized FNA species are discussed.