Veli Cengiz Özalp

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Adenosine 5′-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F0F1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

Aptamer-based switchable nanovalves for stimuli-responsive drug delivery.

Drug-delivery systems with the capability to respond to a given stimulus can dramatically improve therapeutic efficacy. The development of such systems currently relies on responsive polymeric materials that need to be chosen specifically for each application. However, this strategy is limited by the availability of such polymeric systems and thus every new application is a new challenge. One different approach in smart drug-delivery systems has achieved on-demand drug delivery by employing molecular machines as nanovalves. Redox activation; competitive binding; pH, temperatureor light-initiated, and biological triggers have been used for controlled release. In this report, we demonstrate that the molecular recognition capacity of aptamers can be used to design reversible, stimuli-responsive polymeric hybrid systems, such that any polymeric nanocarrier can be functionalized to respond to potentially any kind of trigger molecule. The incorporation of aptamers as nanovalves would bring about a generic system in drug delivery, since aptamers can be selected to a wide variety of targets from ions to whole cells in vitro. Aptamers are nucleic acids with biorecognition properties that can go along with a switching of their structure. These two properties of aptamers can be combined to improve the performance of drug-delivery nanocarriers in two aspects: 1) better control of the release kinetics and 2) the range of potential trigger stimuli can be extended. Nevertheless, the potential of aptamers for creating stimulus-responsive materials through pore-gating has been limited to only two recent reports. Abelow et al. modified 20 and 65 nm radii glass nanopores with cocaine aptamers in order to control ion transport. Zhu et al. showed that controlled release can be achieved in mesoporous silica nanoparticles with a snaptop design using ATP-binding aptamers attached to gold nanoparticles as a responsive molecular gate. Herein we report on a switchable nanovalve directly using an ATPbinding aptamer sequence (Figure 1) that was covalently attached onto the surface of nanoparticles. This system was found to be highly reversible, which means that partial delivery of drug molecules can be controlled better instead of a sudden release as is the case in snap-top designs. Our drug-delivery system was centred on the conversion of an aptamer sequence into a molecular-beacon-type hair-

Aptamers Embedded in Polyacrylamide Nanoparticles: A Tool for in Vivo Metabolite Sensing

We describe a new type of aptamer-based optical nanosensor which uses the embedding of target responsive oligonucleotides in porous polyacrylamide nanoparticles to eliminate nuclease instability. The latter is a common problem in the use of aptamer sensors in biological environments. These aptamers embedded in nanoparticles (AptaNPs) are proposed as a tool in real-time metabolite measurements in living cells. The AptaNPs comprise 30 nm polyacrylamide nanoparticles, prepared by inverse microemulsion polymerization, which contain water-soluble aptamer switch probes (ASPs) trapped in the porous matrix of the nanoparticles. The matrix acts as a molecular fence allowing rapid diffusion of small metabolites into the particles to interact with the aptamer molecules, but at the same time it retains the larger aptamer molecules inside the nanoparticles providing protection against intracellular degradation. We tested the ability of the AptaNPs to measure the adenine-nucleotide content in yeast cells. Our results successfully demonstrate the potential for monitoring any metabolite of interest in living cells by selecting specific aptamers and embedding them in nanoparticles.

Single-Step Purification of Recombinant Thermus aquaticus DNA Polymerase Using DNA-Aptamer Immobilized Novel Affinity Magnetic Beads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq‐polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5′‐thiol labeled DNA‐aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq‐polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq‐polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq‐polymerase from crude extracts was also evaluated. For this case, the enzyme “recombinant Taq‐DNA polymerase” was cloned and expressed using an Amersham E. coli GST‐Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag‐polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one‐step purification protocol. Apparently, the system was found highly effective as one step for the low‐cost purification of Taq‐polymerase in bacterial crude extract.

Aptamer-Gated Nanoparticles for Smart Drug Delivery

Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas. Moreover, nucleic acids are recognized as an attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. A active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles. In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained. The main focus will be on aptamer-incorporated nanoparticle systems for drug delivery purposes in order to assess the future potential of aptamers in the therapeutic area. Special emphasis will be given to the very recent progress in controlled drug release based on molecular gating achieved with aptamers.

Pathogen detection in complex samples by quartz crystal microbalance sensor coupled to aptamer functionalized core–shell type magnetic separation

A quartz crystal microbalance sensor (QCM) was developed for sensitive and specific detection of Salmonella enterica serovar typhimurium cells in food samples by integrating a magnetic bead purification system. Although many sensor formats based on bioaffinity agents have been developed for sensitive and specific detection of bacterial cells, the development of robust sensor applications for food samples remained a challenging issue. A viable strategy would be to integrate QCM to a pre-purification system. Here, we report a novel and sensitive high throughput strategy which combines an aptamer-based magnetic separation system for rapid enrichment of target pathogens and a QCM analysis for specific and real-time monitoring. As a proof-of-concept study, the integration of Salmonella binding aptamer immobilized magnetic beads to the aptamer-based QCM system was reported in order to develop a method for selective detection of Salmonella. Since our magnetic separation system can efficiently capture cells in a relatively short processing time (less than 10 min), feeding captured bacteria to a QCM flow cell system showed specific detection of Salmonella cells at 100 CFU mL(-1) from model food sample (i.e., milk). Subsequent treatment of the QCM crystal surface with NaOH solution regenerated the aptamer-sensor allowing each crystal to be used several times.

Targeting cancer cells with controlled release nanocapsules based on a single aptamer

Molecular gates have received considerable attention as drug delivery systems. More recently, aptamer-based gates showed great potential in overcoming major challenges associated with drug delivery by means of nanocapsules. Based on a switchable aptamer nanovalves approach, we herein report the first demonstration of an engineered single molecular gate that directs nanoparticles to cancer cells and subsequently delivers the payload in a controllable fashion.

Acoustic quantification of ATP using a quartz crystal microbalance with dissipation

A quartz crystal microbalance with a dissipation monitoring (QCM-D) sensor was developed for highly sensitive and specific detection of adenosine-5′-triphosphate (ATP) by using an aptamer. The binding of ATP molecules on the aptamer films could be calculated as accurate mass changes using multiple frequency and dissipation measurements. The detection is achieved by calculating the mass changes from conformational rearrangements of the sensor surface upon interaction with the target. The sensor was demonstrated to respond to changes in ATP concentrations in real time suitable for continuous monitoring applications. This sensor showed excellent selectivity toward ATP compared with other chemically similar nucleotide GTP. The feasibility of the sensor was demonstrated by analyzing ATP concentrations in cell culture media with serum. The maximum frequency change was about −2 Hz after injection of 500 μM ATP. The affinity constant of the aptamer was determined to be 49 ± 7.59 μM. The proposed sensor can extend the application of the QCM-D system in medical diagnosis, and could be adopted for the detection of other small molecules with the use of specific aptamers.

Antimicrobial aptamers for detection and inhibition of microbial pathogen growth

Discovery of alternative sources of antimicrobial agents are essential in the ongoing battle against microbial pathogens. Legislative and scientific challenges considerably hinder the discovery and use of new antimicrobial drugs, and new approaches are in urgent demand. On the other hand, rapid, specific and sensitive detection of airborne pathogens is becoming increasingly critical for public health. In this respect affinity oligonucleotides, aptamers, provide unique opportunities for the development of nanotechnological solutions for such medical applications. In recent years, aptamers specifically recognizing microbial cells and viruses showed great potential in a range of analytical and therapeutic applications. This article describes the significant advances in the development of aptamers targeting specific pathogens. Therapeutic application of aptamers as neutralizing agents demonstrates great potential as a future source of antimicrobial agent.

An Aptamer-Based Nanobiosensor for Real-Time Measurements of ATP Dynamics

ATP (adenosine-5’-triphosphate) is central to cellular metabolism as a multifunctional intermediate in cellular processes. Many cellular reactions depend on the hydrolysis of ATP, for example, ion transport across membranes, cell motility, and biosynthetic reactions. In cell-signaling cascades, protein kinases transfer a phosphate group from ATP to key regulatory proteins that serve in the control of cell metabolism, growth, and differentiation. Thus, assays that monitor ATP concentration in both bioassays and in cellular environments have wide applications in biochemical and biomedical research. Despite the important role played by ATP in biological systems, only a handful of sensors that can monitor ATP in real-time exist and several of them have limitations in intracellular usage. In most ATP-utilizing metabolic reactions, ATP is converted to ADP (adenosine-5’-diphosphate) and only to a lesser extent to AMP (adenosine-5’-monophosphate). ADP is recycled to ATP through phosphorylation reactions. Therefore, the challenge in developing a specific ATP sensor is to produce one that can differentiate between ATP and ADP. However, only a few such sensors have been reported. Furthermore, most of the available sensors suffer from the fact that they have high affinity for ATP and therefore will only have a limited use in many bioassays and in cellular environments where the ATP concentration is in the millimolar range. Recently, four kinds of biosensor were reported for real-time monitoring of ATP : 1) A sandwich stacking of pyrene–adenine–pyrene was designed to measure ATP concentrations in HeLa cells, 2) a protein-based biosensor for ADP was developed by engineering the bacterial actin ParM to measure ATPase and kinase activity, 3) two genetically encoded biosensors have been reported: i : the e-subunit of F0F1-ATP synthase was sandwiched between two fluorescent proteins to develop a FRET-type indicator of ATP and ii : the bacterial regulatory protein, GlnK1 was combined with GFP to measure ATP:ADP ratios, 4) enzyme activity-coupled sensors in the form of a luciferase–carbon nanotube ATP sensor. An alternative approach to designing an ATP sensor is to develop a functional aptamer sensor for simple and accurate real-time ATP detection in bioassays and in cellular environments. Aptamers are single-stranded nucleic acids with specific affinity for their targets. In vitro selection can provide an aptamer for almost any kind of target with preselected affinity that thus may be considered an attractive diagnostic and sensing molecule. Moreover, once an aptamer has been selected, it can be chemically synthesized and used directly as a probe— unlike genetically encoded sensors, which often require molecular cloning and cellular expression procedures. In developing a nanobiosensor specific for ATP we selected a new DNA aptamer sequence that preferentially binds ATP. A selection method based on Flu-Mag SELEX was designed to obtain ATP-specific enrichment through a total of 14 cycles of negative–positive selections and counter selections. The selected aptamer sequence was converted to a switch probe based on a design by Tang et al. The probe is an intramolecular signal-transduction aptamer design that consists of the aptamer sequence and a short-stem DNA sequence complementary to the other end of the aptamer sequence and separated by a PEG linker. A fluorophore (Texas Red) and quencher (Black Hole 2) are covalently attached at the two ends of the sequence. The interaction of ATP with the aptamer sequence disrupts the hairpin structure and leads to rearrangement of the structure separating the quencher and fluorophore from each other. This leads to an increase in fluorescence that depends on the concentration of ATP (Figure 1 A). The instant and reversible response can be obtained upon addition of ATP to solutions containing the probe; this indicates the usefulness of the new sensor in monitoring biological reactions involving ATP (Figure 1 B). An important selection criterion was to identify aptamers with affinity in the millimolar range given that many enzymes catalyzing ATP consumption have KM values for ATP in this range. The intracellular ATP concentration is also in this range. Thus, the selection procedure used here was designed to seek a specific ATP aptamer with a dissociation constant of 1– 10 mm by following a series of negative selections to eliminate ADP and AMP binding. One of the selected sequences was further characterized by surface plasmon resonance analysis, which indicated a Kd of 0.7 mm (Figure S2 in the Supporting Information). In the early years after the discovery of aptamers, RNA and DNA aptamers that target ATP were selected. Both aptamers were selective for ATP over other nucleotides (GTP, CTP, and UTP), but did not discriminate well between adenine nucleotides. The issue of selection on the phosphate moiety was later addressed by selecting a second RNA aptamer for ATP by applying selective pressure for the triphosphate group of the molecule as well as an RNA aptamer for ADP. However, there are difficulties in designing an aptamer-based sensor by using the existing aptamer sequences. RNA aptamers have high affinities and generally are less desirable in rational designs of sensors because of nuclease susceptibility. Moreover, chemical modifications can be challenging during the synthesis of RNA oligomers. While DNA aptamers are physically more [a] Prof. V. C. zalp, L. J. Nielsen, Prof. L. F. Olsen Department of Biochemistry and Molecular Biology University of Southern Denmark Campusvej 55, 5230 Odense (Denmark) Fax: (+ 45) 65502467 E-mail : cengiz_ozalp@ehu.es Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000500.