Specific Molecular Binding Research Articles

An optical thin film assay incorporating rhinovirus protease inhibitors as detector reagents

Human rhinoviruses (HRV) are the main cause of the common cold. Viral replication utilizes the activity of the HRV3C protease (3CP) enzyme [Antimicrob. Agents Chemother. 43 (1999) 2444; Antimicrob. Agents Chemother. 44 (2000) 1236]. Therefore, 3CP is an attractive target for antiviral drug development, and a new class of orally bioavailable irreversible 3CP inhibitors has been designed [J. Med. Chem. 45 (2002) 1607]. We have used related inhibitors to develop a rapid test for rhinovirus. The optical immuno assay (OIA ®) thin film detection technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. The purpose of this study was to develop a rapid assay for the determination of 3CP combining the Thermo Electron Bio Star ® OIA technology and the newly designed inhibitor compounds. The advantage of this assay was in its approach, in which therapeutic and diagnostic targets are the same thus allowing patients with detected rhinoviruses to receive optimal treatment. Three different biotinylated inhibitor compounds were synthesized. The length of the spacer between the inhibitor and biotin core was 5, 10, and 15 atoms. These compounds were incorporated into the OIA format for the HRV assay development. A rapid (20 min) OIA test was developed using a 15 atom spacer biotinylated inhibitor (4). Forty different HRV serotypes were studied and thirty three serotypes of these 40 were detected (80%).

Imprinted Polymers—Tailor‐Made Mimics of Antibodies and Receptors

AbstractFor Abstract see ChemInform Abstract in Full Text.

Imprinted polymers-tailor-made mimics of antibodies and receptors.

The technique of molecular imprinting allows the formation of specific recognition sites in synthetic polymers through the use of templates or imprint molecules. These recognition sites mimic the binding sites of antibodies and other biological receptor molecules. Molecularly imprinted polymers can therefore be used in applications relying on specific molecular binding events. The stability, ease of preparation and low cost of these materials make them particularly attractive. This review focuses on recent developments and advances in the field of molecularly imprinted materials, with special emphasis on applications in immunoassays and sensors recently developed by our group and by others.

Stathmin Family Proteins Display Specific Molecular and Tubulin Binding Properties

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3'/RB3") are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific "stathmin-like domain" and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two alpha/beta tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3'. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.

Radiopharmaceuticals for Targeted Tumor Diagnosis and Therapy

Radiopharmaceutical research and development is carried out by the Center for Radiopharmaceutical Science as part of the PSI Life Science Department, the Department of Applied BioSciences at the Swiss Federal Institute of Technology Zürich and the University Hospital Zürich. The common theme is the search for radioactive-labeled tracer molecules, which bind to specific targets in the body. Such radiopharmaceuticals are applied either systemically into the blood stream or locally to patients. Due to their specific molecular binding properties combined with the emitted radiation, they can be used for non-invasive imaging of tumors and the destruction of tumor cells. In this first of two articles, we will present exemplified topics from the research activities of the groups involved with tumor targeting.

Aspects of the bioinorganic chemistry of silicon in conjunction with the biometals calcium, iron and aluminium

Silicon, in its various forms (silicic acid (Si(OH) 4) through to hydrated amorphous silica (SiO n (OH) 4−2 n , n=2–4) is important in geological and biological processes. Silicon is considered to be an `essential' element for some plants and higher animals and when present as silica imparts structural, defensive and photosynthetic advantages to many plants. For the majority of essential elements specific molecular binding sites have been identified, but this is not the case for silicon. This has lead to the proposal that silicon may not act directly on biological systems but may exert its influence via interactions with biometals. Metals for which this may be a viable option include aluminium, iron and calcium, all of which can be found co-located with silicon in both minerals and living organisms. This article briefly reviews the pertinent solution and solid state chemistry of silicon in relation to aluminium, iron and calcium. Examples where silicon and an additional metal are found in solid state phases are described. The biological systems where these deposits are found are extremely complex and preliminary results from a model system designed to study silicon–biometal interactions are described.

Local unbinding of pinched membranes.

Loosely bound membranes exhibit an unusual elastic response when pinched together by optical tweezers, locally unbinding to a large intermembrane distance. Tweezing a stack of many bound membranes produces extreme swelling in the vicinity of the tweezing point. We introduce a model that incorporates bending elasticity, fluctuations, and intermembrane interactions to calculate the membrane profiles subject to a pinch. Theoretically, we find strongly overshooting profiles in agreement with experiment. We predict scaling behavior of the overshoot with the pinch strength and size. PACS numbers: 82.65.Dp, 68. 10.Et, 87.22.Bt Adhesion of biological membranes involves both the attraction of two homogeneous surfaces [1] and the interactions of specific molecular binding sites [2,3]. Experiments suggest that binding sites are dominant locally. However, the overall binding of two membranes with localized chemical stickers ' is strongly influenced by the elastic response and fluctuations of the embedding bilayer [4]. To study the overall membrane response to a single binding site, we study a simplified model system by locally pinching together two membranes with optical tweezers [5,6]. This system can also model local collisions, which are important in the study of interacting, flexible membranes and the understanding of their unbinding transition [7,8]. Previously, we showed that laser tweezers remove area from the membrane, inducing tension in membranes with limited area and thus driving shape transitions. In the present work the tweezers are used primarily as a mechanical tool, clamping at a point two large planar membranes, which effectively have unlimited area and allow only a negligible buildup of tension. We studied bilayers composed of dimyristoyl-phosphatidylcholine (DMPC) (Sigma) produced by a standard technique [9]. Our experimental setup is described elsewhere [10,11]. Typical working conditions were 30 C and laser intensities from 30 up to 150 mW. We selected sets of two or more nearly Oat membranes, which were sections either of huge vesicles or of lamellar sheets that extended from the bottom of the cell to its top. We chose sections of membranes that appeared to be bound or loosely bound, at a distance of -1 p, m. The membranes are pinched by the laser tweezers in the — 0.5 p, m region of the trap. Regardless of the

Sequence Specific Molecular Recognition and Binding by a GC Recognizing Hoechst 33258 Analogue to the Decadeoxyribonucleotide d-[CATGGCCATG]2: Structural and Dynamic Aspects Deduced from High Field1H-NMR Studies

The non-exchangeable and imino proton NMR resonances have been assigned of the 1:1 complex of an analogue 2 of Hoechst 33258 1 bound to the decadeoxyribonuycleotide d-[CATGGCCATG]2 by a combination of NOE difference, COSY and NOESYPH techniques. In contrast to Hoechst 33258 which recognizes 5'-AATT sequences exclusively, analogue 2 possesses structural features designed to permit the recognition of GC sites. The NOESY and 1D-NOE experiments place the drug in the minor groove and it is located on the 5'-CCAT sequence. The orientation of the drug in the groove is such as to place the N-methylpiperazine terminus at a GC site. Cross-correlation peaks in the NOESY experiment show that the DNA duplex retains its right-handed B form, similar to that in the free decamer. Specific NOEs locate the benzoxazole moiety on the 5'-CCAT and are consistent with the pyridine nitrogen forming a new hydrogen bond to G(4)-2NH2 at 5'-CCAT. The drug appears to undergo rotation around the C9-C10 bond, at a rate comparable with NMR time scale, even after binding. Variable temperature 1H-NMR studies established that the DNA is thermally stabilized as a result of the drug binding. The drug binding is a dynamic process involving exchange between the equivalent 5'-CCAT sites at approximately 60s-1 with delta G degree of 65 kJ mol-1 at 308K. The experimental evidence is in accord with a slide-swing mechanism for this process.

Sequence Specific Molecular Recognition and Binding of a Monocationic Bis-Imidazole Lexitropsin to the Decadeoxyribonucleotide d-[(GATCCGTATG) (CATACGGATC)]: Structural and Dynamic Aspects of Intermolecular Exchange Studied by1H-NMR

The non-exchangeable and imino proton NMR resonances of the non self-complementary decadeoxyribonucleotide d-[(GATCCGTATG).(GATACGGATC)] as well as those of the 1:1 complex of the monocatonic bis-imidazole lexitropsin 1 to this sequence have been assigned by using a combination of NOE difference, COSY and NOESY techniques. Confirmation of complete annealing of the two non self-complementary decamer strands to give the duplex decadeoxyribonucleotide is obtained by the detection of ten imino protons. It is established that the sugar-base orientations of all the bases in the duplex decamer are anti. From NOE studies, it is concluded that the duplex oligomer is right-handed and adopts a conformation in solution that belongs to the B family. A population analysis reveals that the sugar moieties exist predominantly in the S-form (2'-endo-3'-exo). Addition of 1 to the DNA solution leads to doubling of the resonances for CH6(4,5), GH8(6), TH6(7) and T-CH3(7). The base, anomeric H1' and imino proton signals for the base sequence 5'-CCGT undergo the most marked drug-induced chemical shift changes. These results provide evidence that the lexitropsin is bound to the sequence 5'-CCGT in the minor groove of the DNA. NOE measurements between the amide protons (NH1 and NH4) and the imino proton (IV and V) signals confirmed the location and orientation of 1 in the 1:1 complex, with the imino terminus oriented to C(4). The specific binding of 1 to the sequence 5'-CCGT-3' deduced in this study is in agreement with the footprinting data obtained using the Hind III/Nci fragment from pBR322 DNA [Kissinger et al. 1987 (13)]. Intramolecular NOEs observed between H4 and H9 of the lexitropsin suggest that the molecule is not planar, but subjected to propeller twisting, in both the free and bound forms. Furthermore, NOE measurements permit assignment of the DNA duplex in the 1:1 complex to the B-form, which is similar to that of the free DNA. The [(T7A8T9).(A12T13A14)] segment of the DNA shows better stacking, by propeller twisting, compared to the rest of the molecule in the free as well as the complex forms. The intermolecular rate of exchange of 1 between the equivalent 5'-CCGT sites, at a concentration of 12 mM, is estimated to be approximately 88s-1 at 308 degrees K with delta G not equal to of 63 +/- 5 KJ mol-1.

Detailed mechanics of membrane-membrane adhesion and separation. II. Discrete kinetically trapped molecular cross-bridges

In general, membrane-membrane adhesion involves specific molecular binding and cross-bridging reactions. The ideal, classical view is that near equilibrium the forces required to separate adhesive contacts are essentially equal to those induced in the membrane when the contact is formed. In contrast to the classical view, experimental observations often show that negligible levels of tension are induced by the adhesive contact even though the tension required to separate the contact is large enough to rupture the membrane. The deviation in tension levels associated with contact formation and separation appears to be due to the sparse distribution of strong molecular cross-bridges. Here, the mechanics of membrane-membrane adhesion and separation is developed for the case of discrete, kinetically trapped cross-bridges. The solution is obtained by numerical computation of the membrane contour that minimizes the total free energy (membrane elastic energy of deformation plus cross-bridge energies) in the contact zone. This solution is matched with the analytical solution for membrane stresses and geometry derived for the adjacent, unbridged zone. The results yield specific values of the macroscopic tension applied to the membrane in the plane region away from the contact zone and the microscopic angle at the edge of the contact zone. Two disparate values of the macroscopic tension are found: (a) the minimum tension required to separate the adherent membranes; and (b) the maximum tension induced in the membranes when the contact is formed (i.e., the level of tension at which the contact will just begin to spread). The results show that the deviation between these two tensions can be very large and depends strongly on the surface density of cross-bridges. In addition, the results provide an estimate of the restraining forces that anchor receptors within the plane of the membrane.