Specific affinity interactions

Au NPs functionalized with biomolecules can be synthesized using different methods, depending on factors inducing the interactions promoted between the nanoparticle and the biomolecule. These interactions can be classified as electrostatic adsorption, chemisorption and covalent binding and, finally, specific affinity interactions. Some examples are given in the following paragraphs (Scheme 3.23). 

The third group of Au NP-biomolecule interactions are referred to as specific affinity interactions and include nanoparticles functionalized with groups that provide affinity sites for the binding of bio molecules such as proteins and oligonucleotides. 

Probably the best means of protein attachment is through highly specific affinity interactions (e.g., streptavidin-biotin or His-tag-nickel-chelates) [111]. Proteins fused with a high-affinity tag, at their amino or carboxyl terminus, are finked to the surface of the chip via this tag, and hence all the attached proteins should orient uniformly away from the surface. Using this method, immobilized proteins are more likely to remain in their native conformation, while the analytes have easier access to the active sites of the proteins. 

Electrode surfaces modified with a multilayered surface architecture prepared by a layer-by-layer repeated deposition of several enzyme mono-layers show a modulated increase of surface-bound protein with a subsequent increase in output current, which is directly correlated with the number of deposited protein layers. The versatility of this approach allows alternate layers of different proteins for the manufacture of electrode surfaces, which are the basis for multianalyte sensing devices with multiple substrate specificities. The surface chemistry used for the manufacture of multilayered electrode surfaces is similar to that previously described for the preparation of affinity sensors, and is based on the stabilization of self-assembled multilayer assemblies by specific affinity interactions, electrostatic attraction, or covalent binding between adjacent monolayers. 

In order to quantitate protein capture and binding capacity in the TBA coated capillary, eluate was collected after each step (load, wash, and elnte), and total amonnts of protein in each were determined using flnorescence spectroscopy. The experiment was also performed using a bare (unmodified) capillary and a capillary coated with an oligonucleotide that has the same base composition as the TBA bnt in a scrambled sequence that does not form a G-qnadrnplex strnctnre and does not bind with thrombin (Bock et ah, 1992). The resnlts (Table 11.1) showed that the aptamer-coated capillary captures approximately three times as much thrombin as the bare and scrambled oligonucleotide-coated capillaries. The scrambled oligonucleotide yielded no more captnre than the bare snrface, indicating that thrombin capture at the TBA surface is due to specific affinity interactions. Since thrombin capture occurs only if the TBA is in the G-qnadrnplex conformation, we conld further conclude that the immobilized TBA at the surface is able to form the G-quadruplex. 

Functionalization of Nanoparticles with Biomolecules by Specific Affinity Interactions... 

Affinity chromatography A form of chromatography in which separation is achieved by utilizing highly specific biochemical interactions, such as steric-or charge-related conditions, between me analyte and a molecule immobilized on a column. It is different from most forms of chromatography in mat analytes do not continuously elute from me column - only mose mat interact wim me stationary phase are retained and mus separated from omer components of me mixture under investigation. These immobilized materials are eluted from me column after all omer materials have been removed. 

The predictable nature of DNA and RNA base pairing make their interactions the most defined of any biological system. The specific affinity of one strand for its complementary... 

Coupling of affinity molecules to surfaces also can be enhanced by the use of discrete PEG linkers. Nishimura et al. (2005) modified an amino surface with a NHS-PEG -maleimide crosslinker to create a hydrophilic self-assembled monolayer (SAM) surface that was thiol reactive for the conjugation of sulfhydryl-modified RNAs. This array then was used to investigate the binding specificity of synthetic kanamycins with selected RNA sequences to prove the specific interaction of ribosomal RNA with this molecule. The PEG linkers on surfaces provide lower nonspecific binding character than alkyl linkers, when preparing SAM surfaces for affinity interactions. 

Transition metals such as iron can catalyze oxidation reactions in aqueous solution, which are known to cause modification of amino acid side chains and damage to polypeptide backbones (see Chapter 1, Section 1.1 Halliwell and Gutteridge, 1984 Kim et al., 1985 Tabor and Richardson, 1987). These reactions can oxidize thiols, create aldehydes and other carbonyls on certain amino acids, and even cleave peptide bonds. The purposeful use of metal-catalyzed oxidation in the study of protein interactions has been done to map interaction surfaces or identify which regions of biomolecules are in contact during specific affinity binding events. 

The fundamental behaviour of stationary phase materials is related to their solubility-interaction properties. A hydrophobic phase acts as a partner to a hydrophobic interaction. An ionic phase acts as a partner for ion-ion interactions, and surface metal ions as a partner for ligand complex formation. A chiral phase partners chiral recognition, and specific three-dimensional phases partner affinity interactions. 

Nucleolin lacks a characteristic DNA binding domain (Ginisty et al, 1999). Its non-specific affinity for DNA is conferred by two different domains its four RNA binding domains, particularly the 3rd and the 4th ones, and its C-terminal GAR domain (Hanakahi et al, 1999 Sapp et al, 1989). Of importance, these properties were determined in vitro with the native protein purified from cell extracts or recombinant truncated proteins they are likely to be altered in vivo by interaction with other DNA binding factors (Dempsey et al, 1998) and/or by post-translational modifications. 

One other class of biochemical proteins that offers a similar advantage in clinical biochemistry is antibodies, which are highly specific for interaction with other proteins (i.e. with antigens) and bind with a high affinity. The combination of antibodies with radioisotopes produces very sensitive and very specific assays. This system has been used particularly for measuring the concentrations of hormones (see below) peptides and cytokines. 

In the case of HI variants, linker histones selectively bind ADPr homopolymers over competitor DNA [223]. Furthermore, Hit displays a high degree of affinity for the ADPr subunits even in the presence of salt [223]. Interestingly, this testis specific variant interacts with DNA the least tightly, and has been implicated in fiber decondensation [25,226,227]. This result suggests that potential interactions between HI molecules and ADPr are specific and not just the bi-product of electrostatic attractions. In this regard, specificity for the ADPr subunits may facilitate removal of HI from chromatosomal DNA, and initiate an unraveling of the 30 nm fiber required for DNA activation or repair. Unfortunately, the... 

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