Spacer arms functional groups

The unique properties of oligonucleotides create crosslinking options that are far different from any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically duplicated in vitro by enzymatic means. The addition of modified nucleoside triphosphates to an existing DNA strand by the action of polymerases or transferases allows addition of spacer arms or detection components at random or discrete sites along the chain. Alternatively, chemical methods that modify nucleotides at selected functional groups can be used to produce spacer arm derivatives or activated intermediates for subsequent coupling to other molecules. 

Perhaps a better design for a bis-hydrazide compound to modify carboxylate particles would include a short PEG spacer arm between the two hydrazide groups. This type of linker would result in a hydrophilic surface due to the presence of the PEG spacers, while providing the terminal hydrazide functionality necessary for coupling to carbonyl compounds. Unfortunately, this type of compound is not currently available, so the aliphatic bis-hydrazides are the only choice. 

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Adsorbents for biomacromolecules such as proteins have special properties. First, they need to have large pore sizes. A ratio of pore radius to molecule radius larger than 5 is desirable to prevent excessive diffusional hindrance (see Intraparticle Mass Transfer in this section). Thus, for typical proteins, pore radii need to be in excess of 10-15 nm. Second, functional groups for interactions with the protein are usually attached to the adsorbent backbone via a spacer arm to provide accessibility. Third, adsorbents based on hydrophilic structures are preferred to limit nonspecific interactions with the adsorbent backbone and prevent global unfolding or denaturation of the protein. Thus, if hydrophobic supports are used, their surfaces are usually rendered hydrophilic by incorporating hydrophilic coatings such as dextran or polyvinyl alcohol. Finally, materials stable in sodium hydroxide solutions (used for clean-in-place) are... 

Succinic anhydride also is a convenient extender for creating spacer arms on chromatography supports. Supports derivatized with amine-terminal spacers may be succinylated to block totally the amine functional groups and form terminal carboxylic acid linkers for coupling amine-containing affinity ligands (Cuatrecasas, 1970). 

An amino-functional spacer arm is introduced at the 5 position of the ODN in the last step of its automated synthesis. ODN can be grafted via various functions available on flat carriers (such as flat silicon surfaces or wafers covered in silane) or on latex particles. Table 2 shows a list of various activation agents used and the reactive group resulting from the activation depending on the compound involved when available, the maximum grafting amount is also reported. 

Fig. 7 Approaches for linking macromolecules to solid surfaces using biotin-strept(avidin) systems. Either biotin or streptavidin may be directly linked to a surface (e.g. Fig. 6). There are a large number of biotin derivatives with different spacer arms (e.g. Fig. 3) that may be used for immobilization. The choice of a specific biotin depends on which active group occurs on the native or derivatized surface. Attachment of strept(avidin) may be done with methods used with other proteins. Note that the deposition of a biotinylated molecules to a surface may also be used (e.g. biotinylated small molecules or large molecules like BSA) to link strept(avidin) to a surface. A DNA covalently attached at one end (3 end) can have a functional biotin at the other end that could bind to strept(avidin) or be covalently linked to streptavidin (see Fig. 4). Single-stranded DNA bound to immobilized streptavidin is available for hybridization to its complementary sequence (See Fig. 4)...

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