Oligonucleotide cross-linking methods

The unique properties of oligonucleotides create cross-linking options that are far different from those of any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically synthesized 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. 

While Merrifield experimented with the polystyrene cross-linked with 2% and later 1% DVB [55], Letsinger adopted the popcorn polystyrene [54] which relies on less cross-linking agent (0.05-0.2%) to achieve complete insolubility in common organic solvents. Ah of his early ohgodeoxyribonucleohde syntheses by the phos-photriester method were performed on this type of support [53, 58, 59], Other researchers have used popcorn PS as well in the syntheses by the phosphodiester method [144] and, later, more advanced phosphotriester method [145], However, with the widespread use of low cross-linked PS and the advent of polyacrylamide and especially silica gel supports (Section 19.2.3), use of popcorn polystyrene in oligonucleotide synthesis has practically ceased. 

Interest in highly cross-linked (20-30% DVB) macroporous polystyrene arose in the early days of oligonucleotide synthesis [158]. It has been argued that this type of support allows for faster reaction kinetics than the low cross-linked gel-type polystyrene. Modified with a trityl-type tinker, it has been tested by Roster and Cramer [159] in the phosphodiester method. The limitations of the chemistry hampered its use for oligomers longer than a few nucleotides. 

While a large number of improvements in procedures for solid phase synthesis of oligonucleotides have been described, the techniques have not altered fundamentally from those described in previous reports. Solid-phase synthesis using a continuous-flow phosphotriester method on a kieselguhr-polyamide support has been described. Other solid phases used as supports for synthesis include derivatized h.p.l.c.-grade silica gel, . derivatized cross-linked poly-... 

Oligonucleotides containing 4-thiothymidine and 4-thiouridine have been previously used for LiV cross-linking studies with a view to the study of nucleic acid structures and DNA-protein interactions. A recent publication describes the synthesis of a. set of deoxyuridine derivatives (174, 175, 176) containing C5-pendant thiothymine residues suitable for incorporation into oligonucleotides using chemical methods. 

Corticosteroid receptor, 76 Corticosterone, 76 Creatine kinase, 21 Cross linking, 90, 641, 642 antibody, 501-504 artifacts, 174, 175 characterization, 172-180 efficiency, 170-172 intermolecular, 90 irradiation, 170-172 methods for, 170-172 nucleic acid-protein, 168-180 of oligonucleotides, 676 quantitation, 171, 178 regions, 175, 176 procedures, 176-178 Curtius-Schmidt rearrangement, 78 w-Cyanobenzyltriphenylphosphonium bromide, 125, 127 Cyanogen bromide, 114 tn-Cyanophenol, 122-124 m-(3-Cyanophenoxy)propylamine hydrobromide, 124... 

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