HOECHST-33258 TETHERED BY A HEXA(ETHYLENE GLYCOL) LINKER TO THE 5'-TERMINI OF OLIGODEOXYNUCLEOTIDE 15-MERS - DUPLEX STABILIZATION AND FLUORESCENCE PROPERTIES

Citation
Sb. Rajur et al., HOECHST-33258 TETHERED BY A HEXA(ETHYLENE GLYCOL) LINKER TO THE 5'-TERMINI OF OLIGODEOXYNUCLEOTIDE 15-MERS - DUPLEX STABILIZATION AND FLUORESCENCE PROPERTIES, Journal of organic chemistry, 62(3), 1997, pp. 523-529
Citations number
31
Categorie Soggetti
Chemistry Inorganic & Nuclear
ISSN journal
00223263
Volume
62
Issue
3
Year of publication
1997
Pages
523 - 529
Database
ISI
SICI code
0022-3263(1997)62:3<523:HTBAHG>2.0.ZU;2-G
Abstract
A fluorescent Hoechst 33258 derivative has been prepared in which a he xa(ethylene glycol) linker is attached to the terminal phenol residue. Conjugation of this derivative to DNA sequences is accomplished by a reversed coupling protocol, one in which the 5'-terminal nucleoside re sidue of a fully protected DNA sequence is converted to a terminal pho sphoramidite. In the presence of the Hoechst derivative and tetrazole the final coupling reaction is achieved to generate the conjugated nuc leic acid. After deprotection and cleavage of the conjugate from the s upport, HPLC analysis indicates that the conjugation reaction proceeds with yields as high as 75%. The presence of the conjugated Hoechst de rivative increases the stability of DNA duplexes typically by 10-16 de grees C. A variety of sequence variants indicate that the tether lengt h is sufficient to reach beyond the terminus of the DNA duplex and bin d to internal A-T rich target sequences as far away as four base pairs from the site of attachment. A four base pair binding site appears to be necessary for effective helix stabilization by the conjugate, but in some cases can include a G-C base pair, which is consistent with a previous X-ray diffraction study regarding the binding of Hoechst 3325 8 to duplex DNA. When A-T base pairs alternate with G-C base pairs, a small but discernible increase is T-m is observed (3.6 degrees C), ind icating that binding to this sequence still occurs, but not in the sam e manner as to A-T rich sequences. Upon formation of the conjugated du plex, an enhanced quantum yield for the fluorescence emission spectrum of the tethered Hoechst derivative is observed. When an A-T rich bind ing site is present, the enhanced quantum yield increases by at least 16- and in some cases to nearly 30-fold relative to the value obtained for the single-stranded DNA-Hoechst conjugate.