Oligodeoxynucleotides with Conjugated Dihydropyrroloindole Oligopeptides: Preparation and Hybridization Properties

Abstract

Synthesis of a new class of conjugates between oligodeoxyribonucleotides (ODNs) and minor groove binders (MGBs) is described. The MGBs are analogs of the potent antibiotic CC-1065 and consist of repeating 1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate (DPI) subunits with N-3 carbamoyl or tert-butyloxycarbonyl groups (CDPI or BocDPI subunits, respectively). The ODN-MGB conjugates were obtained by postsynthetic modification of 5'- or 3'-amino-tailed ODNs with the 2,3,5,6-tetrafluorophenyl (TFP) esters of CDPI1-3 or BocDPI1-2 or by ODN synthesis using a CDPI3-modified controlled pore glass (CPG) support. The hybridization properties of MGB-tailed octathymidylates were determined; they varied with respect to the site of conjugation (3' or 5'), the nature of the linker, the length of the DPI oligopeptide, and the type of N-3 substitution. Optical melting studies showed that the linkage of CDPI1-3 residues to (dTp)8 significantly increased the stability of hybrids formed by the latter with poly(dA). The extent of stabilization increased with the length of the peptide. When CDPI3 was conjugated to either end of (dTp)8, the melting temperature (Tm) of the hybrid formed with poly(dA) was increased by 43-44 degrees C. Free CDPI3 stabilized the (dTp)8-poly(dA) hybrid by only 2 degrees C, thus demonstrating the importance of conjugation. (dTp)8-CDPI1-3 conjugates also formed stabilized duplexes with poly(rA). The extent of stabilization was half that observed with poly(dA).

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove binders

“…These conjugates manifest an enhanced site specificity, which is provided by both oligonucleotide and binder moieties of conjugate [9][10][11][12][13][14]. The use of MGBs with oligonucleotide primers was successful, since it allowed an improved strength and specificity of the primer binding to the target DNA [15].…”

Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.

“…The phosphorothioate ODN was prepared by substituting Beaucage reagent for iodine in the oxidation steps. CDPI3 was conjugated to 5'-or 3'-aminotailed ODNs as described by Lukhtanov et al (20) modified ODNs tested (see Table 1). We conclude that DNA synthesis was arrested only when a blocking downstream hybrid of sufficient stability was formed by a CDPI3-ODN conjugate.…”

Diagnostic System for Rapid and Sensitive Differential Detection of Pathogens

“…gov/ncidod/eid/vol11no02/04-0492_app.htm). Primers, synthesized with a 5′ C6 spacer and aminohexyl modification, were covalently conjugated by a photocleavable link to Masscode tags (Qiagen Masscode technology) (8,9). Masscode tags have a modular structure, including a tetrafluorophenyl ester for tag conjugation to primary amines; an o-nitrobenzyl photolabile linker for photoredox cleavage of the tag from the analyte; a mass spectrometry sensitivity enhancer, which improves the efficiency of atmospheric pressure chemical ionization of the cleaved tag; and a variable mass unit for variation of the cleaved tag mass (8,(10)(11)(12) primer sets are labeled with distinct molecular weight tags.…”