An efficient chemical procedure for the immobilization of carboxylate containing conjugate groups onto controlled pore glass (CPG) is described. The derivatized supports were used in the automated synthesis of an oligodeoxynucleotide (20-mer ODN) containing a 3' phosphodiester linked hexanol, aminohexyl, acridine, or cholesterol group. The stability of the oligomer in a hepatoma cell culture was found to be prolonged two to three fold by the presence of any of the 3' tails. By contrast, an aminohexyl group appended to the 5' terminus of the ODN only marginally improved its nuclease resistance. These data support the notion that antisense ODNs are primarily degraded by 3' exonucleases. Introduction of simple 3' tails which incorporate a normal phosphodiester linkage can increase ODN stability by interfering with these enzymes.
A pair of complementary oligodeoxynucleotides (ODNs) uniformly substituted with 2-amino-adenine (A') in place of adenine and 2-thiothymine (T') in place of thymine did not hybridize to each other but did form very stable hybrids with unmodified complementary ODNs. These unusual properties were a consequence of the hydrogen-bonding properties of the two base analogs. Thermal denaturation studies of short duplexes which contained these bases demonstrated that the A'-T and A-T' doublets formed stable base pairs whereas the A'-T' doublet acted like a mismatch. Complementary ODNs substituted with these base analogs are referred to as SBC or selectively binding complementary ODNs. When used as a pair, these single-stranded ODNs invaded the ends of homologous duplexes and formed stable three-arm junctions under conditions where unmodified ODNs failed to give a product. SBC ODNs have a fundamental thermodynamic advantage in hybridizing to short segments of double-stranded nucleic acid and represent a new approach for the design of oligomeric probes and antisense agents. Many secondary structure features present in long single-stranded nucleic acids should be accessible to these reagents.
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).
Oligodeoxynucleotides (ODNs) were conjugated to chlorambucil and used as affinity labeling reagents to study joint molecule formation by the Escherichia coli recombinase recA. Chlorambucil is a bifunctional nitrogen mustard which alkylates the N-7 position of guanine in the major groove of double-stranded DNA (dsDNA). Incoming ODNs at least 30 nucleotides long cross-linked to a long homologous duplex DNA in the presence of recA and ATP gamma S. Efficient cross-linkage to the complementary recipient strand of the joint occurred preferentially at guanines positioned 5' relative to the appended chlorambucil group. The pattern of recipient strand alkylation was identical to that observed within a protein-free duplex and indicated that strand exchange had occurred prior to alkylation. Modification of the outgoing homologous strand of the joint was less efficient and spanned a 15-20 nucleotide long region offset to the 3' side of the tethered chlorambucil. Alkylation of both recipient and outgoing strands in the same joint molecule occurred with low frequency. By contrast, no affinity alkylation of the displaced strand was observed within a synthetic D-loop. These reaction patterns suggest that the incoming ODN approaches from the minor groove of the duplex to yield a poststrand exchange joint in which the major groove of the newly formed heteroduplex harbors the outgoing strand in an unpaired state. No evidence was obtained for the involvement of a triple-stranded DNA intermediate in recombination.
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