title: A non-nucleotide-based linking method for the preparation of psoralen-derivatized methylphosphonate oligonucleotides.
abstract: A method is reported for conjugating an analog of 4'-(aminomethyl)-4,5',8- trimethylpsoralen to methylphophonate oligonucleotides. This method enables the psoralen moiety to be coupled to the phosphonate backbone between any two desired bases in a sequence. When hybridized to a target mRNA, the psoralen moiety can be directed toward a uridine base and, in turn, can undergo a photo-addition reaction with the target under UV irradiation at 365 nm. Several different non-nucleotide-based amino-linker reagents have been prepared for incorporation into methylphosphonate oligonucleotides by standard phosphonamidite chemistry. In addition, an N-hydroxysuccinimide activated ester analog of 4'-[(3-carboxypropionamido)methyl]-4,5',8- trimethylpsoralen has been synthesized for conjugation to the amino-linker moieties. Using this approach, we have prepared a number of psoralen-methylphosphonate-oligonucleotide conjugates which are complementary to the chimeric bcr/abl mRNA associated with chronic myelogenous leukemia. Solution hybridization studies with a 440-base subfragment of the bcr/abl RNA have shown that the psoralen moiety does not adversely affect duplex stability. Polyacrylamide gel electrophoresis analyses have demonstrated that the psoralen-oligonucleotide conjugates undergo photo-addition to the RNA in a sequence-specific manner. Optimal photo-addition occurs when the psoralen moiety is inserted adjacent to one or more adenine residues in the oligonucleotide sequence, particularly between adenine and thymine (5'-3'). This internal labeling approach greatly increases the number of potential target sites available for photo-cross-linking experiments.
authors.affiliation: Genta Inc., San Diego, California 92121.
authors.forename: M A
authors.initials: MA
authors.lastname: Reynolds
authors.forename: T A
authors.initials: TA
authors.lastname: Beck
authors.forename: R I
authors.initials: RI
authors.lastname: Hogrefe
authors.forename: A
authors.initials: A
authors.lastname: McCaffrey
authors.forename: L J
authors.initials: LJ
authors.lastname: Arnold
authors.forename: M M
authors.initials: MM
authors.lastname: Vaghefi
mesh_terms: D001483:Base Sequence; D002678:Chimera / Q000235:genetics; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D011564:Furocoumarins / Q000138:chemical synthesis*; D007951:Leukemia, Myeloid / Q000235:genetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis*; D009943:Organophosphorus Compounds / Q000138:chemical synthesis*; D010777:Photochemistry; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000138:chemical synthesis
chemical_list: D003432:Cross-Linking Reagents; D011564:Furocoumarins; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; C032627:methylphosphonic acid; C018822:aminomethyltrioxsalen; D014307:Trioxsalen