Photochemistry and Photobiology, 1998, zyxwvut 67(3): 289-294 zyxwvu Mutagenesis Mediated by Triple Helix-Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context Manidipa Raha, Laurent Lacroix and Peter M. Glazer* Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, CT, USA Received 9 June 1997; accepted 11 December 1997 ABSTRACT zyxwvutsr Targeted mutagenesis and gene knock-out can be medi- ated by triple helix-forming oligonucleotides (TFO) linked to mutagenic agents, such as psoralen. However, this strategy is limited by the availability of homopurine/ homopyrimidine stretches at or near the target site be- cause such sequences are required for high-amnity tri- plex formation. To overcome this limitation, we have test- ed TFO conjugated to psoralen zyxwvuts via linker arms of lengths varying from 2 to 86 bonds, thereby designed to deliver the psoralen at varying distances from the third strand binding site present at the 3' end of the zyxwvuts supFGl mutation reporter gene. Following triplex formation and UVA ir- radiation, mutations were detected using an SV40-based shuttle vector assay in human cells. The frequency and distribution of mutations depended on the length of the linker arm. Precise targeting was observed only for link- er arms of length 2 and 6, which also yielded the highest mutation frequencies (3 and 1496, respectively). zyxwvu Psora- len-TFO with longer tethers yielded mutations at mul- tiple sites, with the maximum distance from the triplex site limited by the linker length but with the distribution within that range influenced by the propensity for psor- alen intercalation at A:T base-pair-rich sites. Thus, gene modification can be extended beyond the site of third strand binding but with a decrease in the precision of the targeting. INTRODUCTION Oligonucleotide-mediated triple-helix formation has emerged as a promising tool in molecular biology. Triplex formation has been shown to inhibit transcription factor binding to promoter sites and to block transcription zyxwvu in vitro and in vivu (1). Triplex formation has further been used in strategies to generate unique cleavage sites in DNA in vitru (2). We have recently explored the use of triplex-forming *To whom correspondence should be addressed at: Department of Therapeutic Radiology, Yale University School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, USA. Fax: (203) 737- 2630; e-mail: peter.glazer@qm.yale.edu zyxwvuts 0 1998 American Society for Photobiology 0031-8655/98 $5.00+0.00 oligonucleotides (TFO)? as a method to deliver a tethered mutagen to a selected gene for the site-specific introduction of DNA damage (3,4). Experiments have shown that triple- helix formation can target DNA damage and consequently mutations to specific sites in selected genes in order to pro- duce permanent, heritable changes in gene function and ex- pression (3). Triplex DNA can be formed when oligonucleotides bind as third strands of DNA in the major groove of the double helix in polypurinelpolypyrimidine sequences. Several mo- tifs for third strand binding have been identified. In one, a pyrimidine-rich oligonucleotide binds in a direction parallel to the purine strand in the duplex (5-7). In the alternate purine motif, a polypurine strand binds antiparallel to the purine strand in the Watson-Crick duplex, with A binding to A:T and G binding to G:C (8). In all motifs so far iden- tified, however, third strand formation is limited by the avail- ability of polypurine/polypyrimidine stretches in the target gene. For the purpose of gene knock-out or targeted muta- genesis, it is particularly desirable to have a polypurine bind- ing site immediately adjacent to the base pair to be mutated. In this way, a mutagen conjugated to the 5' or 3' end of the oligonucleotide can be readily positioned for lesion forma- tion at the selected position. In many genes, however, the target site may be at some distance from the optimal triplex binding site. In order to address this problem, we sought to design a reagent capable of forming a triple helix at one region but modifying the gene in another region. For ex- ample, many introns contain polypurine runs, and so the ca- pacity to use a triplex binding site in an intron as part of a strategy to target coding sequences within an exon would be advantageous. To this end, we have synthesized TFO that are attached to psoralen viu linker arms of different lengths. Psoralen, a three-ringed heterocyclic furocoumarin, can in- tercalate into DNA and undergo photoinduced cycloaddition with pyrimidines to generate either monoadducts (MA) or interstrand cross-links (XL). We reasoned that the longer tethers would allow psoralen intercalation and photoreaction at distant sites. We report here that the range of base-pair (bp) modifi- cation and mutation induction can be extended by this strat- tAbbreviations: bp, base pair; MA, monoadduct; TFO, triplex-form- ing oligonucleotide; UVA, long-wavelength UV light; XL, cross- links. 289