Polypurine hairpins directed against the template strand of DNA knock down the expression of mammalian genes.
We analyzed whether polypurine hairpins (PPRHs) had the ability to knock down gene expression. These hairpins are formed by two antiparallel purine domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target polypyrimidine sequence, forming triplex structures. To set up the experimental conditions, the human dhfr gene was used as a model. The PPRHs were designed toward the template strand of DNA. The transfection of the human breast cancer cell line SKBR3 with these template hairpins against the dhfr gene produced higher than 90% of cell mortality. Template PPRHs produced a decrease in DHFR mRNA, protein, and its corresponding enzymatic activity. In addition, the activity of DHFR PPRHs was tested against breast cancer cells resistant to methotrexate, observing high cell mortality. Given the difficulty in finding long polypyrimidine stretches, we studied how to compensate for the presence of purine interruptions in the polypyrimidine target sequence. The stability of PPRH was measured, resulting in a surprisingly long half-life of about 5 days. Finally, to test the generality of usage, template PPRHs were employed against two important genes involved in cell proliferation, telomerase and survivin, producing 80 and 95% of cell death, respectively. Taken together our results show the ability of antiparallel purine hairpins to bind the template strand of double strand DNA and to decrease gene transcription. Thus, PPRHs can be considered as a new type of molecules to modulate gene expression.
de Almagro
M Cristina
MC
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Av. Diagonal 643, E-08028 Barcelona, Spain.
Coma
Silvia
S
Noé
Véronique
V
Ciudad
Carlos J
CJ
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004247:DNA / Q000737:chemistry*; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D011687:Purines / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D000077022:Survivin; D019098:Telomerase / Q000378:metabolism; D014158:Transcription, Genetic
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D011687:Purines; D012333:RNA, Messenger; D000077022:Survivin; D004247:DNA; D019098:Telomerase