Detection of an abasic site in RNA with stem-loop DNA beacons: application to an activity assay for Ricin Toxin A-Chain.
The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the N-glycosidic bond of a specific adenosine in the GAGA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact "GAGA" sequence can be discriminated from the product containing an abasic "GabGA" sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5'- and 3'-ends. In this format the probe-substrate heteroduplex gives a fluorescent signal while the probe-product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5'-end while the quencher is on the probe DNA sequence at its 3'-end. In this format the fluorescence of the substrate-probe heteroduplex is quenched while that of the product-probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.
Roday
Setu
S
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, United States.
Sturm
Matthew B
MB
Blakaj
Dukajgin
D
Schramm
Vern L
VL
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011189:Potassium Chloride; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D012276:Ricin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*
D012313:RNA; D011189:Potassium Chloride; D004247:DNA; D012276:Ricin