Detection of an abasic site in RNA with stem-loop DNA beacons: application to an activity assay for Ricin Toxin A-Chain.

Setu Roday, Matthew B. Sturm, Dukajgin Blakaj, Vern L. Schramm

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)945-953
Number of pages9
JournalJournal of Biochemical and Biophysical Methods
Volume70
Issue number6
DOIs
StatePublished - Apr 24 2008

Fingerprint

Ricin
Assays
RNA
DNA
Inverted Repeat Sequences
Substrates
DNA Probes
Fluorophores
Adenosine
Limit of Detection
Hydrolysis
Fluorescence
DNA sequences

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

Cite this

Detection of an abasic site in RNA with stem-loop DNA beacons : application to an activity assay for Ricin Toxin A-Chain. / Roday, Setu; Sturm, Matthew B.; Blakaj, Dukajgin; Schramm, Vern L.

In: Journal of Biochemical and Biophysical Methods, Vol. 70, No. 6, 24.04.2008, p. 945-953.

Research output: Contribution to journalArticle

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