Participants/Project Abstracts
Summer 2007 Bioanalytical Science REU Participants
DETECTION OF SMALL INTERFERING RNAs BY ROLLING CIRCLE AMPLIFICATION
Sonja
Kress, Ni Li, Wenwan Zhong
Department
of Chemistry, University of California, Riverside, CA 92521 and
Leibniz University, Hanover,Germany
Abstract
It is of great
importance to study the expression profiles of siRNAs in plant samples in order
to understand their functions and regulations, but siRNAs are usually expressed
at low levels. Consequently there is a high demand of
a simple, reliable and sensitive siRNA detection method. Here we present a
liquid based system depending on a padlock probe and rolling circle
amplification to quantitatively analyse siRNAs. The siRNA in interest anneals
to a corresponding ssDNA template and its 3' and 5' ends are linked to the termini of the padlock probe by ligation, which forms a circular DNA. The circular DNA is then amplified isothermally,
generating a long ssDNA product containing multiple copies of the siRNA and
DNAZyme sequence. Upon complexing with hemin, the DNAZyme acts as a peroxidase
domain and catalyzes the oxidation of ABTS by H2O2. The
product can be measured at 405 nm. Such cascade signal amplification should
render sensitive detection of siRNA. Reaction and detection in liquid solutions result in a detection limit
of 1 nM. A solid-based reaction system is under development to enhance the
sensitivity by washing away the free hemin molecules and reactant residues and
lowering down the background.
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