microRNA detection based on in situ activation of a bioluminescent enzyme through protein reassembly

ANYL 169

Kyle A Cissell1, Suresh Shrestha2, and Sapna K Deo2. (1) Department of Chemistry, Indiana University Purdue University Indianapolis, 402 North Blackford Street, LD 326, Indianapolis, IN 46202, (2) Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, 402 N Blackford St, LD 326, Indianapollis, IN 46202
MicroRNAs are short, 18-24 nucleotide base length RNAs that perform gene regulation in eukaryotes. Currently, there are few methods available for miRNA detection, including hybridization and Northern blotting, which have limited sensitivity and cannot be used in-vivo. Here we have investigated a novel miRNA detection method using an enzyme reporter and protein reassembly. In order to detect miR21, a known miRNA, Renilla luciferase was cleaved into two inactive peptide fragments which were chemically conjugated to two oligonucleotide probes, both complimentary to 5' and 3' portions of miR21. Upon hybridization, the inactive peptide fragments reassemble, thus forming an active enzyme. When the substrate coelenterazine is added, the luciferase oxidizes the coelenterazine, releasing light. The photon release will only occur upon hybridization of the oligos to the miRNA. This is a mix-and-measure type of assay suitable for intracellular analysis. Furthermore, the use of a bioluminescent enzyme reporter allows for detection of miRNA in the femto-attomole range.
 

General Papers
7:00 PM-9:00 PM, Sunday, 10 September 2006 Moscone Center -- Hall D, Poster

Division of Analytical Chemistry

The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006