Nucleic Acids Research, 2000, Vol. 28, No. 21 4306-4316
© 2000 Oxford University Press
Characterization of the interaction between 
CP2 and the 3'-untranslated region of collagen 1(I) mRNA
Department of Medicine and Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-7038, USA and 1Bayer Pharmaceutical Division, Pharmaceutical Research Center, Whuppertal, Germany
Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein–RNA interactions in the 3'-UTR of the collagen 
1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is CP2. Recombinant CP2 is sufficient for binding to the 3'-UTR of collagen 1(I). To characterize the binding affinity of and specificity for CP2, we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3'-UTR of collagen 1(I) as probe. The binding affinity of CP2 for the 3'-UTR sequence is 
2 nM in vitro and the wild-type 3' sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein–nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA–DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant CP2 to the wild-type 3' sequence, although the kinetics of binding were slower.
* To whom correspondence should be addressed. Tel: +1 919 966 0650; Fax: +1 919 966 7468; Email: dab@med.unc.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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