Restriction Inhibition
Assay: A Qualitative and Quantitative Method to Screen
Sequence Specific DNA Binder from Herbal Plants
Syed A Hassan1*,
Ritu Barthwal2, Shree R Padmadeo3
and Omar M Barukab4
1Faculty of Computing and
Information Technology Rabigh, King Abdulaziz
University, PO Box 344, Rabigh-21911, Saudi Arabia,
2Molecular Biophysics Laboratory, Department of
Biotechnology, Indian Institute of Technology Roorkee,
Roorkee 247667, Uttrakhand, 3Department of
Biochemistry, Patna Science College, Patna
University-110067, India, 4Faculty of
Computing and Information Technology Rabigh, King
Abdulaziz University, PO Box 344, Rabigh-21911, Saudi
Arabia.
*For correspondence:
Email:
asif_srmcbt@yahoo.com; Tel:
+966544894526
Received: 4 December 2012
Revised accepted: 30
December 2013
Tropical Journal of
Pharmaceutical Research, January 2014;
13(2):
267-273
http://dx.doi.org/10.4314/tjpr.v13i2.16
Abstract
Purpose: To employ restriction
inhibition assay (RIA) to screen phytochemical-rich
fractions (PRFs) with high affinity for EcoRI and
HindIII restriction sequences and correlate their
interaction to an anticancer activity.
Methods: pBR322 linear plasmid DNA
was used as a template to screen the sequence-selective
inhibition of aqueous extracts of Cinnamomum zeylanicum
and Picrorhiza kurroa, respectively. The template was
further incubated with different concentrations of PRFs
prior to digestion with restriction endonucleases
HindIII and EcoRI. The Expressed Sequence Tags (ESTs)
and Sequence Tag Sites (STS) of oncogenes were screened
for the presence of EcoRI and HindIII restriction
sequences to associate an anticancer property to PRF.
Results: The inhibitory
concentrations of Cinnamomum zeylanicum aqueous extract
against HindIII and EcoRI endonucleases were
approximately 2.5 and 5 µg/µl, respectively. No binding
was observed for Picrorhiza kurroa at both HindIII and
EcoRI restriction sites. The saponin-rich fractions of
Cinnamomum zeylanicum showed significant (p < 0.001)
inhibition as compared to control at concentrations of
0.28±1.45 µg/µl for EcoRI and 0.11±2.68 µg/µl for
HindIII endonucleases. Both EcoRI and HindIII
restriction sites were found repeatedly in the STS and
ESTs of BRCA2, the early onset oncogene.
Conclusion:
The inhibition of
endonucleases by phytochemical-rich fractions provides
direct evidence of the use of RIA for screening as well
as demonstrating the binding specificity of these PRFs.
The presence of 5’-AAGCTT-3’ & 5’-GAATTC-3’ in the ESTs
of BRAC2 provides an insight into the use of screened
components as leads in the search for novel anticancer
compounds.
Keywords: Restriction
endonucleases, Restriction sites, Phytochemicals,
Restriction inhibition assay (RIA), Binding specificity,
Oncogenes, Sequence tag sites, Expressed sequence tag,
Anticancer
