Exploring Ligand-DNA Space Using Type IIS Restriction Enzymes

Author(s): Brian Ward

Journal Name: Combinatorial Chemistry & High Throughput Screening
Accelerated Technologies for Biotechnology, Bioassays, Medicinal Chemistry and Natural Products Research

Volume 5 , Issue 4 , 2002

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Investigating ligand-DNA interactions using type IIS restriction enzymes (IISRE) as footprinting reagents is reviewed and contemplated. Ligand binding at a IISREs cleavage but not sequence recognition site protects DNA from strand scission. This spatial arrangement has been exploited in the development of qualitative (combinatorial) and quantitative ligand-DNA investigative methods collectively termed Type IIS Restriction Enzyme Footprinting (cIISREF and qIISREF respectively). In cIISREF, the consensus binding sequence of a ligand is sought by using a IISRE to segregate combinatorial library members that are bound by ligand from those that are not. A PCR is performed following the segregation step to enrich the library in ligand binding (i.e. uncut) sequences. It might be possible that diversities approaching 1030 unique sequences could be simultaneously searched using this homogeneous and biologically relevant method. For qIISREF, a ligand-DNA equilibrium constant is measured by quantifying the amounts of target and control DNA IISRE cleavage products as a function of ligand concentration. The control sequence is engineered to not bind ligand. Along with illustrating these methods by reviewing published works, current concerns and future prospects for IISREF are discussed.

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Article Details

Year: 2002
Published on: 01 March, 2012
Page: [271 - 287]
Pages: 17
DOI: 10.2174/1386207023330309
Price: $65

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