Sequence-Enabled Reassembly of β-Lactamase (SEER-LAC):  A Sensitive Method for the Detection of Double-Stranded DNA

• Published in: Biochemistry (Easton) Vol. 45; no. 11; pp. 3620 - 3625
• Main Authors: Ooi, Aik T, Stains, Cliff I, Ghosh, Indraneel, Segal, David J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.03.2006
• Subjects: Base Pair Mismatch; Base Sequence; beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; Cephalosporins - metabolism; Cephalosporins - pharmacology; DNA - analysis; DNA - metabolism; DNA Probes - chemistry; DNA Probes - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Enzyme Activation; Eukaryotic Cells - enzymology; Eukaryotic Cells - metabolism; Feasibility Studies; Hydrolysis; Indicators and Reagents - metabolism; Indicators and Reagents - pharmacology; Molecular Sequence Data; Mutation; Prokaryotic Cells - enzymology; Prokaryotic Cells - metabolism; Protein Structure, Tertiary; Structure-Activity Relationship; Zinc Fingers
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0517032

This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), was demonstrated in vitro to produce a DNA-concentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM-1 β-lacatamase. This system could distinguish target DNA from nontarget DNA in less than 5 min, representing a more than 1000-fold improvement over our previous SEER design. A single base-pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic DNA when present in equal mass to the target DNA. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences.