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. 2006 Mar 21;45(11):3620-5.
doi: 10.1021/bi0517032.

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

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

Aik T Ooi et al. Biochemistry. .
Free PMC article

Abstract

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 beta-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.

Figures

Figure 1
Figure 1
The SEER-LAC strategy. LacA-Zif268 comprises residues 26-196 of β-lactamase (green) fused by a 15-aa linker (orange) to the DNA binding ZF Zif268 (blue). PBSII-LacB comprises the ZF PBSII (red) fused by a 15-aa linker (magenta) to residues 198-290 of β-lactamase (yellow). In the presence of target DNA containing binding sites for Zif268 (cyan) and PBSII (pink) with an appropriate spacer (0-bp spacer is shown), SEER fragments reassemble to form an active reporter enzyme.
Figure 2
Figure 2
The DNA concentration-dependent SEER signal. A) Digital image of triplicate nitrocefin assays after 30 minutes incubation. DNA target oligonucleotides (with target site spacings of 0, 6 and 10 bp) and their concentrations are indicated above the image; SEER fragments (0.5 μM each) are indicated to the left. B) Relative Signal vs. DNA concentration plot for the assay shown in A. Relative Signal (normalized and background subtracted) after 20 minutes of incubation was calculated on Zif-0-PBSII DNA (blue diamonds), Zif-6-PBSII DNA (gray squares), and Zif-10-PBSII DNA (purple circles) as described in the Experimental Procedures. C) Kinetic data for the reactions with Zif-0-PBSII DNA. Absorbance at 486 nm was measured at 3 minutes and every 2 minutes after. Raw data were plotted for LacA-Zif268 & PBSII-LacB with 1 μM DNA (red diamonds), 20 nM (orange diamonds), 200 pM (yellow diamonds), and no DNA (black triangles), as well as the non-cognate LacA-Zif268 & PE1A-LacB (green diamonds) with 1 μM Zif-0-PBSII DNA. Hydrolysis rates were calculated as the slope of the linear fit of the kinetic data (solid lines).
Figure 3
Figure 3
Sensitivity of SEER to mutations in the target DNA. A) Digital image of triplicate nitrocefin assays after 30 minutes incubation. A series of modified Zif-0-PBSII target oligonucleotides were used at 1 μM, containing 1, 2, 3 or 5 G to T substitutions (boxed) in the Zif268 (blue) or PBSII (red) binding sites, as indicated. SEER fragments LacA-Zif268 & PBSII-LacB were used at 0.5 μM each. B) Graphical representation of the relative signal intensities after 20 minutes of incubation for the assay shown in A.
Figure 4
Figure 4
SEER activity using various combinations of ZF binding domains and DNA targets. The relative signal intensities after 20 minutes of incubation for triplicate nitrocefin assays are shown. Target oligonucleotides at 1 μM are indicated above the graph (black and white bars); SEER fragments at 0.5 μM each are indicated below.
Figure 5
Figure 5
SEER binding in the presence of genomic DNA. LacA-Zif268 & PBSII-LacB at 0.5 μM each were incubated with 1 μM Zif-0-PBSII (black bars) or 1 μM Zif-0-PE1A (white bars) for 20 minutes in the presence or absence (as indicated) of 3.2 μg of sheared, double-stranded Herring Sperm DNA. This concentration is equal in moles of base pairs (5.2 nmoles bp) to 1μM of the target oligonucleotide.

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