DNA Sequence-Enabled Reassembly of the Green Fluorescent Protein

Abstract

We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers.

Bimolecular Fluorescence Complementation: Visualization of Molecular Interactions in Living Cells

“…Using this principle, RNA binding by the human zipcode-binding protein 1 ortholog (IMP1), the iron regulatory protein (IRP1), the fragile X mental retardation protein (FMRP), and the human Staufen homologue (hStau1) was visualized in living cells (Rackham and Brown, 2004). Likewise, co-occupancy by zinc-finger DNA binding proteins on oligonucleotides has been detected in vitro (Stains, et al, 2005). Complexes between the exon junction complex components Y14 and NXF1 were also detected only in the presence of newly synthesized transcripts (Schmidt, et al, 2006).…”

“…The association of such fragments can be conditional on their molecular proximity even if the fragments can associate independent of tethering when they are present at sufficiently high concentrations. The conditional association of protein fragments is a powerful reporter of molecular proximity, and can be used to investigate many biological processes that involve changes in molecular proximity, including protein interactions, nucleoprotein complex formation and covalent protein modifications (Hu, et al, 2002, Rackham and Brown, 2004, Fang and Kerppola, 2004, Stains, et al, 2005.…”

Novel In Vitro Protein Fragment Complementation Assay Applicable to High-Throughput Screening in a 1536-Well Format

“…[16][17][18] Furthermore, in vitro PCAs with split GFP have also been reported; however, these assays can be cumbersome because they require the purification, denaturing, and refolding of split GFP-fused proteins expressed in Escherichia coli. 14,19,20 In the present study, we developed an in vitro PCA using mKG that is applicable to HTS; the system is advantageous in that it is very easy and simple to handle and does not require protein purification and refolding (Fig. 1).…”

Recent progress in the construction methodology of fluorescent biosensors based on biomolecules

“…(Ghosh, I. et al 2000) Based on this strategy, a receptor composed of two subunits that are associated by binding to the analyte can be converted into a fluorescent biosensor by connecting each of the two subunits with each split AFP fragment (Figure 2). Actually, several types of biosensors have been developed for fluorescent detection of specific DNA sequences (Stains, C. I. et al 2005;Demidov, V. V. et al 2006), DNA methylation (Stains, C. I. et al 2006), mRNA (Ozawa, T. et al 2007;Valencia-Burton, M. et al 2007) and protein interactions (Nyfeler, B. et al 2005;Hu, C. -D. et al 2003;Wilson, C. G. et al, 2004). Unlike the above-mentioned split AFP reconstitution, in which split AFP halves reform into a fluorescent structure via noncovalent association, another reconstitution strategy, inteinmediated reconstitution, has been developed by Ozawa and co-workers .…”