Fluorescent microplate-based analysis of protein-DNA interactions II: immobilized DNA

Zhang, Zhan-Ren; Hughes, Marcus D.; Morgan, Leonie J.; Santos, Albert F.; Hine, Anna V.

doi:10.2144/03355st07

Cited by 9 publications

(13 citation statements)

References 20 publications

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“…2 C , 36 h cleavable). (Note that it is unlikely that GFP fluorescence would be detected after release from the microspheres, because the protein would be diffuse within the cells, and thus its cytoplasmic concentration would be too low; in optimized, extracellular experiments, the lowest concentration of free GFP that we have been able to detect is 0.4 n m (33). ) In contrast, when GFP was attached to the microspheres via the non-cleavable linker, green fluorescence remained unchanged after 36 h (Fig.…”

Section: Resultsmentioning

confidence: 88%

Polymeric Microspheres as Protein Transduction Reagents

Nagel

Behrendt

Chimonides

et al. 2014

Molecular & Cellular Proteomics

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Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere–protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.

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Section: Resultsmentioning

confidence: 88%

Polymeric Microspheres as Protein Transduction Reagents

Nagel

Behrendt

Chimonides

et al. 2014

Molecular & Cellular Proteomics

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“…Resulting libraries were accepted only if the accompanying plate contained a minimum of 120 colonies, which equates to 6120 individual clones within the liquid-culture library. Libraries were expressed and clarified lysates were prepared as described previously (31). …”

Section: Methodsmentioning

confidence: 99%

“…Randomized, GFP-labeled protein libraries were screened against immobilized double stranded DNA of sequence 5′-T 10 GGGXXXGCTT 10 -3′ and 5′ biotinylated complement (where XXX refers to the appropriate DNA triplet) as described previously (31). …”

Section: Methodsmentioning

confidence: 99%

“…The encoding strategy is designed to allow simple and rapid deconvolution of the data to elucidate sequences that bind optimally to each DNA triplet, thereby eliminating the need for either purification or for a linked sequence tag. Our studies focus on the randomization of three principal DNA-contacting residues in the second finger of a three-zinc finger protein, ZFH, described previously (30), fused to green fluorescent protein (GFP) to generate His 6 -ZFH-GFP (31). The principal DNA-contacting residues in fingers 1 and 3 were left unchanged to provide a constant framework within which to analyze binding of the second finger to any desired DNA triplet.…”

Section: Introductionmentioning

confidence: 99%

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Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

Hughes

Zhang²,

Sutherland³

et al. 2005

Nucleic Acids Research

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We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using ‘MAX’ randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40 000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.

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“…In another report, a direct "read out" of ZF-DNA interactions was performed directly in a bacterial lysate by expression of the ZF libraries fused with GFP with simultaneous biopanning with the DNA-target site [41][42]. Binding was measured in a high-throughput system by measuring GFP in a plate reader.…”

Section: In Vitro and In Vivo Strategies For The Se-lection Zf Domainmentioning

confidence: 99%

Rational Design, Selection and Specificity of Artificial Transcription Factors (ATFs): The Influence of Chromatin in Target Gene Regulation

Blancafort¹,

Beltran²

2008

CCHTS

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Artificial Transcription Factors (ATFs) are engineered DNA-binding proteins designed to bind specific sequences of DNA. ATFs made of Zinc Finger (ZF) domains have been developed to regulate specific genes and phenotypes both in cells and whole organisms. Recently, an emerging application of engineered DNA-binding domains include the specific editing of the genome, the ability to specifically cut, recombine, modify DNA and image protein-nucleic acid interactions in living cells. In this review we will summarize the techniques used for the rational design, screening and functional selection of ZF proteins in mammalian cell systems and their applications in areas of biotechnology, functional genomics and molecular therapeutics. The in vivo specificity of the engineered ATFs will be discussed, with particular emphasis on epigenetic modifications influencing ATF-DNA interactions.

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Fluorescent microplate-based analysis of protein-DNA interactions II: immobilized DNA

Cited by 9 publications

References 20 publications

Polymeric Microspheres as Protein Transduction Reagents

Polymeric Microspheres as Protein Transduction Reagents

Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

Rational Design, Selection and Specificity of Artificial Transcription Factors (ATFs): The Influence of Chromatin in Target Gene Regulation

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