Donor-Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light.

Tryptophan (Trp) plays a variety of critical functional roles in protein biochemistry; however, owing to its low natural frequency and poor nucleophilicity, the design of effective methods for both single protein bioconjugation at Trp as well as for in situ chemoproteomic profiling remains a challenge. Here, we report a method for covalent Trp modification that is suitable for both scenarios by invoking photo-induced electron transfer (PET) as a means of driving efficient reactivity. We have engineered biaryl N-carbamoyl pyridinium salts that possess a donor-acceptor relationship that enables optical triggering with visible light whilst simultaneously attenuating the probe's photo-oxidation potential in order to prevent photodegradation. This probe was assayed against a small bank of eight peptides and proteins, where it was found that micromolar concentrations of the probe and short irradiation times (10-60 min) with violet light enabled efficient reactivity toward surface exposed Trp residues. The carbamate transferring group can be used to transfer useful functional groups to proteins including affinity tags and click handles. DFT calculations and other mechanistic analyses reveal correlations between excited state lifetimes, relative fluorescence quantum yields, and chemical reactivity. Biotinylated and azide-functionalized pyridinium salts were used for Trp profiling in HEK293T lysates and in situ in HEK293T cells using 440 nm LED irradiation. Peptide-level enrichment from live cell labeling experiments identified 290 Trp modifications, with 82% selectivity for Trp modification over other π-amino acids, demonstrating the ability of this method to identify and quantify reactive Trp residues from live cells.

An engineered calmodulin-based allosteric switch for Peptide biosensing.

This work describes the development of a new platform for allosteric protein engineering that takes advantage of the ability of calmodulin to change conformation upon binding to peptide and protein ligands. The switch we have developed consists of a fusion protein in which calmodulin is genetically inserted into the sequence of TEM1 ß-lactamase. In this approach, calmodulin acts as the input domain, whose ligand-dependent conformational changes control the activity of the ß-lactamase output domain. The new allosteric enzyme exhibits up to 120 times higher catalytic activity in the activated (peptide bound) state compared to the inactive (no peptide bound) state in vitro. Activation of the enzyme is ligand-dependent-peptides with higher affinities for wild-type calmodulin exhibit increased switch activity. Calmodulin's ability to "turn on" the activity of ß-lactamase makes this a potentially valuable scaffold for the directed evolution of highly specific biosensors for detecting toxins and other clinically relevant biomarkers.

Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells.

The ability to target methylation to specific genomic sites would further the study of DNA methylation's biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5'-YGGCCR-3', we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.

An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity.

The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency.

Targeted DNA methylation in human cells using engineered dCas9-methyltransferases.

Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.

Partial acetylation of polyethylenimine enhances in vitro gene delivery.

PURPOSE: Polyethylenimine (PEI) is a highly effective gene delivery vector, but because it is an off-the shelf material, its properties may not be optimal. To investigate the effects of the protonation properties of the polymer, we generated PEI derivatives by acetylating varying fractions of the primary and secondary amines to form secondary and tertiary amides, respectively. METHODS: Reaction of PEI with increasing amounts of acetic anhydride at 60 degrees C for 4.5 h yielded polymers with 15%, 27%, and 43% of the primary amines modified with acetyl groups. Polymer-DNA complexes were characterized by dynamic light scattering and zeta potential measurements. Cytotoxicity of the polymers was assessed by XTT assay for metabolic activity, and gene delivery efficiency was determined as the relative expression of a luciferase gene in MDA-MB-231 and C2C12 cell lines. RESULTS: Acetylation of PEI decreased the "physiological buffering capacity," defined as the moles of protons absorbed per mole of nitrogen on titration from pH 7.5 to 4.5, from 0.29 mol H+/mol N to 0.17 mol H+/mot N, 0.12 mol H+/mol N, and 0.090 mol H+/mol N for PEI-Ac15, PEI-Ac27, and PEI-Ac43, respectively. In addition, acetylation decreased the zeta potential of polyplexes from 14 mV to 8-11 mV and increased the polyplex diameter by two- to threefold. Surprisingly, acetylation had a negligible effect on cytotoxicity of the polymers and increased gene delivery effectiveness by up to 21-fold compared to unmodified PEI, both in the presence and absence of serum. CONCLUSIONS: Reduction of the buffering capacity of PEI greatly enhanced the gene delivery activity of the polymer. The mechanism is not yet understood, but the enhancement may be caused by more effective polyplex unpackaging, altered endocytic trafficking, and/or increased lipophilicity of acetylated PEI-DNA complexes. Future studies will address these possibilities in more detail.