Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates

Z-Lysine (ZLys) is a lysine derivative with a benzyloxycarbonyl group linked to the ε-nitrogen. It has been genetically encoded with the UAG stop codon, using the pair of an engineered variant of pyrrolysyl-tRNA synthetase (PylRS) and tRNA^Pyl. In the present study, we designed a novel Z-lysine derivative (AmAzZLys), which is doubly functionalized with amino and azido substituents at the meta positions of the benzyl moiety, and demonstrated its applicability for creating protein conjugates. AmAzZLys was incorporated into proteins in Escherichia coli, by using the ZLys-specific PylRS variant. AmAzZLys was then site-specifically incorporated into a camelid single-domain antibody specific to the epidermal growth factor receptor (EGFR). A one-pot reaction demonstrated that the phenyl amine and azide were efficiently linked to the 5 kDa polyethylene glycol and a fluorescent probe, respectively, through specific bio-orthogonal chemistry. The antibody was then tested for the ability to form a photo-cross-link between its phenylazide moiety and the antigen, while the amino group on the same ring was used for chemical labeling. When incorporated at a selected position in the antibody and exposed to 365 nm light, AmAzZLys formed a covalent bond with the EGFR ectodomain, with the phenylamine moiety labeled fluorescently prior to the reaction. The present results illuminated the versatility of the ZLys scaffold, which can accommodate multiple reactive groups useful for protein conjugation

Inhibition of DNA Methylation at the <i>MLH1</i> Promoter Region Using Pyrrole–Imidazole Polyamide

Aberrant DNA methylation causes major epigenetic changes and has been implicated in cancer following the inactivation of tumor suppressor genes by hypermethylation of promoter CpG islands. Although methylated DNA regions can be randomly demethylated by 5-azacytidine and 5-aza-2′-deoxycytidine, site-specific inhibition of DNA methylation, for example, in the promoter region of a specific gene, has yet to be technically achieved. Hairpin pyrrole (Py)–imidazole (Im) polyamides are small molecules that can be designed to recognize and bind to particular DNA sequences. In this study, we synthesized the hairpin polyamide MLH1_–16 (Py-Im-β-Im-Im-Py-γ-Im-Py-β-Im-Py-Py) to target a CpG site 16 bp upstream of the transcription start site of the human <i>MLH1</i> gene. <i>MLH1</i> is known to be frequently silenced by promoter hypermethylation, causing microsatellite instability and a hypermutation phenotype in cancer. We show that MLH1_–16 binds to the target site and that CpG methylation around the binding site is selectively inhibited in vitro. MLH1_non, which does not have a recognition site in the <i>MLH1</i> promoter, neither binds to the sequence nor inhibits DNA methylation in the region. When MLH1_–16 was used to treat RKO human colorectal cancer cells in a remethylating system involving the <i>MLH1</i> promoter under hypoxic conditions (1% O₂), methylation of the <i>MLH1</i> promoter was inhibited in the region surrounding the compound binding site. Silencing of the <i>MLH1</i> expression was also inhibited. Promoter methylation and silencing of <i>MLH1</i> were not inhibited when MLH1_non was added. These results indicate that Py–Im polyamides can act as sequence-specific antagonists of CpG methylation in living cells

Chromatin accessibility of KR12.

<p>(A) Position-coverage plot of KR12 binding (9 bp per point, black and red) in relation to DHS regions (10,000 bp per point, gray) per chromosome. Horizontal axes, relative position along chromosome; vertical axes, relative coverage of a feature; for DHS regions, a coverage of 100% indicates that all 10,000 bp of a genomic region have DHS, while for KR12 sites, black spots (0%) indicate that a particular site is outside the boundaries of a DHS region, in contrast to the red sites (> 0%). (B) Gene expressions (vertical,–log₂FC) as a function of KR12 site proximity (horizontal, 1000 bp) to the histone modification feature H3K27Ac. Spearman’s correlation coefficient (<i>ρ</i> = –0.171) suggests a weak correlation with histone modification. (C) Comparison of predicted binding site counts in coding regions (CDS) and in the hg19 genome for KR12 (red) against 100 randomly selected motifs (black, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165581#pone.0165581.s010" target="_blank">S4 Table</a>); horizontal axis, ratio of binding sites per motif/KR12 binding sites; vertical axis, ratio of binding sites in CDS per motif/KR12. Dashed line with slope of 1 (gray) is provided as reference.</p

Genome-wide effect of KR12 binding and implications of mutant codon 12 <i>KRAS</i> as a driver gene.

<p>(A) Mean expressions (vertical axis,–log₂FC) from RNA microarray analysis. Left: genome-wide KR12 binding (KR12-bound ‘+’, black; otherwise ‘–’, gray); middle: genes with computationally predetermined motif matches (‘+’) and those without (‘–’); right: expressions of genes with identified sites (‘+’) compared to genes with motif matches but no binding (‘–’) as determined by the workflow. <i>**</i>, <i>p</i> < .01 from two-sample Welch’s <i>t</i>-test; error bars indicate ±1 SEM. (B) Mean expressions (–log₂FC) of identified KR12-bound genes (“Post-validation”) compared to candidate genes following sliding-window determination (“Pre-validation”); “Difference” indicates pre-validation candidates not found in the Post-validation group. Error bars, ±1 SEM; <i>**</i>, <i>p</i> < .01 from two-sample Welch’s <i>t</i>-test. (C) Interaction network of down-regulated KR12-bound genes. <i>KRAS</i> (black) and its first neighbors (blue, marked with green arrows) are linked by solid black edges. Blue dashed edges indicate direct interactions with first neighbors of <i>KRAS</i>.</p

KR12 binding in the human colorectal carcinoma LS180 genome.

<p>(A) Workflow to identify KR12 binding sites in the LS180 genome by IonTorrent sequencing. Cells are administered with KR12 (500 nM, 6 h) prior to genomic DNA extraction and fragmentation by sonication. Enrichment by streptavidin allows the capture of KR12-bound nucleotides. A computational routine maps candidate regions in sequencing data, followed by site calling via motif matching and statistical validation. Subsequent microarray analyses provide the means to confirm binding data with genome-level changes. (B) Sample sequencing coverage of <i>PIK3CA</i> and <i>KRAS</i>, genes with KR12 binding sites (“KR12+”), and for reference, a predicted site by motif matching but non-binding (“KR12–”) in <i>GUSB</i>; windows centered around a KR12 site (black arrow, dashed line) are within –500 to +500 bp; blue and orange arrows indicate cumulative coverage for the pulldown and input tracks, respectively. (C) Semi-quantitative PCR of <i>RPS18</i>, <i>KRAS</i>, <i>PIK3CA</i> and <i>GUSB</i> in the presence of 500 nM KR12 or DMSO as control. “Motif match” and “binding” indicate whether a particular gene contains a computationally predetermined match to the KR12 motif in the hg19 genome or a KR12 binding site determined by sequencing analysis, respectively.</p

Statistically overrepresented pathways for KR12-bound genes, compared to members in the Ras pathway (hsa04014) alone.

<p>Fold enrichment between two groups have a <i>χ</i>^<i>2</i> statistic of 30.958 (<i>p</i> ~ 1.43 × 10⁻⁴).</p

Next-generation sequencing with biotinylated KR12.

<p>(A) Synthetic scheme of KR12 from the non-biotinylated precursor KR12 N/B, with pyrroles and imidazoles colored in blue and red, respectively. (B) HPLC retention time diagram (upper right) and mass spectrum (LC-MS) of KR12. (C) WST assay of LS180 cells at 300 and 1000 nM dosage of KR12 (black) compared to the non-biotinylated precursor (KR12 N/B, gray) and DMSO only (white); error bars indicate ±1 SEM; n.s., no significance by two-sample Welch’s <i>t</i>-test.</p

Effects of injection of pyrrole-imidazole (PI) polyamide targeting human transforming growth factor (TGF)-β1 promoter (GB1101) on development of hypertrophic scars.

<p>Hypertrophic scars were created on marmoset abdomen skin by scalpel incision. A mixture of 100 μg of GB1101 and mismatch PI polyamides dissolved in 500 μl of H₂O (Water) was subcutaneously injected into the wound site just before incision. (A) Typical macroscopic changes on post-incision days 21 and days 35 (two experiments) from 5 experiments. Arrows indicate hypertrophic scars. Bar = 5 mm. (B) Sections of 6 μm in thickness at 35 days post-incision from 4 experiments were stained with hematoxylin-eosin. Arrows indicate dermal thickness of hypertrophic scars. Bar = 500 μm. (C) Comparison of water, mismatch PI polyamide and GB1101 injection on epidermal thickness of hypertrophic scars at 35 days post-incision. Values are expressed as mean ± SE (n = 4). * <i>p</i> < 0.01 vs. Mismatch PI polyamide.</p

Binding sites of synthetic pyrrole-imidazole (PI) polyamides (GB1101-1107) on human transforming growth factor-β1 promoter sequence.

<p>Arrow: Major transcription initiation points, Open box: transcription factor binding sites, Underlines: PI polyamide binding sites. FSE2: fat-specific element 2, AP-1: activating protein-1, NF-1: nuclear factor-1.</p

Structures and target sequences of pyrrole-imidazole polyamides (GB1101, GB1105 and GB1106) targeting to the human transforming growth factor-β1 promoter and structure of mismatch polyamide.

<p>Box indicates fat-specific element 2 (FSE2) binding element or activating protein-1 (AP-1) binding element.</p