Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer.

Climate change and rapid urbanization are important factors restricting future land use. Situational analysis, as an important foundation for the optimization of land use, needs to focus on the impact of climate factors and socio-economic factors. In this paper, the Markov model and the DLS (Simulation of Land System Dynamics) model are combined for the first time, and the land use pattern in 2020 is simulated based on the data of land use in 2000 and 2010 as well as the climate, soil, topography and socio-economic factors of Yunnan Province. In his paper, we took Yunnan Province as the case study area, and selected 12 driving factors by logistic regression method, then the land use demands and layout of Yunnan Province in 2020 has been forecasted and simulated under business as usual (BAU) scenario and farmland protection (FP) scenario and the changes in ecosystem service value has been calculated. The result shows that: (1) after the regression analysis and ROC (Relative Operating Characteristics) test, the 12 factors selected in this paper have a strong ability to explain the land use change in Yunnan Province. (2) Under the two scenarios, the significant reduction of arable land area is a common feature of land use change in Yunnan Province in the future, and its main land use type will be construction land. However, under FP scenario, the current situation where construction land encroach on arable land will be improved. Compared with the change from 2000 to 2010, the trend of arable land, forest land, water area, construction land and unused land will be the same under the two scenarios, whereas the change trend of grassland was opposite. (3) From 2000 to 2020, the value of ecosystem services in Yunnan Province is on the rise, but the ecosystem service value under FP scenario is higher than that of the ecosystem services under BAU scenario. In general, land use in 2020 in Yunnan Province continues the pattern of 2010, but there are also significant spatial differences. Under the BAU scenario, the construction land is mainly in the south of Lijiang City and the northeastern part of Kunming. Under the FP scenario, the new construction land is concentrated near the Lashi dam in northern Yunnan Province, and the high-quality arable land in the valley will be better protected. The research results can provide reference for the optimization of land use pattern in Yunnan Province, and provide scientific basis for land use management and planning. Based on the value of ecosystem services, we should implement the policy of strict protection of arable land, both to ensure food supply and promote the healthy development of ecological environment.

1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet-to-be-reported side products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show that the trends established with model peptides hold for more complex systems.

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LF N ) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore.

Mirror image peptides have unique stability and immunogenic properties in mammals, making them attractive agents to investigate.

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript.

Antibody mimics have significant scientific and therapeutic utility for the disruption of protein-protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein-protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.

Mirror image peptides have unique stability and immunogenic properties in mammals, making them attractive agents to investigate. Their properties inside cells have been mostly unexplored because biopolymers are difficult to transport across cellular membranes. Here, we used protective antigen (PA) from anthrax toxin to deliver mirror image polypeptide cargo into the cytosol of mammalian cells when conjugated to the C-terminus of the PA-binding domain of lethal factor, LFN. We found mirror image polypeptides and proteins were translocated as efficiently into cells as their L counterparts. Once in the cytosol, by the use of western blot, we found that d peptides at the C-terminus of LFN were able to achieve higher steady state concentrations when compared to the l-peptide conjugate. With this platform, we delivered a d-peptide MDM2 antagonist to disrupt the p53/MDM2 interaction in cancer cells. For the first time, we show the PA/LFN system is adaptable for the intracellular delivery of mirror image peptides and proteins.

Sortase-mediated ligation (sortagging) is a versatile, powerful strategy for protein modification. Because the sortase reaction reaches equilibrium, a large excess of polyglycine nucleophile is often employed to drive the reaction forward and suppress sortase-mediated side reactions. A flow-based sortagging platform employing immobilized sortase A within a microreactor was developed that permits efficient sortagging at low nucleophile concentrations. The platform was tested with several reaction partners and used to generate a protein bioconjugate inaccessible by solution-phase batch sortagging.

Mirror image peptides have unique stability and immunogenic properties in mammals, making them attractive agents to investigate. Their properties inside cells have been mostly unexplored because biopolymers are difficult to transport across cellular membranes. Here, we used protective antigen (PA) from anthrax toxin to deliver mirror image polypeptide cargo into the cytosol of mammalian cells when conjugated to the C-terminus of the PA-binding domain of lethal factor, LFN. We found mirror image polypeptides and proteins were translocated as efficiently into cells as their L counterparts. Once in the cytosol, by the use of western blot, we found that D peptides at the C-terminus of LFN were able to achieve higher steady state concentrations when compared to the L-peptide conjugate. With this platform, we delivered a D-peptide MDM2 antagonist to disrupt the p53/MDM2 interaction in cancer cells. For the first time, we show the PA/LFN system is adaptable for the intracellular delivery of mirror image peptides and proteins.

The post-translational modification of proteins is controlled by the relative activities of two opposing enzymes. For example, the extent of phosphorylation of tyrosine residues reflects the balance of a kinase and a phosphatase enzyme. The present article uses as a model system a self-assembled monolayer that presents a peptide that can be phosphorylated by Abl kinase and subsequently dephosphorylated by Lambda phosphatase. Treatment of monolayers with a reaction mixture containing both enzymes reveals that the steady-state level of peptide phosphorylation is dependent on the density of the peptide. Using identical reaction mixtures, surfaces that presented the substrate at high density led to a phosphorylated peptide at steady-state, whereas surfaces that presented the substrate at low density led to unphosphorylated peptide at steady-state. This dependence owes to an autocatalytic phosphorylation reaction that operates at high densities of substrate. This work provides an example of an interfacial reaction that has properties that have no analogue in the corresponding solution phase reaction. It also provides a model system that is relevant to understanding mechanisms that regulate signaling at the cellular membrane.

Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal (α)thioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an (α)thioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation.

Surfing with the kinase: A soluble kinase phosphorylates an immobilized peptide substrate most rapidly at the boundary between product and substrate, and gives rise to a “wave” of propagating product. Such systems offer an approach to engineering reactions to give spatio-temporal control over progression of reactions. Unlike reactions performed in the laboratory—which use homogeneous solutions having spatially uniform concentration profiles of each reactant—reactions that occur in biological systems often involve reactants that are present in concentration gradients and can display unexpected kinetic properties.1–5 Such biological reactions can generate spatio-temporal structures that derive from the inclusion of feedback, autocatalysis, and other non-linear influences on rate.6–9 While these examples are prevalent in biology, it is still challenging to engineer such systems in the laboratory.10–13 Here, we demonstrate a reaction wherein a soluble kinase enzyme phosphorylates a peptide that is immobilized to a self-assembled monolayer (SAM) and that proceeds with a spatially organized propagation of the product. This spatial control over the reaction derives from an autocatalytic feature that operates at the boundary between the substrate and product. Our system is based on Abelson tyrosine kinase (Abl) which has both a catalytic domain and a Src homology 2 (SH2) domain that binds to the phosphopeptide product of the phosphorylation reaction. For example, Abl phosphorylates the substrate peptide AIYENPFARKC to give AIpYENPFARKC (which we abbreviate as Y and pY, respectively), and the kinase can then bind to pY. Previously, we used SAMs of alkanetiolates on gold to demonstrate that Abl can operate autocatalytically on an immobilized substrate (Figure 1 a).14 The SAMs presented a peptide substrate for Abl kinase against a background of tri(ethylene glycol) groups, which render the monolayers inert to protein adsorption,15 and the peptides could be characterized with matrix-assisted laser desorption–ionization mass spectrometry (i.e., the SAMDI method).16–18 The autocatalytic kinetic property arises due to the binding of the phosphopeptide product to the SH2 domain of the kinase, which then recruits the kinase to the surface, giving an approximately 30-fold increase in the rate for phosphorylation of nearby peptides.14 Hence, the phosphorylation reaction is fastest in those regions of the monolayer that are directly adjacent to the regions that present product. Here, we explicitly characterize the spatial propagation of the reaction product and we also show that patterned monolayers display initial rates that depend on the geometrical features of the pattern, but not the relative amounts of substrate and product. a) Treatment of a monolayer that presents a peptide with Abl kinase and ATP results in phosphorylation of the peptide, which can be detected by mass spectrometry that reveals the corresponding mass change of 80 m/z. b) Schematic of the reaction front propagating away from regions initially patterned with the phosphopeptide, where the reaction front is indicated in green, the SH2 domain of Abl kinase in purple, and the catalytic domain in red. c) Regions of phosphopeptide (green) were initially patterned on the surface using an agarose stamp. The remainder of the surface was backfilled with the unphosphorylated peptide (black). d) The patterned surface was treated with Abl kinase for different times, and the reaction propagation was visualized using an antiphosphotyrosine antibody. Scale bar=25 μm. We patterned a monolayer into alternate regions that presented the phosphopeptide and the peptide substrate. We reasoned that those peptide substrates located immediately adjacent to regions that present phosphopeptide would be phosphorylated most rapidly, because only at the boundary can tethered enzyme interact with the substrate. Hence, we expected that the reaction would show a spatial propagation in time (Figure 1 b). To test this hypothesis and to characterize the spatial velocity with which the reaction proceeds, we used an agarose stamp that was inked with the phosphopeptide to pattern a maleimide-terminated monolayer with a parallel set of striped regions that presented the peptide. We removed the stamp and treated the monolayer with the unphosphorylated peptide to backfill the remaining regions. We then treated the entire surface with a solution containing Abl kinase and ATP. Parallel reactions were performed and individual reactions were stopped at different times and treated with an antibody that binds phosphotyrosine and a fluorescently labeled secondary antibody (Figure 1 c). In this way, we could spatially resolve the phosphorylated peptide on the surface and characterize the time course of the reaction. The bright regions correspond to the phosphorylated peptide and black regions correspond to the unphosphorylated peptide. As expected, the stripe having product broadens with increasing reaction times, and is consistent with a propagation of the reaction from the initially patterned boundary between phosphorylated and unphosphorylated peptide (Figure 1 d). The reaction propagated over distances greater than 10 µm. We measured a velocity of 0.87 μm min−1 for a surface presenting the peptide substrate at a density of 1 % relative to total alkanethiolates. The experiment was repeated using a monolayer that presented the peptide at a density of 0.5 % and we measured a propagation velocity for the reaction of 0.51 μm min−1 (Figure 2 a). This lower velocity is consistent with our previous observation that the autocatalytic reaction has a strong dependence on substrate density, and shows a greater rate increase for monolayers that present the substrate at higher density.14 We have not determined whether sequential phosphorylation reactions are catalyzed by a single kinase—where the enzyme repeatedly dissociates and reassociates to the newly formed phosphopeptide—or whether new kinase enzymes are recruited from the bulk region. Therefore, we do not know whether the use of an SH2 domain having a higher dissociation rate constant will lead to a larger propagation velocity. In contrast to molecular motors, which move directionally along a pattern of ligand, in our system, the propagating species is not the enzyme itself, but the product peptides, and the propagation direction is determined by the spatial organization of the product and the substrate on the surface.19, 20 We also tested whether the rate of spatial propagation depends on the intrinsic activity of the kinase enzyme. We used a constant concentration of ATP (200 μM) in the reaction buffer but included increasing concentrations of the non-hydrolyzable ATP analogue adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) which is a competitive inhibitor for ATP and therefore reduces the turnover number of the kinase. Indeed, we found that the propagation rate decreases with increasing concentrations of AMP-PNP (Figure 2 b), demonstrating that the kinase activity was partially inhibited. a) The reaction velocity depends on the density of the immobilized peptide substrate. Reactions were stopped after different times and imaged with fluorescence microscopy. A plot of the distance of reaction propagation versus time was linear and has a slope that corresponds to the reaction velocity. ▪: 0.5 % substrate density, dx/dt=0.51 µm min−1, R2=0.965; •: 1.0 % substrate density, dx/dt=0.87 µm min−1, R2=0.997. b) The reaction velocity decreases in the presence of the non-hydrolyzable ATP analogue AMP-PNP. The finding that the reaction proceeds most rapidly at the boundary between phosphorylated and unphosphorylated peptides suggests that relative rates for a reaction on patterned surfaces would depend on the spatial arrangement of the pattern and not only on the ratio of the two peptides on the surface. If the rate is averaged over a region of the surface that is large compared to the feature sizes of the patterns, we reasoned that it would increase with the “boundary density” of the pattern, defined as the integrated length of the perimeter of phosphopeptide-terminated regions of the monolayer per unit area of the monolayer. To explore this possibility, we utilized four patterns that had the phosphopeptide organized in circular features or zig-zag lines, each at two different sizes (Figure 3 a). The small circles had a larger boundary density than did the large circles, and likewise for the zig-zag lines. We treated the patterned monolayers with kinase for times ranging from 0 to 30 min and then used SAMDI mass spectrometry to analyze the monolayers to determine the average yield of the reaction. We found that the initial rate of reaction (rinitial, again which represent a rate averaged over the patterned region) increased linearly with the boundary density (Figure 3 b). We also investigated how the binding affinity of the phosphopeptide for the SH2 domain affects the rate. We compared the reaction on two patterned monolayers that each presented the same peptide substrate but with one of two different phosphopeptides that differed in their affinity for the SH2 domain. The peptide sequence AIpYENPFARKC (black curve) has a higher affinity for the SH2 domain than does the pYAAPFKC (red curve) (Figure 3 b). We found that the higher affinity phosphopeptide led to a greater initial rate. These results demonstrate that the rate of the reaction can be manipulated by controlling the spatial organization of product and substrate on the monolayer and by adjusting the affinity of the phosphopeptide for the SH2 domain. a) Monolayers were patterned with four distinct geometries that had a similar overall ratio of substrate to product, but with varying boundary densities as described in the text. The icon in the lower left of each image corresponds to its position on the graph. b) The reaction kinetics are sensitive to the initial geometry of patterned phosphopeptide, where the initial rate for patterned surfaces (rinitial) increases with the boundary density (r0 is the initial rate of non-patterned surface, serving as the background rate). The black curve represents the high affinity phosphopeptide and the red curve represents the lower affinity phosphopeptide. Scale bar=100 μm. This study demonstrates a reaction system that shows a spatial propagation of product, despite the uniform concentration of enzyme catalyst throughout the system. This example is significant because it describes an approach to engineering reactions to give spatio-temporal control over the products and progression of reactions in a system. The use of self-assembled monolayers was important to this work for several reasons.21 First, they allow the placement of reactive groups in a uniform environment on the surface and offer good control over the densities and geometric patterns of the reactants. The compatibility of monolayers with a variety of analytical techniques, including mass spectrometry and fluorescence microscopy, allows reaction kinetics and spatial structure to be analyzed, and the use of immobilized reactants is important for eliminating diffusion and effectively stabilizing the spatial distributions of the products. We believe that this and related approaches offer a new opportunity to create reaction systems that share the emergent properties that are so common in biological reaction networks.22–26 Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

This paper describes a model system for studying the autocatalytic phosphorylation of an immobilized substrate by a kinase enzyme. This work uses self-assembled monolayers (SAMs) of alkanethiolates on gold to present the peptide substrate on a planar surface. Treatment of the monolayer with Abl kinase results in phosphorylation of the substrate. The phosphorylated peptide then serves as a ligand for the SH2 adaptor domain of the kinase and thereby directs the kinase activity to nearby peptide substrates. This directed reaction is intramolecular and proceeds with a faster rate than does the initial, intermolecular reaction, making this an autocatalytic process. The kinetic non-linearity gives rise to properties that have no counterpart in the corresponding homogeneous phase reaction: in one example, the rate for phosphorylation of a mixture of two peptides is faster than the sum of the rates for phosphorylation of each peptide when presented alone. This work highlights the use of an adaptor domain in modulating the activity of a kinase enzyme for an immobilized substrate and offers a new approach for studying biochemical reactions in spatially inhomogeneous settings.