Generalizable Strategy Research Articles

Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria.

Here we describe a universal approach for plasmid-free genome engineering in cyanobacteria that exploits the polyploidy of their chromosomes as a natural counterselection system. Rather than being delivered via replicating plasmids, genes encoding for DNA modifying enzymes are instead integrated into essential genes on the chromosome by allelic exchange, as facilitated by antibiotic selection, a process that occurs readily and with only minor fitness defects. By virtue of the essentiality of these integration sites, full segregation is never achieved, with the strain instead remaining as a merodiploid so long as antibiotic selection is maintained. As a result, once the desired genome modification is complete, removal of antibiotic selection results in the gene encoding for the DNA modifying enzyme to then be promptly eliminated from the population. Proof of concept of this new and generalizable strategy is provided using two different site-specific recombination systems, CRE-lox and DRE-rox, in the fast-growing cyanobacterium Synechococcus sp. PCC 7002, as well as CRE-lox in the model cyanobacterium Synechocystis sp. PCC 6803. Reusability of the method, meanwhile, is demonstrated by constructing a high-CO2 requiring and markerless Δndh3 Δndh4 ΔbicA ΔsbtA mutant of Synechococcus sp. PCC 7002. Overall, this method enables the simple and efficient construction of stable and unmarked mutants in cyanobacteria without the need to develop additional shuttle vectors nor counterselection systems.

Finding and removing Clever Hans: Using explanation methods to debug and improve deep models

Contemporary learning models for computer vision are typically trained on very large (benchmark) datasets with millions of samples. These may, however, contain biases, artifacts, or errors that have gone unnoticed and are exploitable by the model. In the worst case, the trained model does not learn a valid and generalizable strategy to solve the problem it was trained for, and becomes a “Clever Hans” predictor that bases its decisions on spurious correlations in the training data, potentially yielding an unrepresentative or unfair, and possibly even hazardous predictor. In this paper, we contribute by providing a comprehensive analysis framework based on a scalable statistical analysis of attributions from explanation methods for large data corpora. Based on a recent technique — Spectral Relevance Analysis — we propose the following technical contributions and resulting findings: (a) a scalable quantification of artifactual and poisoned classes where the machine learning models under study exhibit Clever Hans behavior, (b) several approaches we collectively denote as Class Artifact Compensation, which are able to effectively and significantly reduce a model’s Clever Hans behavior, i.e., we are able to un-Hans models trained on (poisoned) datasets, such as the popular ImageNet data corpus. We demonstrate that Class Artifact Compensation, defined in a simple theoretical framework, may be implemented as part of a neural network’s training or fine-tuning process, or in a post-hoc manner by injecting additional layers, preventing any further propagation of undesired Clever Hans features, into the network architecture. Using our proposed methods, we provide qualitative and quantitative analyses of the biases and artifacts in, e.g., the ImageNet dataset, the Adience benchmark dataset of unfiltered faces, and the ISIC 2019 skin lesion analysis dataset. We demonstrate that these insights can give rise to improved, more representative, and fairer models operating on implicitly cleaned data corpora.

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

A generalizable strategy for constructing ultralight three-dimensional hierarchical network heterostructure as high-efficient microwave absorber

Using previous models and theories to construct and develop high-efficient microwave absorbers (MAs) should be a strategic and effective ways to optimize the electromagnetic wave attenuation. Herein, the ultralow density and flexible graphene oxide foam (GOF) and reduced graphene oxide foam (RGOF)/MoS2 nanosheets were designed and fabricated by the method of chemical vapor deposition and hydrothermal reaction. The obtained GOF and RGOF/MoS2 samples exhibited very excellent microwave absorption properties while their densities were merely 0.0082 and 0.0084 g•cm−3, respectively. More importantly, benefiting from the excellent synergistic effect between RGOF and MoS2, the designed RGOF/MoS2 well inherited the combined advantages of GOF and MoS2 in terms of strong absorption abilities, broad absorption bandwidth and thin matching thicknesses. The values of minimum reflection loss and effective frequency bandwidth for RGOF/MoS2 sample could reach up to −62.92 dB with the matching thickness of 2.27 mm and 4.48 GHz with the matching thickness of 2.12 mm, which were very desirable for high-performance MAs. Moreover, the obtained results indicated that the microwave absorption properties of RGOF/MoS2 sample could be further optimized by regulating the MoS2 content. Therefore, a new and effective strategy was proposed to develop high efficiency MAs with ultra-lightweight, wide-band, thin thickness and strong absorption capabilities.

Defining Nutrient Colocation Typologies for Human-Derived Supply and Crop Demand To Advance Resource Recovery.

Resource recovery from human excreta can advance circular economies while improving access to sanitation and renewable agricultural inputs. While national projections of nutrient recovery potential provide motivation for resource recovery sanitation, elucidating generalizable strategies for sustainable implementation requires a deeper understanding of country-specific overlap between supply and demand. For 107 countries, we analyze the colocation of human-derived nutrients (in urine) and crop demands for nitrogen, phosphorus, and potassium. To characterize colocation patterns, we fit data for each country to a generalized logistic function. Using fitted logistic curve parameters, three typologies were identified: (i) dislocated nutrient supply and demand resulting from high density agriculture (with low population density) and nutrient islands (e.g., dense cities) motivating nutrient concentration and transport; (ii) colocated nutrient supply and demand enabling local reuse; and (iii) diverse nutrient supply-demand proximities, with countries spanning the continuum between (i) and (ii). Finally, we explored connections between these typologies and country-specific contextual characteristics via principal component analysis and found that the Human Development Index was clustered by typology. By providing a generalizable, quantitative framework for characterizing the colocation of human-derived nutrient supply and agricultural nutrient demand, these typologies can advance resource recovery by informing resource management strategies, policy, and investment.

Targeting the Epigenetic and Metabolic Vulnerabilities of Ezh2-Deficiency-Induced Myeloid Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal, progressive hematopoietic malignancies characterized by alterations of multiple signaling and epigenetic pathways in hematopoietic stem cells (HSCs). The identification of driver oncogenic mutations in JAK2, CALR or MPL has transformed our knowledge of MPNs; however, patients with non-mutated JAK2, CALR and MPL (so-called ‘triple-negative‘) have the highest incidence of leukemic transformation and the lowest overall survival (Rumi et al., 2014 Blood 124:1062-1069), indicating that other factors may contribute to MPNs. The molecular processes controlling MPN progression from chronic to acute leukemic transformation remain unknown. This is a major impediment for developing target-based therapeutics to selectively eliminate the mutant stem cells to prevent disease progression and/or relapse. Therefore, the challenge is twofold: 1) to identify and characterize the genetic complexity of MPN pathophysiology, and 2) to understand and control the progression of MPNs from chronic to life-threatening stages.We have developed a new mouse model of MPNs by hematopoietic-selective (Mx1-cre) activation of oncogenic Ras (NRasG12D+/-) and inactivation of Ezh2, the enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2), which mimics concurrent mutations in human MPNs. While NRasG12D+/- alone led to an indolent myeloproliferative disorder, Ezh2 loss markedly accelerated disease progression to life-threatening stages, resulting in thrombocytosis, neutrophilia, splenomegaly, primary myelofibrosis, leukemic transformation and mortality. Strikingly, while loss of Ezh1, a homolog of Ezh2, had no effect on normal hematopoiesis, concurrent inactivation of Ezh1 and Ezh2 with NRasG12D+/- completely abolished MPN development and myelofibrosis in vivo . These results establish distinct functions of Ezh1 and Ezh2 in myeloid neoplasms, and identify a selective epigenetic vulnerability for MPNs induced by Ezh2-deficiency. Follow-up studies of Ezh1/2-mediated chromatin (by ChIP-seq) and transcriptomic (by RNA-seq) changes in normal HSCs and leukemia-initiating cells (LICs) revealed non-overlapping gene targets and cellular pathways required for the distinct cellular phenotypes.Reprogrammed cellular metabolism underlies the progression of various human cancers including leukemia. We noted by RNA-seq transcriptomic profiling that Bcat1, the first and rate-limiting enzyme catalyzing the transamination of branched-chain amino acids (BCAAs), was aberrantly activated in Ezh2-deficient leukemia-initiating cells (LICs). By ChIP-seq analysis, we observed that the Bcat1 promoter is highly enriched with tri-methylated H3-Lys27 (H3K27me3), suggesting that it is epigenetically silenced by Ezh2-PRC2 in normal HSCs. Furthermore, genetic or pharmacologic inhibition of BCAT1 activity selectively abrogated the clonogenic activity of Ezh2-deficient LICs in vitro and the leukemogenic potential in transplanted recipients in vivo, highlighting a metabolic dependency for Ezh2-mutant myeloid neoplasms. Follow-up targeted metabolomic analysis of Ezh2-deficiency-induced metabolic changes established the critical role of Bcat1 and the associated BCAA metabolism in the progression of myeloid neoplasms and leukemia.Collectively, our results not only elucidate the mechanisms by which Ezh1 is specifically required for Ezh2-deficient LICs, but also validate an important epigenetic vulnerability for MPN patients carrying loss-of-function Ezh2 mutations. Moreover, in-depth analysis of BCAA metabolism in myeloid neoplasms identifies a new metabolic dependency for Ezh2-deficient LICs. Because normal hematopoietic stem cells are unaffected by loss of Ezh1 or Bcat1, the selective vulnerabilities raise the possibility of leveraging Ezh1 or BCAA metabolism as targeted therapies to specifically eradicate Ezh2-mutant LICs. Hence, our study promises to provide critical insights into developing new and generalizable therapeutic strategies to selectively eliminate leukemia-initiating cells harboring alterations of epigenetic pathways. DisclosuresDeberardinis:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Promiscuous molecules for smarter file operations in DNA-based data storage

DNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent biomolecular structure of DNA which differentiates it from conventional storage media. The unique molecular architecture of DNA storage also prompts important discussions on how data should be organized, accessed, and manipulated and what practical functionalities may be possible. Here we leverage thermodynamic tuning of biomolecular interactions to implement useful data access and organizational features. Specific sets of environmental conditions including distinct DNA concentrations and temperatures were screened for their ability to switchably access either all DNA strands encoding full image files from a GB-sized background database or subsets of those strands encoding low resolution, File Preview, versions. We demonstrate File Preview with four JPEG images and provide an argument for the substantial and practical economic benefit of this generalizable strategy to organize data.

Rationally Designed Molecularly Imprinted Polymers for Trace Electroanalysis of Environmental Micropollutants

The undetected discharge of solubilized micropollutants into natural waterways constitutes a major public health concern. Thus, generalizable strategies to detect various micropollutant species coupled with deployable sensing platforms are paramount for indicating the presence of contamination at its earliest onset. Benchmark chromatographic methods paired with mass spectrometry to quantify micropollutants are expensive and difficult to deploy into waterways as passive environmental probes. In contrast, electrochemical sensors are inexpensive and easily miniaturized. We report the electrochemical detection of two compounds, 2,4,6-trinitrotoluene (TNT), a prevalent munition, and 2,4-dichlorophenoxyacetic acid (2,4-D), a common fertilizer, using molecularly imprinted polymers (MIPs). MIPs were rationally designed using DFT-level molecular simulations to determine an ideal functional monomer with maximal affinity for each micropollutant species. NMR titration methods were used to verify the simulation results and optimize the monomer/micropollutant ratio. Following optimization, MIPs were synthesized by anodic electropolymerization onto gold substrates in the presence of the micropollutant, which was subsequently stripped away via solvent washing to reveal micropollutant-specific binding sites. Upon micropollutant association with the MIP when placed in contaminated water, surface sites were blocked, which was tracked electrochemically. Each sensor exhibited a limit of detection in the sub-parts-per-billion regime, demonstrating the amenability of this sensing method for trace analysis. Combining the sensitivity of electroanalysis with the selectivity provided by the MIP recognition element constitutes a powerful platform for the detection of micropollutants in environmental matrices.FIGURE CAPTION:Electroanalytical signal obtained following the adsorption of TNT or 2,4-D molecules to an electropolymerized molecularly imprinted polymer (MIP) recognition element. MIPs may be rationally designed using density functional theory (DFT, inset) to maximize the monomer/toxin binding affinity, enhancing the analytical response and permitting the sub-ppb quantification of these toxins in environmental matrices. Figure 1

Regulation of TDP-43 phosphorylation in aging and disease

Insoluble inclusions of phosphorylated TDP-43 occur in disease-affected neurons of most patients with amyotrophic lateral sclerosis (ALS) and about half of patients with frontotemporal lobar degeneration (FTLD-TDP). Phosphorylated TDP-43 potentiates a number of neurotoxic effects including reduced liquid–liquid phase separation dynamicity, changes in splicing, cytoplasmic mislocalization, and aggregation. Accumulating evidence suggests a balance of kinase and phosphatase activities control TDP-43 phosphorylation. Dysregulation of these processes may lead to an increase in phosphorylated TDP-43, ultimately contributing to neurotoxicity and neurodegeneration in disease. Here we summarize the evolving understanding of major regulators of TDP-43 phosphorylation as well as downstream consequences of their activities. Interventions restoring kinase and phosphatase balance may be a generalizable therapeutic strategy for all TDP-43 proteinopathies including ALS and FTLD-TDP.

Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development

Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.

Effect of External Surface Diffusion Barriers on Platinum/Beta‐Catalyzed Isomerization of n‐Pentane

AbstractWe have developed a generalizable strategy to quantify the effect of surface barriers on zeolite catalysis. Isomerization of n‐pentane, catalyzed by Pt/Beta, is taken as a model reaction system. Firstly, the surface modification by chemical liquid deposition of SiO2 was carried out to control the surface barriers on zeolite Beta crystals. The deposition of SiO2 leads to a very slight change in the physical properties of Beta crystals, but an obvious reduction in Brønsted acid sites. Diffusion measurements by the zero‐length column (ZLC) method show that the apparent diffusivity of n‐pentane can be more than doubled after SiO2 deposition, indicating that the surface barriers have been weakened. Catalytic performance was tested in a fixed‐bed reactor, showing that the apparent catalytic activity improved by 51–131 % after SiO2 deposition. These results provide direct proof that reducing surface barriers can be an effective route to improve zeolite catalyst performance deteriorated by transport limitations.

Effect of External Surface Diffusion Barriers on Platinum/Beta-Catalyzed Isomerization of n-Pentane.

We have developed a generalizable strategy to quantify the effect of surface barriers on zeolite catalysis. Isomerization of n‐pentane, catalyzed by Pt/Beta, is taken as a model reaction system. Firstly, the surface modification by chemical liquid deposition of SiO2 was carried out to control the surface barriers on zeolite Beta crystals. The deposition of SiO2 leads to a very slight change in the physical properties of Beta crystals, but an obvious reduction in Brønsted acid sites. Diffusion measurements by the zero‐length column (ZLC) method show that the apparent diffusivity of n‐pentane can be more than doubled after SiO2 deposition, indicating that the surface barriers have been weakened. Catalytic performance was tested in a fixed‐bed reactor, showing that the apparent catalytic activity improved by 51–131 % after SiO2 deposition. These results provide direct proof that reducing surface barriers can be an effective route to improve zeolite catalyst performance deteriorated by transport limitations.

Light‐Controlled Tyrosine Nitration of Proteins

AbstractTyrosine nitration of proteins is one of the most important oxidative post‐translational modifications in vivo. A major obstacle for its biochemical and physiological studies is the lack of efficient and chemoselective protein tyrosine nitration reagents. Herein, we report a generalizable strategy for light‐controlled protein tyrosine nitration by employing biocompatible dinitroimidazole reagents. Upon 390 nm irradiation, dinitroimidazoles efficiently convert tyrosine residues into 3‐nitrotyrosine residues in peptides and proteins with fast kinetics and high chemoselectivity under neutral aqueous buffer conditions. The incorporation of 3‐nitrotyrosine residues enhances the thermostability of lasso peptide natural products and endows murine tumor necrosis factor‐α with strong immunogenicity to break self‐tolerance. The light‐controlled time resolution of this method allows the investigation of the impact of tyrosine nitration on the self‐assembly behavior of α‐synuclein.

Light-Controlled Tyrosine Nitration of Proteins.

Tyrosine nitration of proteins is one of the most important oxidative post-translational modifications in vivo. A major obstacle for its biochemical and physiological studies is the lack of efficient and chemoselective protein tyrosine nitration reagents. Herein, we report a generalizable strategy for light-controlled protein tyrosine nitration by employing biocompatible dinitroimidazole reagents. Upon 390 nm irradiation, dinitroimidazoles efficiently convert tyrosine residues into 3-nitrotyrosine residues in peptides and proteins with fast kinetics and high chemoselectivity under neutral aqueous buffer conditions. The incorporation of 3-nitrotyrosine residues enhances the thermostability of lasso peptide natural products and endows murine tumor necrosis factor-α with strong immunogenicity to break self-tolerance. The light-controlled time resolution of this method allows the investigation of the impact of tyrosine nitration on the self-assembly behavior of α-synuclein.

Targeted degradation of transcription factors by TRAFTACs: TRAnscription Factor TArgeting Chimeras

Many diseases, including cancer, stem from aberrant activation or overexpression of oncoproteins that are associated with multiple signaling pathways. Although proteins with catalytic activity can be successfully drugged, the majority of other protein families, such as transcription factors, remain intractable due to their lack of ligandable sites. In this study, we report the development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) as a generalizable strategy for targeted transcription factor degradation. We show that TRAFTACs, which consist of a chimeric oligonucleotide that simultaneously binds to the transcription factor of interest (TOI) and to HaloTag-fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. Application of TRAFTACs to two oncogenic TOIs, NF-κB and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins. Thus, TRAFTAC technology is potentially a generalizable strategy to induce degradation of other transcription factors both invitro and invivo.

Genome-wide enhancer maps link risk variants to disease genes.

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease1. Many of the underlying causal variants may affect enhancers2,3, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types4. Here we apply this ABC model to create enhancer-genemaps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.

A generalizable strategy toward highly tough and heat-resistant stereocomplex-type polylactide/elastomer blends with substantially enhanced melt processability

Sustainable stereocomplex-type polylactide (SC-PLA) has been recognized as a potential substitute for some traditional engineering plastics, however its widespread application still faces several hurdles primarily related to poor melt processability (i.e., the unfavorable melt memory effect in SC crystallization as well as the low melt strength) and insufficient fracture toughness. To overcome these hurdles, an equimolar poly(l-lactide)/poly(d-lactide) (PLLA/PDLA) blend toughened with poly(ethylene-co-vinyl acetate) (EVA) has been chosen as a model system and a facile strategy has been devised by incorporating trace amounts (i.e., 0.5 wt%) of epoxy-functionalized oligo(styrene-acrylic) (ESA) into the PLLA/PDLA/elastomer blends through direct melt-blending at 200 °C. Such a one-pot blending not only facilitates the stereocomplexation between PLLA and PDLA chains but also permits the highly efficient reaction between terminal hydroxyl groups of some enantiomeric PLAs and epoxy groups of ESA before their stereocomplexation. This results in the in situ generation of large amounts of long-chain-branched PLA-graft-ESA copolymer in the SC-PLA matrix. Very impressively, the generation of this PLA-graft-ESA copolymer gives rise to a substantial improvement in the melt memory effect of the SC-PLA matrix. This copolymer behaves as a compatibilizer to stabilize regular PLLA/PDLA chain clusters in blend melts and hence to stimulate the exclusive formation of SC crystallites in melt-processed blend products. In addition, long PLA branches endow the matrix with a considerably enhanced melt viscosity owing to the increased interchain entanglement density. Consequently, highly stereocomplexed SC-PLA/EVA products with exceptional impact toughness and heat resistance as well as robust mechanical strength have been prepared by injection molding. More notably, this strategy can be generalized to various SC-PLA-based blends, irrespective of whether the elastomers have reactive groups. Overall, these exciting findings illustrate a promising avenue toward the design and applications of high-performance SC-PLA-based materials with excellent melt-processability via the generation of unique graft copolymers having long PLLA and PDLA branches.

Titrating Polyarginine into Nanofibers Enhances Cyclic-Dinucleotide Adjuvanticity in Vitro and after Sublingual Immunization.

Effective sublingual peptide immunization requires overcoming challenges of both delivery and immunogenicity. Mucosal adjuvants, such as cyclic-dinucleotides (CDN), can promote sublingual immune responses but must be codelivered with the antigen to the epithelium for maximum effect. We designed peptide-polymer nanofibers (PEG-Q11) displaying nona-arginine (R9) at a high density to promote complexation with CDNs via bidentate hydrogen-bonding with arginine side chains. We coassembled PEG-Q11 and PEG-Q11R9 peptides to titrate the concentration of R9 within nanofibers. In vitro, PEG-Q11R9 fibers and cyclic-di-GMP or cyclic-di-AMP adjuvants had a synergistic effect on enhancing dendritic cell activation that was STING-dependent and increased monotonically with increasing R9 concentration. The polyvalent display of R9 on assembled nanofibers was significantly more effective at promoting CDN-mediated DC activation in vitro than mixing nanofibers with an equimolar concentration of unassembled R9 peptide. The sublingual administration of nanofibers revealed a bell-shaped trend between increasing R9 concentration and enhancements to antigen trafficking and the activation of DCs in the draining lymph nodes. Intermediate levels of R9 within sublingually administered PEG-Q11 fibers were optimal for immunization, suggesting a balance between polyarginine's ability to sequester CDNs along the nanofiber and its potentially detrimental mucoadhesive interactions. These findings present a potentially generalizable biomaterial strategy for enhancing the potency of CDN adjuvants and reveal important design considerations for the nascent field of sublingual biomaterial immunization.

Tumor-Targeting Cholesterol-Decorated DNA Nanoflowers for Intracellular Ratiometric Aptasensing.

Probing endogenous molecular profiles is of fundamental importance to understand cellular function and processes. Despite the promise of programmable nucleic-acid-based aptasensors across the breadth of biomolecular detection, target-responsive aptasensors enabling intracellular detection are as of yet infrequently realized. Several challenges remain, including the difficulties in quantification/normalization of quencher-based intensiometric signals, stability issues of the probe architecture, and complex sensor operations often necessitating extensive structural modeling. Here, the biomimetic crystallization-empowered self-assembly of a tumor-targetable DNA-inorganic hybrid nanocomposite aptasensor is presented, which enables Förster resonance energy transfer (FRET)-based quantitative interpretation of changes in the cellular target abundance. Leveraging the design programmability and high-throughput fabrication of rolling circle amplification-driven DNA nanoarchitecture, this designer platform offers a method to self-assemble a robust nanosensor from a multifunctionality-encoded template that includes a cell-targeting aptamer, a ratiometric aptasensor, and a cholesterol-decorating element. Taking prostate cancer cells and intracellular adenosine triphosphate molecules as a model system, a synergistic effect in the targeted delivery by cholesterol and aptamers, and the feasibility of quantitative intracellular aptasensing are demonstrated. It is envisioned that this approach provides a highly generalizable strategy across wide-ranging target systems toward a biologically deliverable nanosensor that enables quantitative monitoring of the abundance of endogenous biomolecules.

Photopatterned biomolecule immobilization to guide three-dimensional cell fate in natural protein-based hydrogels

Hydrogel biomaterials derived from natural biopolymers (e.g., fibrin, collagen, decellularized extracellular matrix) are regularly utilized in three-dimensional (3D) cell culture and tissue engineering. In contrast to those based on synthetic polymers, natural materials permit enhanced cytocompatibility, matrix remodeling, and biological integration. Despite these advantages, natural protein-based gels have lagged behind synthetic alternatives in their tunability; methods to selectively modulate the biochemical properties of these networks in a user-defined and heterogeneous fashion that can drive encapsulated cell function have not yet been established. Here, we report a generalizable strategy utilizing a photomediated oxime ligation to covalently decorate naturally derived hydrogels with bioactive proteins including growth factors. This bioorthogonal photofunctionalization is readily amenable to mask-based and laser-scanning lithographic patterning, enabling full four-dimensional (4D) control over protein immobilization within virtually any natural protein-based biomaterial. Such versatility affords exciting opportunities to probe and direct advanced cell fates inaccessible using purely synthetic approaches in response to anisotropic environmental signaling.