Intrinsically disordered sequences can tune fungal growth and the cell cycle for specific temperatures.

Temperature can impact every reaction essential to a cell. For organisms that cannot regulate their own temperature, adapting to temperatures that fluctuate unpredictably and on variable timescales is a major challenge. Extremes in the magnitude and frequency of temperature changes are increasing across the planet, raising questions as to how the biosphere will respond.

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation.

Phase transition of GvpU regulates gas vesicle clustering in bacteria.

Gas vesicles (GVs) are microbial protein organelles that support cellular buoyancy. GV engineering has multiple applications, including reporter gene imaging, acoustic control and payload delivery. GVs often cluster into a honeycomb pattern to minimize occupancy of the cytosol.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles.

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus.

Grand Challenges at the Interface of Engineering and Medicine.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease.

Addressing Signal Drift and Screening for Detection of Biomarkers with Carbon Nanotube Transistors.

Electrical biosensors, including transistor-based devices (i.e., BioFETs), have the potential to offer versatile biomarker detection in a simple, low-cost, scalable, and point-of-care manner.

Encoding Structure in Intrinsically Disordered Protein Biomaterials.

In nature, proteins range from those with highly ordered secondary and tertiary structures to those that completely lack a well-defined three-dimensional structure, termed intrinsically disordered proteins (IDPs). IDPs are generally characterized by one or more segments that have a compositional bias toward small hydrophilic amino acids and proline residues that promote structural disorder and are called intrinsically disordered regions (IDRs). The combination of IDRs with ordered regions and the interactions between the two determine the phase behavior, structure, and function of IDPs.

Modulating hierarchical self-assembly in thermoresponsive intrinsically disordered proteins through high-temperature incubation time.

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium.

Enzymatic Synthesis of Aptamer-Polynucleotide Nanoparticles with High Anticancer Drug Loading for Targeted Delivery.

We report a targeted prodrug delivery platform that can deliver a cytostatic nucleobase analog with high drug loading. We chose fluorouracil (5FU), a drug used to treat various cancers, whose active metabolite 5-fluorodeoxyuridine monophosphate (5-FdUMP) is the antineoplastic agent. We use terminal deoxynucleotidyl transferase (TdT) to polymerize 5-fluorodeoxyuridine triphosphate (5-FdUTP) onto the 3'-end of an aptamer.

Global control of cellular physiology by biomolecular condensates through modulation of electrochemical equilibria.

Control of the electrochemical environment in living cells is typically attributed to ion channels. Here we show that the formation of biomolecular condensates can modulate the electrochemical environment in cells, which affects processes globally within the cell and interactions of the cell with its environment. Condensate formation results in the depletion or enrichment of certain ions, generating intracellular ion gradients.

A gravity-driven droplet fluidic point-of-care test.

We report a simple droplet fluidic point-of-care test (POCT) that uses gravity to manipulate the sequence, timing, and motion of droplets on a surface. To fabricate this POCT, we first developed a surface coating toolbox of nine different coatings with three levels of wettability and three levels of slipperiness that can be independently tailored. We then fabricated a device that has interconnected fluidic elements-pumps, flow resistors and flow guides-on a highly slippery solid surface to precisely control the timing and sequence of motion of multiple droplets and their interactions on the surface.

Modulating Hierarchical Self-Assembly In Thermoresponsive Intrinsically Disordered Proteins Through High-Temperature Incubation Time.

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium.

IsoTagAAV: an innovative, scalable & non-chromatographic method for streamlined AAV manufacturing.

Demand for gene therapies capable of treating previously inaccessible targets has risen precipitously in the past decade. Adeno-associated viruses (AAVs) are the preferred vector for gene delivery because of their favorable safety profile and tissue tropism, but they have significant manufacturing challenges, with end-to-end yields as low as 10-30%. To combat these low yields, we developed IsoTagAAV, a novel purification technology for AAV that is a departure from the chromatographic paradigm in downstream processing.

Modulating Hierarchical Self-Assembly In Thermoresponsive Intrinsically Disordered Proteins Through High-Temperature Incubation Time.

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium.

Interface of biomolecular condensates modulates redox reactions.

Biomolecular condensates mediate diverse cellular processes. The density transition process of condensate formation results in selective partitioning of molecules, which define a distinct chemical environment within the condensates. However, the fundamental features of the chemical environment and the mechanisms by which such environment can contribute to condensate functions have not been revealed.

Engineering synthetic biomolecular condensates.

The concept of phase-separation-mediated formation of biomolecular condensates provides a new framework to understand cellular organization and cooperativity-dependent cellular functions. With growing understanding of how biological systems drive phase separation and how cellular functions are encoded by biomolecular condensates, opportunities have emerged for cellular control through engineering of synthetic biomolecular condensates. In this Review, we discuss how to construct synthetic biomolecular condensates and how they can regulate cellular functions.

Engineering Innovative Interfaces for Point-of-Care Diagnostics.

The ongoing Coronavirus disease 2019 (COVID-19) pandemic illustrates the need for sensitive and reliable tools to diagnose and monitor diseases. Traditional diagnostic approaches rely on centralized laboratory tests that result in long wait times to results and reduce the number of tests that can be given. Point-of-care tests (POCTs) are a group of technologies that miniaturize clinical assays into portable form factors that can be run both in clinical areas --in place of traditional tests-- and outside of traditional clinical settings --to enable new testing paradigms.

Environmentally Resilient Microfluidic Point-of-Care Immunoassay Enables Rapid Diagnosis of Talaromycosis.

Point-of-care tests (POCTs) are increasingly being used in field settings, particularly outdoors. The performance of current POCTs─most commonly the lateral flow immunoassay─can be adversely affected by ambient temperature and humidity. We developed a self-contained immunoassay platform─the D4 POCT─that can be conducted at the POC by integrating all reagents in a capillary-driven passive microfluidic cassette that minimizes user intervention.

COVID-19 Diagnosis and SARS-CoV-2 Strain Identification by a Rapid, Multiplexed, Point-of-Care Antibody Microarray.

Antigen tests to detect SARS-CoV-2 have emerged as a promising rapid diagnostic method for COVID-19, but they are unable to differentiate between variants of concern (VOCs). Here, we report a rapid point-of-care test (POC-T), termed CoVariant-SPOT, that uses a set of antibodies that are either tolerant or intolerant to spike protein mutations to identify the likely SARS-CoV-2 strain concurrent with COVID-19 diagnosis using antibodies targeting the nucleocapsid protein. All reagents are incorporated into a portable, multiplexed, and sensitive diagnostic platform built upon a nonfouling polymer brush.

Programmable synthetic biomolecular condensates for cellular control.

The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube.

An injectable PEG-like conjugate forms a subcutaneous depot and enables sustained delivery of a peptide drug.

Many biologics have a short plasma half-life, and their conjugation to polyethylene glycol (PEG) is commonly used to solve this problem. However, the improvement in the plasma half-life of PEGylated drugs' is at an asymptote because the development of branched PEG has only had a modest impact on pharmacokinetics and pharmacodynamics. Here, we developed an injectable PEG-like conjugate that forms a subcutaneous depot for the sustained delivery of biologics.

Mixed-surface polyamidoamine polymer variants retain nucleic acid-scavenger ability with reduced toxicity.

Nucleic acid-binding polymers can have anti-inflammatory properties and beneficial effects in animal models of infection, trauma, cancer, and autoimmunity. PAMAM G3, a polyamidoamine dendrimer, is fully cationic bearing 32 protonable surface amines. However, while PAMAM G3 treatment leads to improved outcomes for mice infected with influenza, at risk of cancer metastasis, or genetically prone to lupus, its administration can lead to serosal inflammation and elevation of biomarkers of liver and kidney damage.

Brachytherapy via a depot of biopolymer-bound I synergizes with nanoparticle paclitaxel in therapy-resistant pancreatic tumours.

Locally advanced pancreatic tumours are highly resistant to conventional radiochemotherapy. Here we show that such resistance can be surmounted by an injectable depot of thermally responsive elastin-like polypeptide (ELP) conjugated with iodine-131 radionuclides (I-ELP) when combined with systemically delivered nanoparticle albumin-bound paclitaxel. This combination therapy induced complete tumour regressions in diverse subcutaneous and orthotopic mouse models of locoregional pancreatic tumours.

Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures.

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation.