Sequence-independent, site-specific incorporation of chemical modifications to generate light-activated plasmids.

Plasmids are ubiquitous in biology, where they are used to study gene-function relationships and intricate molecular networks, and hold potential as therapeutic devices. Developing methods to control their function will advance their application in research and may also expedite their translation to clinical settings. Light is an attractive stimulus to conditionally regulate plasmid expression as it is non-invasive, and its properties such as wavelength, intensity, and duration can be adjusted to minimise cellular toxicity and increase penetration. Herein, we have developed a method to site-specifically introduce photocages into plasmids, by resynthesising one strand in a manner similar to Kunkel mutagenesis. Unlike alternative approaches to chemically modify plasmids, this method is sequence-independent at the site of modification and uses commercially available phosphoramidites. To generate our light-activated (LA) plasmids, photocleavable biotinylated nucleobases were introduced at specific sites across the T7 and CMV promoters on plasmids and bound to streptavidin to sterically block access. These LA-plasmids were then successfully used to control expression in both cell-free systems (T7 promoter) and mammalian cells (CMV promoter). These light-activated plasmids might be used to remotely control cellular activity and reduce off-target toxicity for future medical use. Our simple approach to plasmid modification might also be used to introduce novel chemical moieties for advanced function.

Engineering cellular communication between light-activated synthetic cells and bacteria.

Gene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells with life-like functions. By incorporating gene regulatory motifs into their encapsulated DNA templates, in situ gene expression and, thereby, synthetic cell function can be controlled according to specific stimuli. In this work, cell-free protein synthesis within synthetic cells was controlled using light by encoding genes of interest on light-activated DNA templates. Light-activated DNA contained a photocleavable blockade within the T7 promoter region that tightly repressed transcription until the blocking groups were removed with ultraviolet light. In this way, synthetic cells were activated remotely, in a spatiotemporally controlled manner. By applying this strategy to the expression of an acyl homoserine lactone synthase, BjaI, quorum-sensing-based communication between synthetic cells and bacteria was controlled with light. This work provides a framework for the remote-controlled production and delivery of small molecules from nonliving matter to living matter, with applications in biology and medicine.

Orthogonal Light-Activated DNA for Patterned Biocomputing within Synthetic Cells.

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and use with synthetic cells, particularly using remote, nondamaging stimuli such as visible light. Here, we have synthesized blue light-activatable DNA parts that tightly regulate cell-free RNA and protein synthesis. We found that this blue light-activated DNA could initiate expression orthogonally to our previously generated ultraviolet (UV) light-activated DNA, which we used to generate a dual-wavelength light-controlled cell-free AND-gate. By encapsulating these orthogonal light-activated DNAs into synthetic cells, we used two overlapping patterns of blue and UV light to provide precise spatiotemporal control over the logic gate. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.

Accessible light-controlled knockdown of cell-free protein synthesis using phosphorothioate-caged antisense oligonucleotides.

Controlling cell-free expression of a gene to protein with non-invasive stimuli is vital to the future application of DNA nanodevices and synthetic cells. However, little emphasis has been placed on developing light-controlled 'off' switches for cell-free expression. Light-activated antisense oligonucleotides have been developed to induce gene knockdown in living cells; however, they are complicated to synthesise and have not been tested in cell-free systems. Developing simple, accessible methods to produce light-activated antisense oligonucleotides will be crucial for allowing their application in cell-free biology and biotechnology. Here, we report a mild, one-step method for selectively attaching commercially-available photoremovable protecting groups, photocages, onto phosphorothioate linkages of antisense oligonucleotides. Using this photocaging method, upon illumination, the original phosphorothioate antisense oligonucleotide is reformed. Photocaged antisense oligonucleotides, containing mixed phosphorothioate and phosphate backbones, showed a drastic reduction in duplex formation and RNase H activity, which was recovered upon illumination. We then demonstrated that these photocaged antisense oligonucleotides can be used to knock down cell-free protein synthesis using light. This simple and accessible technology will have future applications in light-controlled biological logic gates and regulating the activity of synthetic cells.

Handcuffed antisense oligonucleotides for light-controlled cell-free expression.

Developing simple methods to silence antisense oligonucleotides (ASOs) using photocages opens up the possibility of precise regulation of biological systems. Here, we have developed a photocaging strategy based on 'handcuffing' two ASOs to a protein. Silencing was achieved by divalent binding of two terminally photocleavable biotin-modified ASOs to a single streptavidin. These 'handcuffed' oligonucleotides showed a drastic reduction in gene knockdown activity in cell-free protein synthesis and were unlocked through illumination, regaining full activity.

Precise, Orthogonal Remote-Control of Cell-Free Systems Using Photocaged Nucleic Acids.

Cell-free expression of a gene to protein has become a vital tool in nanotechnology and synthetic biology. Remote-control of cell-free systems with multiple, orthogonal wavelengths of light would enable precise, noninvasive modulation, opening many new applications in biology and medicine. While there has been success in developing ON switches, the development of OFF switches has been lacking. Here, we have developed orthogonally light-controlled cell-free expression OFF switches by attaching nitrobenzyl and coumarin photocages to antisense oligonucleotides. These light-controlled OFF switches can be made from commercially available oligonucleotides and show a tight control of cell-free expression. Using this technology, we have demonstrated orthogonal degradation of two different mRNAs, depending on the wavelength used. By combining with our previously generated blue-light-activated DNA template ON switch, we were able to start transcription with one wavelength of light and then halt the translation of the corresponding mRNA to protein with a different wavelength, at multiple timepoints. This precise, orthogonal ON and OFF remote-control of cell-free expression will be an important tool for the future of cell-free biology, especially for use with biological logic gates and synthetic cells.

Reaction-Diffusion Patterning of DNA-Based Artificial Cells.

Biological cells display complex internal architectures with distinct micro environments that establish the chemical heterogeneity needed to sustain cellular functions. The continued efforts to create advanced cell mimics, namely, artificial cells, demands strategies for constructing similarly heterogeneous structures with localized functionalities. Here, we introduce a platform for constructing membraneless artificial cells from the self-assembly of synthetic DNA nanostructures in which internal domains can be established thanks to prescribed reaction-diffusion waves. The method, rationalized through numerical modeling, enables the formation of up to five distinct concentric environments in which functional moieties can be localized. As a proof-of-concept, we apply this platform to build DNA-based artificial cells in which a prototypical nucleus synthesizes fluorescent RNA aptamers that then accumulate in a surrounding storage shell, thus demonstrating the spatial segregation of functionalities reminiscent of that observed in biological cells.

No increased risk of Alzheimer's disease among people with immune-mediated inflammatory diseases: findings from a longitudinal cohort study of U.S. older adults.

OBJECTIVE: Immune-mediated inflammatory diseases (IMID) are characterized by systemic inflammation affecting the joints and bodily organs. Studies examining the association between individual IMIDs and the risk of Alzheimer's disease (AD) have yielded inconsistent findings. This study examines AD risk across a group of IMIDs in a large population-based sample of older adults. METHODS: Data on a national sample of US adults over age 50 was drawn from the Health and Retirement Study (HRS) and linked Medicare claims from 2006 to 2014. IMIDs include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, and related conditions. We identified IMIDs from 2006 to 2009 Medicare claims using International Classification of Diseases (ICD9-CM) codes. The date of incident AD was derived from Chronic Conditions Warehouse (CCW) identifiers. We examined the risk of AD from 2009 to 2014 using Cox proportional hazards models, both unadjusted and adjusted for age, gender, education, race, and the genetic risk factor APOE-e4. RESULTS: One hundred seventy-one (6.02%) of the 2842 total HRS respondents with Medicare coverage and genetic data were classified with IMIDs. Over the subsequent 6 years, 9.36% of IMID patients developed AD compared to 8.57% of controls (unadjusted hazard ratio (HR): 1.09, 95% CI .66-1.81, p = 0.74). Adjusted HR 1.27 (95% CI 0.76-2.12, p = 0.35). Age (HR for 10-year increment 3.56, p < .001), less than high school education (HR 1.70, p = .007), and APOE-e4 (HR 2.61, p < .001 for one or two copies), were also statistically significant predictors of AD. CONCLUSION: HRS respondents with common IMIDs do not have increased risk of Alzheimer's disease over a 6-year period.

A Lipid-Based Droplet Processor for Parallel Chemical Signals.

A key goal of bottom-up synthetic biology is to construct cell- and tissue-like structures. Underpinning cellular life is the ability to process several external chemical signals, often in parallel. Until now, cell- and tissue-like structures have been constructed with no more than one signaling pathway. Many pathways rely on signal transport across membranes using protein nanopores. However, such systems currently suffer from the slow transport of molecules. We have optimized the application of these nanopores to permit fast molecular transport, which has allowed us to construct a processor for parallel chemical signals from the bottom up in a modular fashion. The processor comprises three aqueous droplet compartments connected by lipid bilayers and operates in an aqueous environment. It can receive two chemical signals from the external environment, process them orthogonally, and then produce a distinct output for each signal. It is suitable for both sensing and enzymatic processing of environmental signals, with fluorescence and molecular outputs. In the future, such processors could serve as smart drug delivery vehicles or as modules within synthetic tissues to control their behavior in response to external chemical signals.

No association between rheumatoid arthritis and cognitive impairment in a cross-sectional national sample of older U.S. adults.

BACKGROUND: Studies suggest an increased prevalence of cognitive impairment (CI) among people with rheumatoid arthritis (RA). However, most prior studies have used convenience samples which are subject to selection biases or have failed to adjust for key confounding variables. We thus examined the association between CI and RA in a large national probability sample of older US adults. METHODS: Data were from interviews with 4462 participants in the 2016 wave of the nationally representative U.S. Health and Retirement Study with linked Medicare claims. RA diagnoses were identified via a minimum of two ICD-9CM or ICD-10 codes in Medicare billing records during the prior 2 years. The Langa-Weir Classification was used to classify cognitive status as normal, cognitively impaired non-dementia (CIND), or dementia based on a brief neuropsychological battery for self-respondents and informant reports for proxy respondents. We compared the odds of CI between older adults with and without RA using logistic regression, adjusted for age, education, gender, and race. RESULTS: Medicare records identified a 3.36% prevalence of RA (150/4462). While age, gender, education, and race independently predicted CI status, controlling for these covariates we found no difference in CI prevalence according to RA status (prevalent CI in 36.7% of older adults with RA vs. 34.0% without RA; adjusted OR = 1.08, 95% CI 0.74-1.59, p = .69). CONCLUSION: There was no association between RA and CI in this national sample of older U.S. adults.

Reduced Bisulfite Sequencing: Quantitative Base-Resolution Sequencing of 5-Formylcytosine.

The generation of tools to study mammalian epigenetics is vital to understanding normal biological function and to identify how it is dysregulated in disease. The well-studied epigenetic DNA modification 5-methylcytosine can be enzymatically oxidized to 5-formylcytosine (5fC) in vivo. 5fC has been demonstrated to be an intermediate in demethylation, but recent evidence suggests that 5fC may have an epigenetic function of its own. We have developed reduced bisulfite sequencing (redBS-seq), which can quantitatively locate 5fC bases at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), 5fC is converted to uracil, as happens to unmodified cytosine (C), and thus cannot be discriminated from C. However, in redBS-seq, a specific reduction of 5fC to 5-hydroxymethylcytosine (5hmC) stops this conversion, allowing its discrimination from C. 5fC levels are inferred by comparison of a redBS-Seq run with a BS-seq run.

Validation of Self-Reported Rheumatoid Arthritis Using Medicare Claims: A Nationally Representative Longitudinal Study of Older Adults.

OBJECTIVE: To determine the validity of self-reported physician diagnosis of rheumatoid arthritis (RA) using multiple gold-standard measures based on Medicare claims in a nationally representative sample of older adults and to verify whether additional questions about taking medication and having seen a physician in the past two years for arthritis can improve the positive predictive value (PPV) and other measures of the validity of self-reported RA. METHODS: A total of 3768 Medicare-eligible respondents with and without incident self-reported RA were identified from the 2004, 2008, and 2012 waves of the United States Health and Retirement Study. Self-reported RA was validated using the following three claims-based algorithms: 1) a single International Classification of Diseases, ninth edition, Clinical Modification claim for RA, 2) two or more claims no greater than 2 years apart, and 3) two or more claims with at least one diagnosis by a rheumatologist. Additional self-report questions of medication use and having seen a doctor for arthritis in the past two years were validated against the same criteria. RESULTS: A total of 345 respondents self-reported a physician diagnosis of RA. Across all three RA algorithms, the PPV of self-report ranged from 0.05 to 0.16., the sensitivity ranged from 0.23 to 0.55., and the κ statistic ranged from 0.07 to 0.15. Additional self-report data regarding arthritis care improved the PPV and other validity measures of self-report; however, the values remained low. CONCLUSION: Most older adults who self-report RA do not have a Medicare claims history consistent with that diagnosis. Revisions to current self-reported RA questions may yield more valid identification of RA in national health surveys.

Controlling Synthetic Cell-Cell Communication.

Synthetic cells, which mimic cellular function within a minimal compartment, are finding wide application, for instance in studying cellular communication and as delivery devices to living cells. However, to fully realise the potential of synthetic cells, control of their function is vital. An array of tools has already been developed to control the communication of synthetic cells to neighbouring synthetic cells or living cells. These tools use either chemical inputs, such as small molecules, or physical inputs, such as light. Here, we examine these current methods of controlling synthetic cell communication and consider alternative mechanisms for future use.

Controlling gene expression with light: a multidisciplinary endeavour.

The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

Transmembrane protein rotaxanes reveal kinetic traps in the refolding of translocated substrates.

Understanding protein folding under conditions similar to those found in vivo remains challenging. Folding occurs mainly vectorially as a polypeptide emerges from the ribosome or from a membrane translocon. Protein folding during membrane translocation is particularly difficult to study. Here, we describe a single-molecule method to characterize the folded state of individual proteins after membrane translocation, by monitoring the ionic current passing through the pore. We tag both N and C termini of a model protein, thioredoxin, with biotinylated oligonucleotides. Under an electric potential, one of the oligonucleotides is pulled through a α-hemolysin nanopore driving the unfolding and translocation of the protein. We trap the protein in the nanopore as a rotaxane-like complex using streptavidin stoppers. The protein is subjected to cycles of unfolding-translocation-refolding switching the voltage polarity. We find that the refolding pathway after translocation is slower than in bulk solution due to the existence of kinetic traps.

Multi-responsive hydrogel structures from patterned droplet networks.

Responsive hydrogels that undergo controlled shape changes in response to a range of stimuli are of interest for microscale soft robotic and biomedical devices. However, these applications require fabrication methods capable of preparing complex, heterogeneous materials. Here we report a new approach for making patterned, multi-material and multi-responsive hydrogels, on a micrometre to millimetre scale. Nanolitre aqueous pre-gel droplets were connected through lipid bilayers in predetermined architectures and photopolymerized to yield continuous hydrogel structures. By using this droplet network technology to pattern domains containing temperature-responsive or non-responsive hydrogels, structures that undergo reversible curling were produced. Through patterning of gold nanoparticle-containing domains into the hydrogels, light-activated shape change was achieved, while domains bearing magnetic particles allowed movement of the structures in a magnetic field. To highlight our technique, we generated a multi-responsive hydrogel that, at one temperature, could be moved through a constriction under a magnetic field and, at a second temperature, could grip and transport a cargo.

Light-patterning of synthetic tissues with single droplet resolution.

Synthetic tissues can be generated by forming networks of aqueous droplets in lipid-containing oil. Each droplet contains a cell-free expression system and is connected to its neighbor through a lipid bilayer. In the present work, we have demonstrated precise external control of such networks by activating protein expression within single droplets, by using light-activated DNA to encode either a fluorescent or a pore-forming protein. By controlling the extent of activation, synthetic tissues were generated with graded levels of protein expression in patterns of single droplets. Further, we have demonstrated reversible activation within individual compartments in synthetic tissues by turning a fluorescent protein on-and-off. This is the first example of the high-resolution patterning of droplet networks, following their formation. Single-droplet control will be essential to power subsets of compartments within synthetic tissues or to stimulate subsets of cells when synthetic tissues are interfaced with living tissues.

Functional aqueous droplet networks.

Droplet interface bilayers (DIBs), comprising individual lipid bilayers between pairs of aqueous droplets in an oil, are proving to be a useful tool for studying membrane proteins. Recently, attention has turned to the elaboration of networks of aqueous droplets, connected through functionalized interface bilayers, with collective properties unachievable in droplet pairs. Small 2D collections of droplets have been formed into soft biodevices, which can act as electronic components, light-sensors and batteries. A substantial breakthrough has been the development of a droplet printer, which can create patterned 3D droplet networks of hundreds to thousands of connected droplets. The 3D networks can change shape, or carry electrical signals through defined pathways, or express proteins in response to patterned illumination. We envisage using functional 3D droplet networks as autonomous synthetic tissues or coupling them with cells to repair or enhance the properties of living tissues.

Light-Patterned Current Generation in a Droplet Bilayer Array.

We have created a 4 × 4 droplet bilayer array comprising light-activatable aqueous droplet bio-pixels. Aqueous droplets containing bacteriorhodopsin (bR), a light-driven proton pump, were arranged on a common hydrogel surface in lipid-containing oil. A separate lipid bilayer formed at the interface between each droplet and the hydrogel; each bilayer then incorporated bR. Electrodes in each droplet simultaneously measured the light-driven proton-pumping activities of each bio-pixel. The 4 × 4 array derived by this bottom-up synthetic biology approach can detect grey-scale images and patterns of light moving across the device, which are transduced as electrical current generated in each bio-pixel. We propose that synthetic biological light-activatable arrays, produced with soft materials, might be interfaced with living tissues to stimulate neuronal pathways.