Wearable materials with embedded synthetic biology sensors for biomolecule detection.

Integrating synthetic biology into wearables could expand opportunities for noninvasive monitoring of physiological status, disease states and exposure to pathogens or toxins. However, the operation of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightweight, flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chemicals and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aqueous exposure events and report the presence of specific molecular targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current laboratory methods such as quantitative PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temperature within 90 min, requiring no user intervention other than the press of a button.

A single combination gene therapy treats multiple age-related diseases.

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes (fibroblast growth factor 21 [FGF21], αKlotho, soluble form of mouse transforming growth factor-ß receptor 2 [sTGFßR2]) delivered using adeno-associated viruses and explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure. Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.

Next-generation biocontainment systems for engineered organisms.

The increasing use of engineered organisms for industrial, clinical, and environmental applications poses a growing risk of spreading hazardous biological entities into the environment. To address this biosafety issue, significant effort has been invested in creating ways to confine these organisms and transgenic materials. Emerging technologies in synthetic biology involving genetic circuit engineering, genome editing, and gene expression regulation have led to the development of novel biocontainment systems. In this perspective, we highlight recent advances in biocontainment and suggest a number of approaches for future development, which may be applied to overcome remaining challenges in safeguard implementation.

Portable, On-Demand Biomolecular Manufacturing.

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.

Synthetic biology devices for in vitro and in vivo diagnostics.

There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.

DNA sense-and-respond protein modules for mammalian cells.

We generated synthetic protein components that can detect specific DNA sequences and subsequently trigger a desired intracellular response. These modular sensors exploit the programmability of zinc-finger DNA recognition to drive the intein-mediated splicing of an artificial trans-activator that signals to a genetic circuit containing a given reporter or response gene. We used the sensors to mediate sequence recognition-induced apoptosis as well as to detect and report a viral infection. This work establishes a synthetic biology framework for endowing mammalian cells with sentinel capabilities, which provides a programmable means to cull infected cells. It may also be used to identify positively transduced or transfected cells, isolate recipients of intentional genomic edits and increase the repertoire of inducible parts in synthetic biology.

Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates.

The posttranscriptional modification of RNA by polyadenylation serves various purposes, among them to assist in RNA degradation (see an alternative protocol for measuring RNA degradation on Method for measuring mRNA decay rate in Saccharomyces cerevisiae). This function, once thought to occur in prokaryotic or organellar systems alone, is now known to operate in the nuclei and cytoplasm of eukaryotes as well (Slomovic et al., 2008; Slomovic et al., 2010; Houseley and Tollervey, 2009; Deutscher, 2006). Poly(A)-assisted RNA decay begins with the endonucleolytic cleavage of the transcript. Following this, a poly(A) or oligo(A) tail is added to the 3' end of the cleavage product. This tag serves as a 'landing pad' for 3'-5' exoribonucleases that then begin to digest the RNA fragment. Truncated RNA molecules that have undergone tail addition but have yet to be degraded are called degradation intermediates. The detection of such intermediates is considered a tell-tale sign that poly(A)-assisted RNA decay occurs in the organism being studied. Determination of the tail nucleotide composition by DNA sequencing often aids the researcher in identifying the enzyme responsible for tail synthesis since tails can be either homopolymeric (exclusively A residues) or heteropolymeric (A-rich tails that may include other nucleotides). The following protocol, based on oligo(dT)-primed reverse transcription, describes the step-by-step detection and isolation of adenylated degradation intermediates in the study of poly(A)-assisted RNA decay.

Circularized RT-PCR (cRT-PCR): analysis of the 5' ends, 3' ends, and poly(A) tails of RNA.

Many techniques that are used to characterize individual RNA molecules can potentially alter the original transcript sequence or its posttranscriptional modifications, such as polyadenylation. Methods that are designed to define the ends of an RNA molecule, for example, oligonucleotide ligation, avoid altering the transcript sequence but can usually fulfill only one objective per experiment (e.g., define the 5' or the 3' end). In contrast, not only does circularized reverse transcription coupled with PCR (cRT-PCR) preserve the original 5' and 3' ends of the transcript and posttranscriptionally added extensions, but also the material from one experimental procedure can be utilized in order to characterize both the 5' and 3' ends. Furthermore, if suitable oligonucleotide primers are designed, cRT-PCR can be used to isolate truncated, adenylated (and nonadenylated) molecules that are intermediates in RNA decay (Slomovic and Schuster, 2008).

Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in Mammalian cells.

Prolonged antibiotic treatment can lead to detrimental side effects in patients, including ototoxicity, nephrotoxicity, and tendinopathy, yet the mechanisms underlying the effects of antibiotics in mammalian systems remain unclear. It has been suggested that bactericidal antibiotics induce the formation of toxic reactive oxygen species (ROS) in bacteria. We show that clinically relevant doses of bactericidal antibiotics-quinolones, aminoglycosides, and ß-lactams-cause mitochondrial dysfunction and ROS overproduction in mammalian cells. We demonstrate that these bactericidal antibiotic-induced effects lead to oxidative damage to DNA, proteins, and membrane lipids. Mice treated with bactericidal antibiotics exhibited elevated oxidative stress markers in the blood, oxidative tissue damage, and up-regulated expression of key genes involved in antioxidant defense mechanisms, which points to the potential physiological relevance of these antibiotic effects. The deleterious effects of bactericidal antibiotics were alleviated in cell culture and in mice by the administration of the antioxidant N-acetyl-l-cysteine or prevented by preferential use of bacteriostatic antibiotics. This work highlights the role of antibiotics in the production of oxidative tissue damage in mammalian cells and presents strategies to mitigate or prevent the resulting damage, with the goal of improving the safety of antibiotic treatment in people.

Exonucleases and endonucleases involved in polyadenylation-assisted RNA decay.

RNA polyadenylation occurs in most forms of life, excluding a small number of biological systems. This posttranscriptional modification undertakes two roles, both of which influence the stability of the polyadenylated transcript. One is associated with the mature 3' ends of nucleus-encoded mRNAs in eukaryotic cells and is important for nuclear exit, translatability, and longevity. The second form of RNA polyadenylation assumes an almost opposite role; it is termed 'transient' and serves to mediate the degradation of RNA. Poly(A)-assisted RNA decay pathways were once thought to occur only in prokaryotes/organelles but are now known to be a common phenomenon, present in bacteria, organelles, archaea, and the nucleus and cytoplasm of eukaryotic cells, regardless of the fact that in some of these systems, stable polyadenylation exists as well. This article will summarize the current knowledge of polyadenylation and degradation factors involved in poly(A)-assisted RNA decay in the domains of life, focusing mainly on that which occurs in prokaryotes and organelles. In addition, it will offer an evolutionary view of the development of RNA polyadenylation and degradation and the cellular machinery that is involved.

Dis3-like 1: a novel exoribonuclease associated with the human exosome.

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine-subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome-associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity-purified human exosome complexes identified a novel exosome-associated exoribonuclease, human Dis3-like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co-immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA-mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)-tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome-associated exoribonuclease in the cytoplasm of human cells.

Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells.

Polyadenylation of RNA is a posttranscriptional modification that can play two somewhat opposite roles: stable polyadenylation of RNA encoded in the nuclear genomes of eukaryote cells contributes to nuclear export, translation initiation, and possibly transcript longevity as well. Conversely, transient polyadenylation targets RNA molecules to rapid exonucleolytic degradation. The latter role has been shown to take place in prokaryotes and organelles, as well as the nucleus of eukaryotic cells. Here we present evidence of hetero- and homopolymeric adenylation of truncated RNA molecules within the cytoplasm of human cells. RNAi-mediated silencing of the major RNA decay machinery of the cell resulted in the accumulation of these polyadenylated RNA fragments, indicating that they are degradation intermediates. Together, these results suggest that a mechanism of RNA decay, involving transient polyadenylation, is present in the cytoplasm of human cells.

Polynucleotide phosphorylase and the archaeal exosome as poly(A)-polymerases.

The addition of poly(A)-tails to RNA is a phenomenon common to almost all organisms. Not only homopolymeric poly(A)-tails, comprised exclusively of adenosines, but also heteropolymeric poly(A)-rich extensions, which include the other three nucleotides as well, have been observed in bacteria, archaea, chloroplasts, and human cells. Polynucleotide phosphorylase (PNPase) and the archaeal exosome, which bear strong similarities to one another, both functionally and structurally, were found to polymerize the heteropolymeric tails in bacteria, spinach chloroplasts, and archaea. As phosphorylases, these enzymes use diphosphate nucleotides as substrates and can reversibly polymerize or degrade RNA, depending on the relative concentrations of nucleotides and inorganic phosphate. A possible scenario, illustrating the evolution of RNA polyadenylation and its related functions, is presented, in which PNPase (or the archaeal exosome) was the first polyadenylating enzyme to evolve and the heteropolymeric tails that it produced, functioned in a polyadenylation-stimulated RNA degradation pathway. Only at a later stage in evolution, did the poly(A)-polymerases that use only ATP as a substrate, hence producing homopolymeric adenosine extensions, arise. Following the appearance of homopolymeric tails, a new role for polyadenylation evolved; RNA stability. This was accomplished by utilizing stable poly(A)-tails associated with the mature 3' ends of transcripts. Today, stable polyadenylation coexists with unstable heteropolymeric and homopolymeric tails. Therefore, the heteropolymeric poly(A)-rich tails, observed in bacteria, organelles, archaea, and human cells, represent an ancestral stage in the evolution of polyadenylation.

Detection and characterization of polyadenylated RNA in Eukarya, Bacteria, Archaea, and organelles.

The posttranscriptional addition of poly(A) extensions to RNA is a phenomenon common to almost all organisms. In eukaryotes, a stable poly(A) tail is added to the 3'-end of most nucleus-encoded mRNAs, as well as to mitochondrion-encoded transcripts in animal cells. In prokaryotes and organelles, RNA molecules are polyadenylated as part of a polyadenylation-stimulated RNA degradation pathway. In addition, polyadenylation of nucleus-encoded transcripts in yeast and human cells was recently reported to promote RNA degradation. Not only homopolymeric poly(A) tails, composed exclusively of adenosines, but also heteropolymeric poly(A)-rich extensions, which include the other three nucleotides as well, have been observed in bacteria, archaea, chloroplasts, and human cells. In most instances, the detection of nonabundant truncated transcripts with posttranscriptionally added poly(A) or poly(A)-rich extensions serves as a telltale sign of the presence of a polyadenylation-stimulated RNA degradation pathway. In this chapter, we describe several methods found to be efficient in detecting and characterizing polyadenylated transcripts in bacteria, archaea, organelles, and nucleus-encoded RNAs. Detailed protocols for the oligo(dT)- and circularized reverse transcription (cRT) PCR methods, as well as the ribonuclease digestion method, are outlined, along with examples of results obtained with these techniques.

Stable PNPase RNAi silencing: its effect on the processing and adenylation of human mitochondrial RNA.

Polynucleotide phosphorylase (PNPase) is a diverse enzyme, involved in RNA polyadenylation, degradation, and processing in prokaryotes and organelles. However, in human mitochondria, PNPase is located in the intermembrane space (IMS), where no mitochondrial RNA (mtRNA) is known to be present. In order to determine the nature and degree of its involvement in mtRNA metabolism, we stably silenced PNPase by establishing HeLa cell lines expressing PNPase short-hairpin RNA (shRNA). Processing and polyadenylation of mt-mRNAs were significantly affected, but, to different degrees in different genes. For instance, the stable poly(A) tails at the 3' ends of COX1 transcripts were abolished, while COX3 poly(A) tails remained unaffected and ND5 and ND3 poly(A) extensions increased in length. Despite the lack of polyadenylation at the 3' end, COX1 mRNA and protein accumulated to normal levels, as was the case for all 13 mt-encoded proteins. Interestingly, ATP depletion also altered poly(A) tail length, demonstrating that adenylation of mtRNA can be manipulated by indirect, environmental means and not solely by direct enzymatic activity. When both PNPase and the mitochondrial poly(A)-polymerase (mtPAP) were concurrently silenced, the mature 3' end of ND3 mRNA lacked poly(A) tails but retained oligo(A) extensions. Furthermore, in mtPAP-silenced cells, truncated adenylated COX1 molecules, considered to be degradation intermediates, were present but harbored significantly shorter tails. Together, these results suggest that an additional mitochondrial polymerase, yet to be identified, is responsible for the oligoadenylation of mtRNA and that PNPase, although located in the IMS, is involved, most likely by indirect means, in the processing and polyadenylation of mtRNA.

Polyadenylation of ribosomal RNA in human cells.

The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3' ends of most nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT-PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles.

Polyadenylation and degradation of human mitochondrial RNA: the prokaryotic past leaves its mark.

RNA polyadenylation serves a purpose in bacteria and organelles opposite from the role it plays in nuclear systems. The majority of nucleus-encoded transcripts are characterized by stable poly(A) tails at their mature 3' ends, which are essential for stabilization and translation initiation. In contrast, in bacteria, chloroplasts, and plant mitochondria, polyadenylation is a transient feature which promotes RNA degradation. Surprisingly, in spite of their prokaryotic origin, human mitochondrial transcripts possess stable 3'-end poly(A) tails, akin to nucleus-encoded mRNAs. Here we asked whether human mitochondria retain truncated and transiently polyadenylated transcripts in addition to stable 3'-end poly(A) tails, which would be consistent with the preservation of the largely ubiquitous polyadenylation-dependent RNA degradation mechanisms of bacteria and organelles. To this end, using both molecular and bioinformatic methods, we sought and revealed numerous examples of such molecules, dispersed throughout the mitochondrial genome. The broad distribution but low abundance of these polyadenylated truncated transcripts strongly suggests that polyadenylation-dependent RNA degradation occurs in human mitochondria. The coexistence of this system with stable 3'-end polyadenylation, despite their seemingly opposite effects, is so far unprecedented in bacteria and other organelles.