The CRISPR effector protein Cas12a has been used for a wide variety of applications such as in vivo gene editing and regulation or in vitro DNA sensing. Here, we add programmability to Cas12a-based ...DNA processing by combining it with strand displacement-based reaction circuits. We first establish a viable strategy for augmenting Cas12a guide RNAs (gRNAs) at their 5' end and then use such 5' extensions to construct strand displacement gRNAs (SD gRNAs) that can be activated by single-stranded RNA trigger molecules. These SD gRNAs are further engineered to exhibit a digital and orthogonal response to different trigger RNA inputs-including full length mRNAs-and to function as multi-input logic gates. We also demonstrate that SD gRNAs can be designed to work inside bacterial cells. Using such in vivo SD gRNAs and a DNase inactive version of Cas12a (dCas12a), we demonstrate logic gated transcriptional control of gene expression in E. coli.
Microplastics are frequently detected in the gastrointestinal tracts of aquatic organisms worldwide. A number of active and passive pathways have been suggested for fish, including the confusion of ...microplastic particles with prey, accidental uptake while foraging and transfer through the food chain, but a holistic understanding of influencing factors is still lacking. The aim of the study was to investigate frequently suggested theories and identify relevant biotic factors, as well as certain plastic properties, affecting microplastic intake in fish. Four species of freshwater fish, each representing a different combination of foraging style (visual/chemosensory) and domestic status (wild/farmed) were exposed to different realistic plastic concentrations and polymer types with and without the provision of genuine food. As most previous investigations of microplastic uptake routes consider only particles large enough to be perceptible to fish, the potential for accidental intake via drinking water has been somewhat neglected. This route is evaluated in the current study using a model approach. The results show that visually oriented fish forage actively on microplastic particles that optically resemble their usual food, while fish with a predominantly chemosensory foraging style are more able to discriminate inedible food items. Even so, the accidental uptake of microplastics while foraging is shown to be relevant pathway, occurring frequently in both visual and chemosensory foragers alike. Several factors were shown to increase plastic uptake, including microplastic concentration in the water, foraging behaviour promoted by availability of genuine food, and fish size. Although both wild and farmed fish ingested microplastic particles, cultured fish showed less discernment in terms of colour and were more likely to forage actively on microplastics when no food was available. Drinking has been identified as a possible source of microplastic intake specifically for large marine fish species. Particles smaller than <5 µm can pass the gastrointestinal tract wall and bioaccumulation could arise when uptake exceeds release or when particles are assimilated in tissues or organs. The effects of accumulation may be significant, especially in long-living species, with implications for food web transfer and fish as food items.
Multicellularity enables the growth of complex life forms as it allows for the specialization of cell types, differentiation and large-scale spatial organization. In a similar way, modular ...construction of synthetic multicellular systems will lead to dynamic biomimetic materials that can respond to their environment in complex ways. To achieve this goal, artificial cellular communication and developmental programs still have to be established. Here, we create geometrically controlled spatial arrangements of emulsion-based artificial cellular compartments containing synthetic in vitro gene circuitry, separated by lipid bilayer membranes. We quantitatively determine the membrane pore-dependent response of the circuits to artificial morphogen gradients, which are established via diffusion from dedicated organizer cells. Utilizing different types of feedforward and feedback in vitro gene circuits, we then implement artificial signalling and differentiation processes, demonstrating the potential for the realization of complex spatiotemporal dynamics in artificial multicellular systems.
Aim
How can we convert biomarkers into reliable, validated laboratory tests? Glomerular filtration rate (GFR) estimators exist for more than a century. The first utilitarian biomarkers were ...endogenously produced urea and creatinine. Clinicians then developed simple tests to determine whether or not renal tubular function was maintained. Are there faster and better tests that reflect decreased renal function and increased acute kidney injury (AKI) risk?
Methods
We inspect earlier, and recently propagated biomarkers. Cystatin C reflects GFR and is not confounded by muscle mass. Direct GFR and plasma volume can now be measured acutely within 3 hours. Better yet would be tests that give information before GFR decreases and prior to urea, creatinine, and cystatin C increases. Prospective tests identifying those persons likely to develop AKI would be helpful. Even more utilitarian would be a test that also suggests a therapeutic avenue.
Results
A number of highly provocative biomarkers have recently been proposed. Moreover the application of big data from huge electronic medical records promise new directions in identifying and dealing with AKI.
Conclusions
Pipedreams are in the pipeline; the novel findings require immediate testing, verification, and perhaps application. Future research promises to make such dreams come true.
Nucleic Acid Based Molecular Devices Krishnan, Yamuna; Simmel, Friedrich C.
Angewandte Chemie (International ed.),
March 28, 2011, Letnik:
50, Številka:
14
Journal Article
Recenzirano
In biology, nucleic acids are carriers of molecular information: DNA’s base sequence stores and imparts genetic instructions, while RNA’s sequence plays the role of a messenger and a regulator of ...gene expression. As biopolymers, nucleic acids also have exciting physicochemical properties, which can be rationally influenced by the base sequence in myriad ways. Consequently, in recent years nucleic acids have also become important building blocks for bottom‐up nanotechnology: as molecules for the self‐assembly of molecular nanostructures and also as a material for building machinelike nanodevices. In this Review we will cover the most important developments in this growing field of nucleic acid nanodevices. We also provide an overview of the biochemical and biophysical background of this field and the major “historical” influences that shaped its development. Particular emphasis is laid on DNA molecular motors, molecular robotics, molecular information processing, and applications of nucleic acid nanodevices in biology.
Nucleic acid power: Molecular machines and devices made from DNA or RNA are becoming more complex and more versatile. Nucleic acids have been shown to be an exquisite material for the rational design of simple molecular switches, and researchers now envision the realization of molecular robots for nanotechnology, computers, as well as nanomachines that operate in vivo.
The use of dynamic, self-assembled DNA nanostructures in the context of nanorobotics requires fast and reliable actuation mechanisms. We therefore created a 55-nanometer-by-55-nanometer DNA-based ...molecular platform with an integrated robotic arm of length 25 nanometers, which can be extended to more than 400 nanometers and actuated with externally applied electrical fields. Precise, computer-controlled switching of the arm between arbitrary positions on the platform can be achieved within milliseconds, as demonstrated with single-pair Förster resonance energy transfer experiments and fluorescence microscopy. The arm can be used for electrically driven transport of molecules or nanoparticles over tens of nanometers, which is useful for the control of photonic and plasmonic processes. Application of piconewton forces by the robot arm is demonstrated in force-induced DNA duplex melting experiments.
Gel‐based artificial organelles have been developed that enable sequence‐specific and programmable localization of cell‐free transcription and translation reactions inside an artificial cellular ...system. To this end, we utilize agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. We show that RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the corresponding DNA addresses. We also demonstrate that mRNA molecules, produced from transcription organelles and controlled by toehold switch riboregulators, are only translated in translation organelles containing their cognate DNA triggers. Spatial confinement of transcription and translation in separate organelles is thus superficially similar to gene expression in eukaryotic cells. Combining communicating gel spheres with specialized functions opens up new possibilities for programming artificial cellular systems at the organelle level.
Chemical processes within biological cells often occur inside organelles. This high degree of intracellular organization has inspired the development of gel‐based artificial organelles that enable sequence‐specific and programmable localization of cell‐free transcription and translation reactions inside an artificial cellular system.
Key message
This study revealed a complex genetic architecture of male floral traits in wheat, and
Rht-D1
was identified as the only major QTL. Genome-wide prediction approaches but also phenotypic ...recurrent selection appear promising to increase outcrossing ability required for hybrid wheat seed production.
Hybrid wheat breeding is a promising approach to increase grain yield and yield stability. However, the identification of lines with favorable male floral characteristics required for hybrid seed production currently poses a severe bottleneck for hybrid wheat breeding. This study therefore aimed to unravel the genetic architecture of floral traits and to assess the potential of genomic approaches to accelerate their improvement. To this end, we employed a panel of 209 diverse winter wheat lines assessed for male floral traits and genotyped with genome-wide markers as well as for
Rht
-
B1
and
Rht
-
D1
. We found the highest proportion of explained genotypic variance for the
Rht
-
D1
locus (11–24 %), for which the dwarfing allele
Rht
-
D1b
had a negative effect on anther extrusion, visual anther extrusion and pollen mass. The genome-wide scan detected only few QTL with small or medium effects, indicating a complex genetic architecture. Consequently, marker-assisted selection yielded only moderate prediction abilities (0.44–0.63), mainly relying on
Rht
-
D1
. Genomic selection based on weighted ridge-regression best linear unbiased prediction achieved higher prediction abilities of up to 0.70 for anther extrusion. In conclusion, recurrent phenotypic selection appears most cost-effective for the initial improvement of floral traits in wheat, while genome-wide prediction approaches may be worthwhile when complete marker profiles are already available in a hybrid wheat breeding program.
Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity ...between components can lead to cross-talk, causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to 1 order of magnitude difference in reaction speed. Finally, we compare two established and one novel technique that speed up TMSD reactions in random sequence pools: a three-letter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.
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