Enzyme-Free Amplified Detection of DNA by an Autonomous Ligation DNAzyme Machinery

The Zn²⁺-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal autocatalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.0 × 10^–11 M. Besides providing a versatile paradigm for the amplified detection of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic mechanism that introduces means for effective programmed synthesis

pH-Programmable DNA Logic Arrays Powered by Modular DNAzyme Libraries

Nature performs complex information processing circuits, such the programmed transformations of versatile stem cells into targeted functional cells. Man-made molecular circuits are, however, unable to mimic such sophisticated biomachineries. To reach these goals, it is essential to construct programmable modular components that can be triggered by environmental stimuli to perform different logic circuits. We report on the unprecedented design of artificial pH-programmable DNA logic arrays, constructed by modular libraries of Mg²⁺- and UO₂²⁺-dependent DNAzyme subunits and their substrates. By the appropriate modular design of the DNA computation units, pH-programmable logic arrays of various complexities are realized, and the arrays can be erased, reused, and/or reprogrammed. Such systems may be implemented in the near future for nanomedical applications by pH-controlled regulation of cellular functions or may be used to control biotransformations stimulated by bacteria

Au Nanoparticle/DNA Rotaxane Hybrid Nanostructures Exhibiting Switchable Fluorescence Properties

The preparation of a DNA rotaxane consisting of a circular nucleic acid interlocked, through hybridization, on a nucleic acid axle and stoppered by two 10-nm-sized Au nanoparticles (NPs) is described. By the tethering of 5-nm- or 15-nm-sized Au NPs on the ring, the supramolecular structure of the rotaxane is confirmed. Using nucleic acids as “fuels” and “anti-fuels”, the cyclic and reversible transition of the rotaxane ring across two states is demonstrated. By the functionalization of the ring with fluorophore-modified nucleic acids in different orientations, the transitions of the rings between the sites are followed by fluorescence quenching or surface-enhanced fluorescence. The experimental results are supported by theoretical modeling

Amplified Analysis of DNA by the Autonomous Assembly of Polymers Consisting of DNAzyme Wires

A systematic study of the amplified optical detection of DNA by Mg²⁺-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10^–9 M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg²⁺-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10^–14 M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene

A Three-Station DNA Catenane Rotary Motor with Controlled Directionality

The assembly of DNA machines represents a central effort in DNA nanotechnology. We report on the first DNA rotor system composed of a two-ring catenane. The DNA rotor ring rotates in dictated directions along a wheel, and it occupies three distinct sites. Hg²⁺/cysteine or pH (H⁺/OH^–) act as fuels or antifuels in positioning the rotor ring. Analysis of the kinetics reveals directional clockwise or anticlockwise population of the target-sites (>85%), and the rotor’s direction is controlled by the shortest path on the wheel

Supporting information file.

Includes figures S1-S10, tables S1-S3, and supporting methods. (PDF)</p

Overview of prototyping strategy and soil samples used.

(a) A stepwise schematic of the pipeline developed in this study. (b) Soil sampling locations and types. Approximate locations and the names of the sampling sites are marked in red over a map of the southeastern Mediterranean shore. White dots represent sample types and names. (c) 16S rDNA gene sequencing analysis data of soil samples showing the Phyla-level relative abundance. The ‘Other’ category includes all phyla with relative abundance (d) Metagenomic data showing the relative abundance of the Bacilli class in soil samples.</p

Bacterial prototyping in replicated environments.

(a) Jungle room photos, showing the location of the B. frigoritolerans A3E1 infused soil samples in flowerpots on a rain event on day six. (b) Simulated environment survival experiment results. Cell-phone pictures of Wildtype and GFP+ B. frigoritolerans A3E1 containing pots under Blue LED flashlight illumination (450 nm) and 505 nm filter (top) and white light (bottom) at three time-points during the experiment. (c) Fluorescent images of GFP+ and WT containing soil flowerpots on day 575 of long-term storage (d) Representative comparison between rehydrated (left) and dry (right) GFP+ containing pots and during the rehydration process. Mountain plots describe GFP fluorescence intensity. The red color represents over-exposure. The experiment has been replicated twice. (e) Quantification of the soil-surface mean fluorescent levels during the rehydration experiment. Day 618 timepoint represents the background fluorescence level before rehydration. The standard error of the mean was calculated and was too small to be observed on the graph. (f) Flow cytometry measurements of GFP+ and WT B. frigoritolerans A3E1 population fluorescence directly after separation from the soil on day 623 (bright green) and after overnight incubation in selective LB media (dark green). As a comparison, the fluorescence of a freshly inoculated B. frigoritolerans A3E1 population is presented (black).</p

Unique biosynthetic genes of <i>B</i>. <i>frigoritolerans</i> A3E1 compared to its reference DSM (8801).

Unique biosynthetic genes of B. frigoritolerans A3E1 compared to its reference DSM (8801).</p

Genomic mutations in <i>B</i>. <i>frigoritolerans</i> strains.

Genomic mutations in B. frigoritolerans strains.</p