Michela Puddu

Robust Chemical Preservation of Digital Information on DNA in Silica with Error‐Correcting Codes

Information, such as text printed on paper or images projected onto microfilm, can survive for over 500 years. However, the storage of digital information for time frames exceeding 50 years is challenging. Here we show that digital information can be stored on DNA and recovered without errors for considerably longer time frames. To allow for the perfect recovery of the information, we encapsulate the DNA in an inorganic matrix, and employ error-correcting codes to correct storage-related errors. Specifically, we translated 83 kB of information to 4991 DNA segments, each 158 nucleotides long, which were encapsulated in silica. Accelerated aging experiments were performed to measure DNA decay kinetics, which show that data can be archived on DNA for millennia under a wide range of conditions. The original information could be recovered error free, even after treating the DNA in silica at 70 °C for one week. This is thermally equivalent to storing information on DNA in central Europe for 2000 years.

Magnetically Recoverable, Thermostable, Hydrophobic DNA/Silica Encapsulates and Their Application as Invisible Oil Tags

A method to encapsulate DNA in heat-resistant and inert magnetic particles was developed. An inexpensive synthesis technique based on co-precipitation was utilized to produce Fe2O3 nanoparticles, which were further functionalized with ammonium groups. DNA was adsorbed on this magnetic support, and the DNA/magnet nanocluster was surface coated with a dense silica layer by sol-gel chemistry. The materials were further surface modified with hexyltrimethoxysilane to achieve particle dispersibility in hydrophobic liquids. The hydrodynamic particle sizes were evaluated by analytical disc centrifugation, and the magnetic properties were investigated by vibrating sample magnetometry. The obtained nanoengineered encapsulates showed good dispersion abilities in various nonaqueous fluids and did not affect the optical properties of the hydrophobic dispersant when present at concentrations lower than 10(3) μg/L. Upon magnetic separation and particle dissolution, the DNA could be recovered unharmed and was analyzed by quantitative real-time PCR and Sanger sequencing. DNA encapsulated within the magnetic particles was stable for 2 years in decalin at room temperature, and the stability was further tested at elevated temperatures. The new magnetic DNA/silica encapsulates were utilized to developed a low-cost platform for the tracing/tagging of oils and oil-derived products, requiring 1 μg/L=1 ppb levels of the taggant and allowing quantification of taggant concentration on a logarithmic scale. The procedure was tested for the barcoding of a fuel (gasoline), a cosmetic oil (bergamot oil), and a food grade oil (extra virgin olive oil), being able to verify the authenticity of the products.

Reversible DNA encapsulation in silica to produce ROS-resistant and heat-resistant synthetic DNA 'fossils'

This protocol describes a method for encapsulating DNA into amorphous silica (glass) spheres, mimicking the protection of nucleic acids within ancient fossils. In this approach, DNA encapsulation is achieved after the ammonium functionalization of silica nanoparticles. Within the glass spheres, the nucleic acid molecules are hermetically sealed and protected from chemical attack, thereby withstanding high temperatures and aggressive radical oxygen species (ROS). The encapsulates can be used as inert taggants to trace chemical and biological entities. The present protocol is applicable to short double-stranded (ds) and single-stranded (ss) DNA fragments, genomic DNA and plasmids. The nucleic acids can be recovered from the glass spheres without harm by using fluoride-containing buffered oxide etch solutions. Special emphasis is placed in this protocol on the safe handling of these buffered hydrogen fluoride solutions. After dissolution of the spheres and subsequent purification, the nucleic acids can be analyzed by standard techniques (gel electrophoresis, quantitative PCR (qPCR) and sequencing). The protocol requires 6 d for completion with a total hands-on time of 4 h.

DNA protection against ultraviolet irradiation by encapsulation in a multilayered SiO2/TiO2 assembly

DNA is protected against UV-induced damage by encapsulation in a core-shell-shell particulate construct. The DNA is hermetically sealed in SiO2 particles coated with TiO2. The TiO2 coating acts as a physical sunscreen and prevents high energy photons from damaging the nucleic acids. DNA can be recovered unharmed from the protection system with fluoride comprising buffers, and then directly analyzed using biochemical standard techniques (quantitative PCR, gel electrophoresis and Sanger sequencing). The coatings increase the DNA UV resistance by 42 times, which is equivalent to the increase in UV resistance obtained by bacteria during sporulation. The attenuation coefficient of the 20 nm titania layer is 1.8 106 cm-1 at 254 nm UV irradiation and optical attenuation is largely attributed to light scattering on the titania surface.

Silica Microcapsules for Long-Term, Robust, and Reliable Room Temperature RNA Preservation

As a consequence of the latest revolutionary discoveries on its functions, RNA is certainly the hottest topic at the moment, being an exceptional tool in biology as well as in medicine. For the various applications, a proper RNA storage is required to prevent the degradation of this extremely unstable molecule. Here a novel freezing-free RNA storage strategy is presented, based on its encapsulation in silica spheres. The silica microcapsules protect the RNA by providing a water-free environment. In this way RNA can be safely stored for prolonged periods of time at ambient and elevated temperatures, maintaining its original integrity, as proved by gel-electrophoresis, capillary electrophoresis, and real-time reverse transcription-polymerase chain reaction (RT-qPCR). The RNA degradation rate at 65 °C in silica microcapsules is approximately ten times smaller in comparison to dry RNA samples or to samples stored in RNAstable matrix, a commercially available product. Moreover, RNA half-life at 65 °C is nearly identical to that of DNA within the silica microcapsules. Samples intended for use in gene expression are compatible with further analysis (RT-qPCR, Sanger sequencing). The novel storage technology permits to safely handle, store, and transport RNA samples, avoiding the expensive shipments and the problems of space presented by freezing-based strategies.

Detecting and Number Counting of Single Engineered Nanoparticles by Digital Particle Polymerase Chain Reaction

The concentrations of nanoparticles present in colloidal dispersions are usually measured and given in mass concentration (e.g. mg/mL), and number concentrations can only be obtained by making assumptions about nanoparticle size and morphology. Additionally traditional nanoparticle concentration measures are not very sensitive, and only the presence/absence of millions/billions of particles occurring together can be obtained. Here, we describe a method, which not only intrinsically results in number concentrations, but is also sensitive enough to count individual nanoparticles, one by one. To make this possible, the sensitivity of the polymerase chain reaction (PCR) was combined with a binary (=0/1, yes/no) measurement arrangement, binomial statistics and DNA comprising monodisperse silica nanoparticles. With this method, individual tagged particles in the range of 60-250 nm could be detected and counted in drinking water in absolute number, utilizing a standard qPCR device within 1.5 h of measurement time. For comparison, the method was validated with single particle inductively coupled plasma mass spectrometry (sp-ICPMS).

Submicrometer-Sized Thermometer Particles Exploiting Selective Nucleic Acid Stability

Encapsulated nucleic acid selective damage quantification by real-time polymerase chain reaction is used as sensing mechanism to build a novel class of submicrometer size thermometer. Thanks to the high thermal and chemical stability, and the capability of storing the read accumulated thermal history, the sensor overcomes some of current limitations in small scale thermometry.

Magnetically deliverable calcium phosphate nanoparticles for localized gene expression

Iron oxide doped tricalcium phosphate (Fe2O3@TCP) nanoparticles were designed as transfection vehicles and prepared by flame spray synthesis. Both components are known to be non-toxic and biocompatible. Calcium phosphate (CaP) facilitates DNA entry into cells without the need for toxic cationic mediators, while magnetic iron oxide allows for particle localization at a target site. Flame spray synthesis ensures easy and low-cost nanoparticle production in a reproducible way. Fe2O3@TCP nanoparticles, exhibiting DNA-binding capacity in the presence of CaCl2, were tested for transfection of a green fluorescent protein (GFP) encoding plasmid with Human Embryonic Kidney 293 (HEK 293) cells. Commercial magnetic agents, polyethylenimine (PEI) and standard calcium phosphate-mediated transfection were used for comparison. Transfection efficiency was estimated by GFP expression detected by fluorescence microscopy, while hoechst/ethidium homodimer-1 staining allowed the evaluation of method toxicity. We were able to efficiently transfect HEK 293 cells, and showed that Fe2O3@TCP particles and bound DNA can be concentrated in specific sites in a culture plate through the application of a magnetic field gradient to achieve localized transfection. While the commercial magnetic controls strongly affected cell growth and morphology, Fe2O3@TCP particles did not show marked toxicity and had only limited effects on cell proliferation. Overall performance in terms of transfection efficiency, cell proliferation and viability, were comparable to that of CaP and PEI, which lack magnetic targeting capability. The newly synthetized Fe2O3@TCP are, therefore, improved tools to deliver nucleic acids into cells and achieve spatial control of transfection.

Silica-Encapsulated DNA-Based Tracers for Aquifer Characterization

Environmental tracing is a direct way to characterize aquifers, evaluate the solute transfer parameter in underground reservoirs, and track contamination. By performing multitracer tests, and translating the tracer breakthrough times into tomographic maps, key parameters such as a reservoirs effective porosity and permeability field may be obtained. DNA, with its modular design, allows the generation of a virtually unlimited number of distinguishable tracers. To overcome the insufficient DNA stability due to microbial activity, heat, and chemical stress, we present a method to encapsulated DNA into silica with control over the particle size. The reliability of DNA quantification is improved by the sample preservation with NaN3 and particle redispersion strategies. In both sand column and unconsolidated aquifer experiments, DNA-based particle tracers exhibited slightly earlier and sharper breakthrough than the traditional solute tracer uranine. The reason behind this observation is the size exclusion effect, whereby larger tracer particles are excluded from small pores, and are therefore transported with higher average velocity, which is pore size-dependent. Identical surface properties, and thus flow behavior, makes the new material an attractive tracer to characterize sandy groundwater reservoirs or to track multiple sources of contaminants with high spatial resolution.