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The ability to regulate, maintain and reproduce fluorogenic properties is a fundamental prerequisite of modern molecular diagnostics, nanotechnology and bioimaging. The sequence-dependence of the fluorescence properties in fluorophores commonly used in nucleic acid labelling is here being exploited to assemble a color scale in 256 shades of green Cy3 fluorescence. Using photolithography, we synthesize microarrays of labeled nucleic acids that can accurately reproduce 8-bit monochrome graphics by mapping color to fluorescence intensity and sequence. This DNA-based painting approach paves the way for a full RGB scale array fabrication process.

The untemplated activity of terminal deoxynucleotidyl transferase (TdT) represents its most appealing feature. Its use is well established in applications aiming for extension of a DNA initiator strand, but a more recent focus points to its potential in enzymatic de novo synthesis of DNA. Whereas its low substrate specificity for nucleoside triphosphates has been studied extensively, here we interrogate how the activity of TdT is modulated by the nature of the initiating strands, in particular their length, chemistry, and nucleotide composition. Investigation of full permutational libraries of mono- to pentamers of d-DNA, l-DNA, and 2'O-methyl-RNA of differing directionality immobilized to glass surfaces, and generated via photolithographic in situ synthesis, shows that the efficiency of extension strongly depends on the nucleobase sequence. We also show TdT being catalytically active on a non-nucleosidic substrate, hexaethylene glycol. These results offer new perspectives on constraints and strategies for de novo synthesis of DNA using TdT regarding the requirements for initiation of enzymatic generation of DNA.

RNA catalytic and binding interactions with proteins and small molecules are fundamental elements of cellular life processes as well as the basis for RNA therapeutics and molecular engineering. In the absence of quantitative predictive capacity for such bioaffinity interactions, high throughput experimental approaches are needed to sufficiently sample RNA sequence space. Here we report on a simple and highly accessible approach to convert commercially available customized DNA microarrays of any complexity and density to RNA microarrays via a T7 RNA polymerase-mediated extension of photocrosslinked methyl RNA primers and subsequent degradation of the DNA templates.

Photolithographic in situ synthesis of nucleic acids enables extremely high oligonucleotide sequence density as well as complex surface patterning and combined spatial and molecular information encoding. No longer limited to DNA synthesis, the technique allows for total control of both chemical and Cartesian space organization on surfaces, suggesting that hybridization patterns can be used to encode, display or encrypt informative signals on multiple chemically orthogonal levels. Nevertheless, cross‐hybridization reduces the available sequence space and limits information density. Here we introduce an additional, fully independent information channel in surface patterning with in situ l‐DNA synthesis. The bioorthogonality of mirror‐image DNA duplex formation prevents both cross‐hybridization on chimeric l‐/d‐DNA microarrays and also results in enzymatic orthogonality, such as nuclease‐proof DNA‐based signatures on the surface. We show how chimeric l‐/d‐DNA hybridization can be used to create informative surface patterns including QR codes, highly counterfeiting resistant authenticity watermarks, and concealed messages within high‐density d‐DNA microarrays., Expanding the toolbox for photolithographic in situ synthesis of microarrays to l‐DNA has opened an additional, independently accessible information channel. The mirror‐image DNA surface‐patterned information, including QR codes, authenticity watermarks and steganography, can thus be accessed in hybridization‐based assays using fluorescently labelled complementary probes without any interference with surface‐bound d‐DNA.

Nucleic acid microarrays are the only tools that can supply very large oligonucleotide libraries, cornerstones of the nascent fields of de novo gene assembly and DNA data storage. Although the chemical synthesis of oligonucleotides is highly developed and robust, it is not error free, requiring the design of methods that can correct or compensate for errors, or select for high-fidelity oligomers. However, outside the realm of array manufacturers, little is known about the sources of errors and their extent. In this study, we look at the error rate of DNA libraries synthesized by photolithography and dissect the proportion of deletion, insertion and substitution errors. We find that the deletion rate is governed by the photolysis yield. We identify the most important substitution error and correlate it to phosphoramidite coupling. Besides synthetic failures originating from the coupling cycle, we uncover the role of imperfections and limitations related to optics, highlight the importance of absorbing UV light to avoid internal reflections and chart the dependence of error rate on both position on the array and position within individual oligonucleotides. Being able to precisely quantify all types of errors will allow for optimal choice of fabrication parameters and array design.

Due to its longevity and enormous information density, DNA is an attractive medium for archival storage. The current hamstring of DNA data storage systems—both in cost and speed—is synthesis. The key idea for breaking this bottleneck pursued in this work is to move beyond the low-error and expensive synthesis employed almost exclusively in today’s systems, towards cheaper, potentially faster, but high-error synthesis technologies. Here, we demonstrate a DNA storage system that relies on massively parallel light-directed synthesis, which is considerably cheaper than conventional solid-phase synthesis. However, this technology has a high sequence error rate when optimized for speed. We demonstrate that even in this high-error regime, reliable storage of information is possible, by developing a pipeline of algorithms for encoding and reconstruction of the information. In our experiments, we store a file containing sheet music of Mozart, and show perfect data recovery from low synthesis fidelity DNA., Nature Communications, 11 (1), ISSN:2041-1723

DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3' terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly efficient reverse synthesis of complex DNA arrays using a photolithographic approach. The method is analogous to conventional solid phase synthesis but makes use of phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3'-hydroxyl group. The use of BzNPPOC, with more than twice the photolytic efficiency of the 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) previously used for 5'→3' synthesis, combined with additional optimizations to the coupling and oxidation reactions results in an approximately 3-fold improvement in the reverse synthesis efficiency of complex arrays of DNA oligonucleotides. The coupling efficiencies of the reverse phosphoramidites are as good as those of regular phosphoramidites, resulting in comparable yields. Microarrays of DNA surface tethered on the 5' end and with free 3' hydroxyl termini can be synthesized quickly and with similarly high stepwise coupling efficiency as microarrays using conventional 3'→5' synthesis. ispartof: SCIENTIFIC REPORTS vol:8 issue:1 ispartof: location:England status: published

Uracil-DNA glycosylase (UDG) is a critical DNA repair enzyme that is well conserved and ubiquitous in nearly all life forms. UDG protects genomic information integrity by catalyzing the excision from DNA of uracil nucleobases resulting from misincorporation or spontaneous cytosine deamination. UDG-mediated strand cleavage is also an important tool in molecular biotechnology, allowing for controlled and location-specific cleavage of single- and double DNA chemically or enzymatically synthesized with single or multiple incorporations of deoxyuridine. Although the cleavage mechanism is well-understood, detailed knowledge of efficiency and sequence specificity, in both single and double-stranded DNA contexts, has so far remained incomplete. Here we use an experimental approach based on the large-scale photolithographic synthesis of uracil-containing DNA oligonucleotides to comprehensively probe the context-dependent uracil excision efficiency of UDG. ispartof: Scientific Reports vol:9 issue:1 ispartof: location:England status: published

Naturally occurring cinnamon compounds such as cinnamaldehyde (CAL) and structurally related constituents have been associated with antiobesity activities, although studies regarding the impact on intestinal fatty acid uptake are scarce. Here, we demonstrate the effects of CAL and structural analogues cinnamyl alcohol (CALC), cinnamic acid (CAC), and cinnamyl isobutyrate on mechanisms regulating intestinal fatty acid uptake in differentiated Caco-2 cells. CAL, CALC, and CAC (3000 μM) were found to decrease fatty acid uptake by 58.0 ± 8.83, 19.4 ± 8.98, and 21.9 ± 6.55%, respectively. While CAL and CALC at a concentration of 300 μM increased serotonin release 14.9 ± 3.00- and 2.72 ± 0.69-fold, respectively, serotonin alone showed no effect on fatty acid uptake. However, CAL revealed transient receptor potential channel A1-dependency in the decrease of fatty acid uptake, as well as in CAL-induced serotonin release. Overall, CAL was identified as the most potent of the cinnamon constituents tested.

Chip-SIP is a stable isotope probing (SIP) method for linking microbial identity and function in mixed communities and is capable of analyzing multiple isotopes (

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid·ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of β-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification. ispartof: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS issue:150 ispartof: location:United States status: published

DNA microarrays have become commonplace in the last two decades, but the synthesis of other nucleic acids biochips, most importantly RNA, has only recently been developed to a similar extent. RNA microarrays can be seen as organized surfaces displaying a potentially very large number of unique sequences and are of invaluable help in understanding the complexity of RNA structure and function as they allow the probing and treatment of each of the many different sequences simultaneously. Three approaches have emerged for the fabrication of RNA microarrays. The earliest examples used a direct, manual or mechanical, deposition of pre-synthesized, purified RNA oligonucleotides onto the surface in a process called spotting. In a second approach, pre-spotted or in situ-synthesized DNA microarrays are employed as templates for the transcription of RNA, subsequently or immediately captured on the surface. Finally, a third approach attempts to mirror the phosphoramidite-based protocols for in situ synthesis of high-density DNA arrays in order to produce in situ synthesized RNA microarrays. In this mini-review, we describe the chemistry and the engineering behind the fabrications methods, underlining the advantages and shortcomings of each, and illustrate how versatile these platforms can be by presenting some of their applications.

Chip-SIP is a stable isotope probing (SIP) method for linking microbial identity and function in mixed communities and is capable of analyzing multiple isotopes (13C, 15N, and 18O) simultaneously. This method uses a high-density microarray to separate taxon-specific 16S (or 18S) rRNA genes and a high sensitivity magnetic sector secondary ion mass spectrometer (SIMS) to determine the relative isotope incorporation of the rRNA at each probe location. Using a maskless array synthesizer (MAS), we synthesize multiple unique sequences to target hundreds of taxa at the ribosomal operational taxonomic unit (OTU) level on an array surface, and then analyze it with a NanoSIMS 50, using its high-spatial resolution imaging capability to generate isotope ratios for individual probes. The Chip-SIP method has been used in diverse systems, including surface marine and estuarine water, rhizosphere, and peat soils, to quantify taxon-specific relative incorporation of different substrates in complex microbial communities. Depending on the hypothesis and experimental design, Chip-SIP allows the user to compare the same community incorporating different substrates, different communities incorporating the same substrate(s), or quantify how a community responds to treatment effects, such as temperature or nutrient concentrations.

The preparation of a polystyrene solid support decorated with a photolabile linker is described. The entire post-synthetic processing of RNA can be carried out in the solid phase in a minimum amount of time. The deprotected RNA is available for "on-support" hybridization and photolysis releases siRNA duplexes under mild, neutral conditions.

Assessing synthesis efficiency, errors, failed deprotections, and chemical and enzymatic degradation of oligonucleotides on microarrays is essential for improving existing in situ synthesis methods, and for the development of new chemistries. We describe the use of LC-MS to analyse DNA and RNA oligonucleotides deprotected and cleaved under basic conditions from microarrays fabricated using light-directed in situ chemistry. The data yield essential information on array quality and sequence identity.

Multiple 2-pyrenyl-C-nucleosides were incorporated into the center of a DNA duplex resulting in stable pyrene self-recognition and excimer formation. This helical pyrene array may find use in DNA-mediated charge transfer and in the creation of DNA-based sensors.

A new bromohexyl phosphoramidite was synthesized and used for the introduction of an azide function at the 5′-end of oligonucleotides after a treatment on solid support with sodium azide and sodium iodide. The corresponding 5′-azido oligonucleotide could be further used for ‘Click’ conjugation with alkyne derivatives or by Staudinger ligation.

Tricyclo-DNA (tc-DNA) is a promising candidate for oligonucleotide-based therapeutic applications exhibiting increased affinity to RNA and increased resistance to nucleases. However, as many other oligonucleotide analogs, tc-DNA does not readily cross cell membranes. We wished to address this issue by preparing a prodrug of tc-DNA containing a metabolically labile group at C(6') that promotes cellular uptake. Two monomeric nucleoside building blocks bearing an ester function at C(6') (tc(ee)-T and tc(hd)-T) were synthesized starting from a known C(6') functionalized bicyclic sugar unit to which the cyclopropane ring was introduced via carbene addition. NIS-mediated nucleosidation of the corresponding glycal with in situ persilylated thymine afforded the β-iodonucleoside exclusively that was dehalogenated via radical reduction. Diversity in the ester function was obtained by hydrolysis and reesterification. The two nucleosides were subsequently incorporated into DNA or tc-DNA by standard phosphoramidite chemistry. The reactivity of the ester function during oligonucleotide deprotection was explored and the corresponding C(6') amide, carboxylic acid, or unchanged ester functions were obtained, depending on the deprotection conditions. Compared to unmodified DNA, these tc-DNA derivatives increased the stability of duplexes investigated with ΔT(m)/mod of +0.4 to +2.0 °C. The only destabilizing residue was tc(hd)-T, most likely due to self-aggregation of the lipophilic side chains in the single stranded oligonucleotide. A decamer containing five tc(hd)-T residues was readily taken up by HeLa and HEK 293T cells without the use of a transfection agent.

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6')-β position is described. Compared to unmodified DNA these oligonucleotides show T(m)-neutral behavior when paired against complementary DNA and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic distribution which was not observed in the case of an unmodified control oligonucleotide.

The synthesis of cyclic, branched, and bicyclic oligonucleotides was performed by copper-catalyzed azide−alkyne cycloaddition assisted by microwaves in solution and on solid support. For that purpose, new phosphoramidite building blocks and new solid supports were designed to introduce alkyne and bromo functions into the same oligonucleotide by solid-phase synthesis on a DNA synthesizer. The bromine atom was then substituted by sodium azide to yield azide oligonucleotides. Cyclizations were found to be more efficient in solution than on solid support. This method allowed the efficient preparation of cyclic (6- to 20-mers), branched (with one or two dangling sequences), and bicyclic (2 × 10-mers) oligonucleotides.