Impaired metal perception and regulation of associated human foliate papillae tongue transcriptome in long-COVID-19.

Chemosensory impairment is an outstanding symptom of SARS-CoV-2 infections. We hypothesized that measured sensory impairments are accompanied by transcriptomic changes in the foliate papillae area of the tongue. Hospital personnel with known SARS-CoV-2 immunoglobulin G (IgG) status completed questionnaires on sensory perception (n = 158). A subcohort of n = 141 participated in forced choice taste tests, and n = 43 participants consented to donate tongue swabs of the foliate papillae area for whole transcriptome analysis. The study included four groups of participants differing in IgG levels (≥ 10 AU/mL = IgG+; < 10 AU/mL = IgG-) and self-reported sensory impairment (SSI±). IgG+ subjects not detecting metallic taste had higher IgG+ levels than IgG+ participants detecting iron gluconate (p = 0.03). Smell perception was the most impaired biological process in the transcriptome data from IgG+/SSI+ participants subjected to gene ontology enrichment. IgG+/SSI+ subjects demonstrated lower expression levels of 166 olfactory receptors (OR) and 9 taste associated receptors (TAS) of which OR1A2, OR2J2, OR1A1, OR5K1 and OR1G1, as well as TAS2R7 are linked to metallic perception. The question raised by this study is whether odorant receptors on the tongue (i) might play a role in metal sensation, and (ii) are potential targets for virus-initiated sensory impairments, which needs to be investigated in future functional studies.

Nonaqueous Oxidation in DNA Microarray Synthesis Improves the Oligonucleotide Quality and Preserves Surface Integrity on Gold and Indium Tin Oxide Substrates.

Nucleic acids attached to electrically conductive surfaces are very frequently used platforms for sensing and analyte detection as well as for imaging. Synthesizing DNA on these uncommon substrates and preserving the conductive layer is challenging as this coating tends to be damaged by the repeated use of iodine and water, which is the standard oxidizing medium following phosphoramidite coupling. Here, we thoroughly investigate the use of camphorsulfonyl oxaziridine (CSO), a nonaqueous alternative to I2/H2O, for the synthesis of DNA microarrays in situ. We find that CSO performs equally well in producing high hybridization signals on glass microscope slides, and CSO also protects the conductive layer on gold and indium tin oxide (ITO)-coated slides. DNA synthesis on conductive substrates with CSO oxidation yields microarrays of quality approaching that of conventional glass with intact physicochemical properties.

A Canvas of Spatially Arranged DNA Strands that Can Produce 24-bit Color Depth.

Nucleic acid microarray photolithography combines density, throughput, and positional control in DNA synthesis. These surface-bound sequence libraries are conventionally used in large-scale hybridization assays against fluorescently labeled, perfect-match DNA strands. Here, we introduce another layer of control for in situ microarray synthesis─hybridization affinity─to precisely modulate fluorescence intensity upon duplex formation. Using a combination of Cy3-, Cy5-, and fluorescein-labeled targets and an ensemble of truncated DNA probes, we organize 256 shades of red, green, and blue intensities that can be superimposed and merged. In so doing, hybridization alone is able to produce a large palette of 16 million colors or 24-bit color depth. Digital images can be reproduced with high fidelity at the micrometer scale by using a simple process that assigns sequence to any RGB value. Largely automated, this approach can be seen as miniaturized DNA-based painting.

Dipodal Silanes Greatly Stabilize Glass Surface Functionalization for DNA Microarray Synthesis and High-Throughput Biological Assays.

Glass is by far the most common substrate for biomolecular arrays, including high-throughput sequencing flow cells and microarrays. The native glass hydroxyl surface is modified by using silane chemistry to provide appropriate functional groups and reactivities for either in situ synthesis or surface immobilization of biologically or chemically synthesized biomolecules. These arrays, typically of oligonucleotides or peptides, are then subjected to long incubation times in warm aqueous buffers prior to fluorescence readout. Under these conditions, the siloxy bonds to the glass are susceptible to hydrolysis, resulting in significant loss of biomolecules and concomitant loss of signal from the assay. Here, we demonstrate that functionalization of glass surfaces with dipodal silanes results in greatly improved stability compared to equivalent functionalization with standard monopodal silanes. Using photolithographic in situ synthesis of DNA, we show that dipodal silanes are compatible with phosphoramidite chemistry and that hybridization performed on the resulting arrays provides greatly improved signal and signal-to-noise ratios compared with surfaces functionalized with monopodal silanes.

In situ enzymatic template replication on DNA microarrays.

DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs phosphoramidite chemistry and yields unmodified DNA generally attached to a surface at the 3' terminus. Having a freely accessible 3'-OH instead of 5'-OH is desirable too, and being able to introduce nucleoside analogs in a combinatorial manner is highly relevant in the context of nucleic acid therapeutics and in aptamer research. Here, we describe an enzymatic approach to the synthesis of high-density DNA microarrays that can also contain chemical modifications. The method uses a standard DNA microarray, to which a DNA primer is covalently bound through photocrosslinking. The extension of the primer with a DNA polymerase yields double-stranded DNA but is also amenable to the incorporation of modified dNTPs. Further processing with T7 exonuclease, which catalyzes the degradation of DNA in a specific (5'→3') direction, results in template strand removal. Overall, the method produces surface-bound natural and non-natural DNA oligonucleotides, is applicable to commercial microarrays and paves the way for the preparation of combinatorial, chemically modified aptamer libraries.

Enzymatic Synthesis of High-Density RNA Microarrays.

Oligonucleotide microarrays are used to investigate the interactome of nucleic acids. DNA microarrays are commercially available, whereas equivalent RNA microarrays are not. This protocol describes a method to convert DNA microarrays of any density and complexity into RNA microarrays using only readily available materials and reagents. This simple conversion protocol will facilitate the accessibility of RNA microarrays to a wide range of researchers. In addition to general considerations for the design of a template DNA microarray, this procedure describes the experimental steps of hybridization of an RNA primer to the immobilized DNA, followed by its covalent attachment via psoralen-mediated photocrosslinking. The subsequent enzymatic processing steps comprise the extension of the primer with T7 RNA polymerase to generate complementary RNA, and finally the removal of the DNA template with TURBO DNase. Beyond the conversion process, we also describe approaches to detect the RNA product either by internal labeling with fluorescently labeled NTPs or via hybridization to the product strand, a step that can then be complemented by an RNase H assay to confirm the nature of the product. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Conversion of a DNA microarray to an RNA microarray Alternate Protocol: Detection of RNA via incorporation of Cy3-UTP Support Protocol 1: Detection of RNA via hybridization Support Protocol 2: RNase H assay.

An 8-bit monochrome palette of fluorescent nucleic acid sequences for DNA-based painting.

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.

Sequence-dependence of Cy3 and Cy5 dyes in 3' terminally-labeled single-stranded DNA.

Fluorescence is an ideal tool to see and manipulate nucleic acids, and engage in their rich and complex biophysical properties. Labeling is the preferred approach to track and quantify fluorescence with nucleic acids and cyanine dyes are emblematic in this context. The fluorescent properties of cyanine dyes are known to be sequence-dependent, with purines in the immediate vicinity increasing the fluorescence intensity of Cy3 and Cy5 dyes, and the ability of nucleobases to modulate the photophysical properties of common fluorophores may influence fluorescence measurements in critical assays such as FISH, qPCR or high-throughput sequencing. In this paper, we comprehensively map the sequence-dependence of Cy3 and Cy5 dyes in 3'-fluorescently labeled single-stranded DNA by preparing the complete permutation library of the 5 consecutive nucleotides immediately adjacent to the dye, or 1024 sequences. G-rich motifs dominate the high fluorescence range, while C-rich motifs lead to significant quenching, an observation consistent with 5'-labeled systems. We also uncover GCGC patterns in the extreme top range of fluorescence, a feature specific to 3'-Cy3 and Cy5 oligonucleotides. This study represents the final piece in linking nucleotide identity to fluorescence changes for Cy3, Cy5 and fluorescein in all 3', 5', single-stranded and double-stranded DNA formats.

Simple synthesis of massively parallel RNA microarrays via enzymatic conversion from DNA microarrays.

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.

Sequence-dependent quenching of fluorescein fluorescence on single-stranded and double-stranded DNA.

Fluorescein is commonly used to label macromolecules, particularly proteins and nucleic acids, but its fluorescence is known to be strongly dependent on its direct chemical environment. In the case of fluorescein-labeled nucleic acids, nucleobase-specific quenching originating in photoinduced charge transfer interactions results in sequence-dependent chemical environments. The resulting sequence specificity of fluorescent intensities can be used as a proximity detection tool, but can also lead to biases when the abundance of labeled nucleic acids is quantified by fluorescence intensity. Here we comprehensively survey how DNA sequences affect fluorescence intensity by preparing permutational libraries containing all possible 5mer contexts of both single-stranded and double-stranded DNA 3' or 5' end labeled with fluorescein (6-carboxyfluorescein, FAM). We observe the expected large quenching of fluorescence with guanine proximity but also find more complex fluorescence intensity changes depending on sequence contexts involving proximity to all four nucleobases. A terminal T (T > A ≈ C ≫ G) in both 3' and 5' labeled single strands results in the strongest fluorescence signal and it changes to a terminal C (C ≫ T > A ≫ G) in double-stranded DNA. Therefore, in dsDNA, the terminal G·C base pair largely controls the intensity of fluorescence emission depending on which of these two nucleotides the dye is attached to. Our data confirms the importance of guanine in fluorescence quenching while pointing towards an additional mechanism beyond the redox potential of DNA bases in modulating fluorescein intensity in both single and double stranded DNA. This study should help in designing better nucleic acid probes that can take sequence-dependent quenching effects into account.

Chemical and photochemical error rates in light-directed synthesis of complex DNA libraries.

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.

Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase.

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.

Low cost DNA data storage using photolithographic synthesis and advanced information reconstruction and error correction.

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.

l-DNA Duplex Formation as a Bioorthogonal Information Channel in Nucleic Acid-Based Surface Patterning.

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.

Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries.

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.

Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage.

Ribonuclease HII (RNase HII) is an essential endoribonuclease that binds to double-stranded DNA with RNA nucleotide incorporations and cleaves 5' of the ribonucleotide at RNA-DNA junctions. Thought to be present in all domains of life, RNase HII protects genomic integrity by initiating excision repair pathways that protect the encoded information from rapid degradation. There is sparse evidence that the enzyme cleaves some substrates better than others, but a large-scale study is missing. Such large-scale studies can be carried out on microarrays, and we employ chemical photolithography to synthesize very large combinatorial libraries of fluorescently labeled DNA/RNA chimeric sequences that self-anneal to form hairpin structures that are substrates for Escherichia coli RNase HII. The relative activity is determined by the loss of fluorescence upon cleavage. Each substrate includes a double-stranded 5 bp variable region with one to five consecutive ribonucleotide substitutions. We also examined the effect of all possible single and double mismatches, for a total of >9500 unique structures. Differences in cleavage efficiency indicate some level of substrate preference, and we identified the 5'-dC/rC-rA-dX-3' motif in well-cleaved substrates. The results significantly extend known patterns of RNase HII sequence specificity and serve as a template using large-scale photolithographic synthesis to comprehensively map landscapes of substrate specificity of nucleic acid-processing enzymes.

Identification of Cinnamaldehyde as Most Effective Fatty Acid Uptake Reducing Cinnamon-Derived Compound in Differentiated Caco-2 Cells Compared to Its Structural Analogues Cinnamyl Alcohol, Cinnamic Acid, and Cinnamyl Isobutyrate.

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.

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries.

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 ß-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.

Multi-level patterning nucleic acid photolithography.

The versatile and tunable self-assembly properties of nucleic acids and engineered nucleic acid constructs make them invaluable in constructing microscale and nanoscale devices, structures and circuits. Increasing the complexity, functionality and ease of assembly of such constructs, as well as interfacing them to the macroscopic world requires a multifaceted and programmable fabrication approach that combines efficient and spatially resolved nucleic acid synthesis with multiple post-synthetic chemical and enzymatic modifications. Here we demonstrate a multi-level photolithographic patterning approach that starts with large-scale in situ surface synthesis of natural, modified or chimeric nucleic acid molecular structures and is followed by chemical and enzymatic nucleic acid modifications and processing. The resulting high-complexity, micrometer-resolution nucleic acid surface patterns include linear and branched structures, multi-color fluorophore labeling and programmable targeted oligonucleotide immobilization and cleavage.

Chip-SIP: Stable Isotope Probing Analyzed with rRNA-Targeted Microarrays and NanoSIMS.

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.