RESUMEN
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.
Asunto(s)
Oro , Oligonucleótidos , Análisis de Secuencia por Matrices de Oligonucleótidos , Oro/química , ADN , Compuestos de Estaño/química , Oxidación-ReducciónRESUMEN
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'â3') 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.
Asunto(s)
ADN Polimerasa Dirigida por ADN , ADN , Análisis de Secuencia por Matrices de Oligonucleótidos , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/genética , Cartilla de ADN/metabolismo , Replicación del ADN/genética , OligonucleótidosRESUMEN
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 synthesisâhybridization affinityâto 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.
Asunto(s)
ADN , Ácidos Nucleicos , ADN/genética , Hibridación de Ácido Nucleico/métodos , Sondas de ADNRESUMEN
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.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Silanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Silanos/química , Hibridación de Ácido Nucleico/métodos , ADN/química , Vidrio/química , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Luz , Nucleótidos/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Procesos FotoquímicosRESUMEN
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.
Asunto(s)
ADN , Propiedades de Superficie , ADN/química , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/químicaRESUMEN
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.
Asunto(s)
ADN/química , ARN/química , Ribonucleasa H/química , ADN/síntesis química , ADN/genética , Escherichia coli/enzimología , Biblioteca de Genes , Hidrólisis , Secuencias Invertidas Repetidas , Análisis por Micromatrices , ARN/síntesis química , ARN/genética , Especificidad por SustratoRESUMEN
In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2Î-Fluoroarabinonucleic acid (2ÎF-ANA) is a prime candidate for such use in microarrays. Indeed, 2ÎF-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2ÎF-ANA and 2ÎF-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2ÎF-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2ÎF-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2ÎF-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2ÎF-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.
Asunto(s)
Aptámeros de Nucleótidos/química , Arabinonucleotidos/química , ADN/química , G-Cuádruplex , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Análisis de Secuencia por Matrices de Oligonucleótidos , Compuestos Organofosforados/químicaRESUMEN
While high-density DNA microarrays have been available for over three decades, the synthesis of equivalent RNA microarrays has proven intractable until now. Herein we describe the first inâ situ synthesis of mixed-based, high-density RNA microarrays using photolithography and light-sensitive RNA phosphoramidites. With coupling efficiencies comparable to those of DNA monomers, RNA oligonucleotides at least 30â nucleotides long can now efficiently be prepared using modified phosphoramidite chemistry. A two-step deprotection route unmasks the phosphodiester, the exocyclic amines and the 2' hydroxyl. Hybridization and enzymatic assays validate the quality and the identity of the surface-bound RNA. We show that high-density is feasible by synthesizing a complex RNA permutation library with 262144 unique sequences. We also introduce DNA/RNA chimeric microarrays and explore their applications by mapping the sequence specificity of RNase HII.
Asunto(s)
Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , ARN/genética , Secuencia de Bases , ADN/química , ADN/genética , Diseño de Equipo , Compuestos Organofosforados/química , ARN/química , Ribonucleasa H/químicaRESUMEN
Nucleic acid photolithography is the only microarray fabrication process that has demonstrated chemical versatility accommodating any type of nucleic acid. The current approach to RNA microarray synthesis requires long coupling and photolysis times and suffers from unavoidable degradation postsynthesis. In this study, we developed a series of RNA phosphoramidites with improved chemical and photochemical protection of the 2'- and 5'-OH functions. In so doing, we reduced the coupling time by more than half and the photolysis time by a factor of 4. Sequence libraries that would otherwise take over 6 hours to synthesize can now be prepared in half the time. Degradation is substantially lowered, and concomitantly, hybridization signals can reach over seven times those of the previous state of the art. Under those conditions, high-density RNA microarrays and RNA libraries can now be synthesized at greatly accelerated rates. We also synthesized fluorogenic RNA Mango aptamers on microarrays and investigated the effect of sequence mutations on their fluorogenic properties.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos , ARN , ARN/química , ARN/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Compuestos OrganofosforadosRESUMEN
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.
Asunto(s)
COVID-19 , SARS-CoV-2 , Lengua , Transcriptoma , Humanos , COVID-19/virología , COVID-19/genética , COVID-19/metabolismo , Masculino , Femenino , Adulto , Persona de Mediana Edad , Lengua/metabolismo , Lengua/virología , Lengua/patología , Inmunoglobulina G , Metales/metabolismo , Papilas Gustativas/metabolismo , Percepción del Gusto/genética , Gusto , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Percepción OlfatoriaRESUMEN
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.
Asunto(s)
Ácidos Nucleicos , ARN , ARN/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Hibridación de Ácido Nucleico , Ribonucleasa HRESUMEN
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 ß-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.
Asunto(s)
ADN/síntesis química , Oligodesoxirribonucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , ARN/química , Emparejamiento Base , Secuencia de Bases , Dicroismo Circular , ADN/química , ADN/metabolismo , Células HEK293 , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Oligodesoxirribonucleótidos/química , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Compuestos Organofosforados/química , TermodinámicaRESUMEN
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.
Asunto(s)
ADN de Cadena Simple , Ácidos Nucleicos , Carbocianinas , Colorantes FluorescentesRESUMEN
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.
Asunto(s)
Nanotecnología , Ácidos Nucleicos , ADNRESUMEN
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 â« G) in both 3' and 5' labeled single strands results in the strongest fluorescence signal and it changes to a terminal C (C â« T > A â« 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.
RESUMEN
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.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , ARN , Secuencia de Bases , Replicación del ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/química , ARN/genéticaRESUMEN
The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6')-ß 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.
Asunto(s)
Oligodesoxirribonucleótidos/química , Timidina/análogos & derivados , Emparejamiento Base , Secuencia de Bases , Línea Celular , ADN/química , ADN/metabolismo , Humanos , Lisina/química , Oligodesoxirribonucleótidos Antisentido/química , ARN/química , ARN/metabolismo , Timidina/síntesis química , Timidina/farmacología , Temperatura de TransiciónRESUMEN
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.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Replicación del ADN , Catálisis , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos/química , Estereoisomerismo , Especificidad por SustratoRESUMEN
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.