Advances in Light‐Directed Synthesis of High‐Density Microarrays and Extension to<scp>RNA</scp>and<scp>2′F‐ANA</scp>Chemistries

Lietard, Jory; Damha, Masad J.; Somoza, Mark M.

doi:10.1002/9783527812103.ch11

Cited by 4 publications

(5 citation statements)

References 54 publications

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“…2) immediately next to the terminal dye. The synthesis is carried out using UV photolithography and DNA phosphoramidites, 21–25 either in the 3′ → 5′ or 5′ → 3′ direction 26 and carrying an appropriate photoprotecting group (BzNPPOC 27 ), followed by a final coupling of a Cy3/Cy5 phosphoramidite.…”

Section: Resultsmentioning

confidence: 99%

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

Kekić

Lietard

2022

Nanoscale

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Section: Resultsmentioning

confidence: 99%

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

Kekić

Lietard

2022

Nanoscale

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“…Oxaziridines readily oxidize P(III) compounds, and the camphorsulfonyl version (CSO) has been successfully applied to oligonucleotide synthesis requiring particular attention to the oxidizing conditions. ,,− A solution of CSO in acetonitrile is commercially available and can be readily diluted. In the context of our expanding chemical toolbox for the synthesis of modified oligonucleotides on microarrays − and our desire to adapt the protocols for microarray photolithography to non-traditional substrates, we explore in this study the synthesis parameters allowing for efficient and reliable CSO-mediated oxidation of oligonucleotides synthesized in situ on glass and on diverse metal-coated substrates. We found that DNA synthesis with stepwise CSO oxidation is as effective as I 2 -mediated oxidation, producing libraries of high quality, and, importantly, that it is efficient and compatible with gold and ITO-coated microscope slides.…”

Section: Introductionmentioning

confidence: 99%

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

Schaudy,

Ibañez-Redín,

Parlar

et al. 2024

Anal. Chem.

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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 I 2 /H 2 O, 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.

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“…Meanwhile, in situ synthesis of RNA microarrays has been found to be a challenging endeavor, with most of the difficulty associated with the protecting group for the 2'-OH function, usually a silyl moiety in standard solid-phase synthesis that is removed with fluorine-based reagents, chemicals that are incompatible with glass or silicon surfaces. Those issues and challenges in DNA and RNA microarray synthesis have lately been the subject of a large body of work, in particular with the photolithography approach 16 .…”

Section: Introductionmentioning

confidence: 99%

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

Lietard

Schaudy

Hölz

et al. 2019

JoVE

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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 mixedbase 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.

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Advances in Light‐Directed Synthesis of High‐Density Microarrays and Extension toRNAand2′F‐ANAChemistries

Cited by 4 publications

References 54 publications

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

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

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

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

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