Ultraviolet
light emitting diodes (UV LEDs) have become widespread
in chemical research as highly efficient light sources for photochemistry
and photopolymerization. However, in more complex experimental setups
requiring highly concentrated light and highly spatially resolved
patterning of the light, high-pressure mercury arc lamps are still
widely used because they emit intense UV light from a compact arc
volume that can be efficiently coupled into optical systems. Advances
in the deposition and p-type doping of gallium nitride
have recently permitted the manufacture of UV LEDs capable of replacing
mercury arc lamps also in these applications. These UV LEDs exceed
the spectral radiance of mercury lamps even at the intense I-line
at 365 nm. Here we present the successful exchange of a high-pressure
mercury arc lamp for a new generation UV LED as a light source in
photolithographic chemistry and its use in the fabrication of high-density
DNA microarrays. We show that the improved light radiance and efficiency
of these LEDs offer substantial practical, economic and ecological
advantages, including faster synthesis, lower hardware costs, very
long lifetime, an >85-fold reduction in electricity consumption
and
the elimination of mercury waste and contamination.
BackgroundDNA microarrays are a core element of modern genomics research and medical diagnostics, allowing the simple and simultaneous determination of the relative abundances of hundreds of thousands to millions of genomic DNA or RNA sequences in a sample. Photolithographic in situ synthesis, using light projection from a digitally-controlled array of micromirrors, has been successful at both commercial and laboratory scales. The advantages of this synthesis method are its ability to reliably produce high-quality custom microarrays with a very high spatial density of DNA features using a compact device with few moving parts. The phosphoramidite chemistry used in photolithographic synthesis is similar to that used in conventional solid-phase synthesis of oligonucleotides, but some unique differences require an independent optimization of the synthesis chemistry to achieve fast and low-cost synthesis without compromising microarray quality.ResultsHigh microarray quality could be maintained while reducing coupling time to a few seconds using DCI activator. Five coupling activators were compared, which resulted in microarray hybridization signals following the order ETT > Activator 42 > DCI ≫ BTT ≫ pyridinium chloride, but only the use of DCI led to both high signal and highly uniform feature intensities. The photodeprotection time was also reduced to a few seconds by replacing the NPPOC photolabile group with the new thiophenyl-NPPOC group. Other chemical parameters, such as oxidation and washing steps were also optimized.ConclusionsHighly optimized and microarray-specific phosphoramidite chemistry, along with the use of the very photosensitive thiophenyl-NPPOC protecting group allow for the synthesis of high-complexity DNA arrays using coupling times of 15 s and deprotection times of 9 s. The resulting overall cycle time (coupling to coupling) of about 50 s, results in a three-fold reduction in synthesis time.
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.
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.
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