Multi-level patterning nucleic acid photolithography

Abstract

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.

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

“…Our studies help in further understanding the intrinsic mechanisms of uracil deglycosylation and help in defining the necessary structural requirements for an efficient enzymatic reaction. These efforts will serve to design and create optimal sequence contexts for UDG-mediated DNA degradation, which will prove useful for the controlled release of DNA, RNA and other nucleic acid libraries synthesized on high density microarrays 54–56 .…”

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

“…33 With mismatched sequences totaling >4000, the chimeric hairpin library contains >9000 unique elements that were synthesized in parallel, with multiple replicates, on a single glass substrate using maskless nucleic acid photolithography and 5′-photoprotected DNA and RNA phosphoramidites. 32,34,35 After deprotection and folding, the hairpins were incubated with E. coli RNase HII, and the array was then subsequently washed and scanned. Fluorescent scanning and subsequent data extraction clearly show differences in the loss of fluorescence, ranging from 0% to ≈45% loss relative to the pure DNA hairpin (Figure 1b and Figure S1).…”

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

“…In situ synthesized microarrays yield the highest oligonucleotide sequence density and, as such, are becoming an ideal source for the digital encoding of information in DNA [6] . In addition, such array fabrication offers complete control over the spatial arrangement of sequences, suggesting that informative surface patterns may be created through simple hybridization‐based assays [7] . Concomitant with the increasing throughput in DNA array synthesis and the decreasing costs of sequencing, there is greater access to DNA‐based information, which raises the potential question of privacy and traceability.…”

“…[6] In addition, such array fabrication offers completec ontrol over the spatial arrangement of sequences, suggesting that informative surfacep atterns may be created through simpleh ybridization-based assays. [7] Concomitant with the increasingthroughput in DNA array synthesis and the decreasing costs of sequencing, there is greater access to DNA-basedi nformation,w hich raises the potential question of privacy and traceability.I tm ay thus soonb ecome an ecessity for data stored in nucleic acid format to provide an encryption layer or atraceability signature that is only availabletothe manufacturer and customer/operator.S uch ak ey or signature could be produced in the form of binary matrices on the array itself and revealed via simple hybridization-based assays, where 0 = no hybridization and 1 = duplexf ormation with ad ye-labelled complementary probe.I deally,t his key should be synthesized alongside the bulk information,b ut not interfere with it. We have recently expanded the method of masklessa rray synthesis (MAS) [8] beyondn ative DNA, allowing for in situ synthesis of complex sequences containing 2'F-ANA [9] and RNA [10] monomers, at high densities.…”