Gene Expression on DNA Biochips Patterned with Strand‐Displacement Lithography

Abstract

Lithographic patterning of DNA molecules enables spatial organization of cell-free genetic circuits under well-controlled experimental conditions. Here, we present a biocompatible, DNA-based resist termed "Bephore", which is based on commercially available components and can be patterned by both photo- and electron-beam lithography. The patterning mechanism is based on cleavage of a chemically modified DNA hairpin by ultraviolet light or electrons, and a subsequent strand-displacement reaction. All steps are performed in aqueous solution and do not require chemical development of the resist, which makes the lithographic process robust and biocompatible. Bephore is well suited for multistep lithographic processes, enabling the immobilization of different types of DNA molecules with micrometer precision. As an application, we demonstrate compartmentalized, on-chip gene expression from three sequentially immobilized DNA templates, leading to three spatially resolved protein-expression gradients.

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

“…Oligonucleotide microarrays are versatile analytical tools where very large numbers of unique sequences are immobilized at precise locations on a planar surface to allow simultaneous access. Originally developed as platforms for gene expression analysis of cell populations, [1] microarrays have recently found new applications in spatial transcriptomics, [2] spatial organization of cell‐free genetic circuits, [3] the generation of large oligonucleotide libraries for genomic applications, [4] DNA circuitry, [5] and others. 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] .…”

Cell-free microcompartmentalised transcription–translation for the prototyping of synthetic communication networks

“…In a follow-up study, Pardatscher et al . developed a lithography technique in order to functionalise a surface with up to three distinct DNA strands on a chip supporting both lysate-based and PURE-based TXTL reaction [43]. This technique will allow the study of complex interactions between multiple, spatially resolved, genes and the formation of more complex spatial patterns.…”

Multi-level patterning nucleic acid photolithography

“…Here we report on multi-patterning nucleic acid photolithography, which combines photolithographic synthesis of cleavable or immobile linear or branched nucleic acid constructs, complex crosslinking photolithography, and sequence-addressable enzymatic cleavage and polymerization. Previous work on photolithographic nucleic acid patterning has been limited to glass or silicon surface immobilization of a single pre-synthesized DNA sequence, followed by a photochemical modification step making use of crosslinking agents, photocleavable spacers or the photo-dimerization potential of thymine homopolymers 21–24 . In these approaches, illumination through a photomask results in either a positive or negative tone transfer that is visualized via addition or subtraction of a fluorescent label or results in surface-bound oligonucleotides which can be further functionalized by hybridization with DNA strands or oligonucleotides to generate biologically functional DNA brushes or DNA circuits.…”