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

Abstract

Photolithographic in situ synthesis of nucleic acids enables extremely high oligonucleotide sequence density as well as complex surfacep atterning and combined spatial and molecular information encoding.N o longer limitedt oD NA synthesis, the technique allows for total controlo fb oth chemical and Cartesian space organization on surfaces, suggesting that hybridizationp atterns can be used to encode, displayo re ncrypti nformatives ignals on multiple chemically orthogonal levels. Nevertheless, cross-hybridization reduces the available sequence space and limits information density.H ere we introduce an additional,f ully independent information channel in surface patterning with in situ l-DNA synthesis. The bioorthogonalityo fm irror-image DNA duplex formation prevents both cross-hybridization on chimeric l-/d-DNA microarrays and also resultsi ne nzymatic orthogonality,s uch 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 resistanta uthenticity watermarks, and concealedm essages within high-density d-DNA microarrays.

Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase

“… 42 We have previously measured the coupling efficiency of most non-RNA phosphoramidites for light-directed synthesis to be ∼99.9%, including DMTr-dT in its role as capping agent; G being the exception at 97–98%. 41 , 43 − 46 After synthesis and deprotection, the surface-bound oligonucleotides serve as initiator sequences for dT homopolymer extension with TdT polymerase. The efficiency of polymerization was evaluated by hybridization to the extension product.…”

“…In order to investigate the ability of TdT to extend terminal hydroxy groups of different nucleic acid chemistries, multiple replicates of each oligonucleotide strand were synthesized on the same array, each present in two versions: one where the final light-sensitive protecting group was removed at the end of the synthesis, exposing an accessible hydroxy group, whereas in the other version, the terminal hydroxy group was capped with a DMTr-dT phosphoramidite . We have previously measured the coupling efficiency of most non-RNA phosphoramidites for light-directed synthesis to be ∼99.9%, including DMTr-dT in its role as capping agent; G being the exception at 97–98%. ,− After synthesis and deprotection, the surface-bound oligonucleotides serve as initiator sequences for dT homopolymer extension with TdT polymerase. The efficiency of polymerization was evaluated by hybridization to the extension product.…”

“…L-DNA 5′-OH Extension. Our recent report establishing photolithographic in situ synthesis for mirror-image DNA (L-DNA) 46 motivated us to investigate the activity of TdT on this non-natural substrate. Surprisingly, we indeed were able to detect significant extension, albeit lower than for 3′-OH terminated D-DNA (Figure 2).…”

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

“…10 Nucleic acid microarrays can also be designed to generate informative molecular patterns for the purpose of authenticity or cryptography. 11,12 All of these approaches rely on standard hybridization following Watson-Crick rules. In most cases, hybridization is detected by recording a fluorescent signal from a dye-labelled complementary strand.…”

A Canvas of Spatially Arranged DNA Strands that Can Produce 24-bit Color Depth

“…DNA hybridization to complementary strands can inform on gene expression levels in a cell sample, and most microarray-based applications revolve around detecting the presence or absence of a fluorescence signal. − Here, fluorescence intensity is a function of thermal stability of the duplex, but the correlation of these two parameters has not been fully explored yet. Still, hybridization alone on patterned nucleic acid surfaces can create simple visual motifs − using multiple dyes to produce color and/or mismatches and truncations to alter intensity. Large-scale probing of the hybridization landscape via the introduction of mismatches and deletions should result in a much richer palette of fluorescence intensities from which a complex color scale can be assembled.…”