Photolithographic in Situ Synthesis

The detailed procedure for photolithographic in situ synthesis has already been described elsewhere.^47,48 Briefly, microscopy glass slides (Schott NEXTERION glass D) were functionalized with a 2% N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (95%; abcr) solution in ethanol/water/acetic acid (95:5:0.1), washed, and cured at 120 °C under a vacuum for 2 h. An Expedite 8909 nucleic acid synthesizer was used to deliver reagents for synthesis to the glass substrate. Anhydrous acetonitrile (Biosolve) and DCI activator (Sigma-Aldrich, L032000) were maintained dry under molecular sieves (Sigma-Aldrich, Z509027). The exposure solvent consisted of 1% imidazole (Sigma-Aldrich, 56750) in anhydrous DMSO (Biosolve). The oxidizer was 20 mM I₂ in H₂O/pyridine/THF (Sigma-Aldrich L060060). Cyanoethyl phosphoramidites were used as 0.03 M solutions in dry acetonitrile and obtained from Orgentis (5′-BzNPPOC d-DNA, 3′-BzNPPOC d-DNA), ChemGenes (5′-NPPOC l-DNA; 3′-NPPOC 2′OMe-RNA; NPPOC-hexaethylene glycol), and LINK (DMTr-dT). Phosphoramidite purity and 3′ phosphitylation selectivity was verified by ³¹P and 2D ¹H–³¹P NMR. Coupling times varied depending on the type of phosphoramidite, between 15 s (d-DNA), 60 s (l-DNA and 2′OMe-RNA), 120 s (DMTr-dT), and 300 s (hexaethylene glycol). After synthesis, cyanoethyl and base protecting groups were removed by treating the array with ethylenediamine/ethanol (1:1) for either 2 or 15 h (3′-BzNPPOC d-DNA, NPPOC-hexaethylene glycol).

An optical system, focusing UV light from a 365 nm high-power UV-LED source (Nichia NVSU333A)⁴⁹ onto a digital micromirror device (Texas Instruments 0.7 XGA DMD) with 1024 × 768 individually addressable micromirrors, and via an Offner optical relay, further onto a functionalized glass slide, allows spatially resolved removal of the photosensitive protecting groups according to a set of digital masks generated by a MATLAB program.