Simple synthesis of massively parallel RNA microarrays via enzymatic conversion from DNA microarrays

Abstract

RNA catalytic and binding interactions with proteins and small molecules are fundamental elements of cellular life processes as well as the basis for RNA therapeutics and molecular engineering. In the absence of quantitative predictive capacity for such bioaffinity interactions, high throughput experimental approaches are needed to sufficiently sample RNA sequence space. Here we report on a simple and highly accessible approach to convert commercially available customized DNA microarrays of any complexity and density to RNA microarrays via a T7 RNA polymerase-mediated extension of photocrosslinked methyl RNA primers and subsequent degradation of the DNA templates.

Nonaqueous Oxidation in DNA Microarray Synthesis Improves the Oligonucleotide Quality and Preserves Surface Integrity on Gold and Indium Tin Oxide Substrates

“…The process of nucleic acid microarray synthesis by maskless photolithography (MAS) has been extensively described elsewhere. ,− For the sake of brevity, only the essential aspects of the process will be discussed here. Oligonucleotide elongation follows the same principle as conventional solid-phase synthesis, meaning that synthesis proceeds through the stepwise addition of nucleotides in a cycle-based approach with repeating chemical reactions following the order 5′-deblocking → coupling → oxidation.…”

Enzymatic Synthesis of High‐Density RNA Microarrays

“… Skirted reagent tubes with screw cap, 50 ml (e.g., Greiner bio‐one, VWR 525‐0396) Microcentrifuge tubes Biopur Safe‐Lock 1.5 ml (VWR 211‐2161) Micropipettes Sterile filter tips (Starlab S1121‐3810, S1120‐1840, and S1126‐7810) Hybridization oven (e.g., Boekel Scientific “Little Shot” 230500) DNA template microarray Self‐adhesive hybridization chambers, whose dimensions are adapted to the layout of the microarray (e.g., Grace Bio‐Labs SecureSeal SA200, RD500958 for Agilent SurePrint 4 × 44 K DNA microarray) Tweezers Adhesive seal tabs (Grace Bio‐Labs ST200) Aluminum foil Timer Slide spinner (Labnet International C1303‐T) Fridge or cold room at ∼4°C High‐power Nichia NVSU333A 365 nm UV LED in a custom‐made setup for photocrosslinking, as previously described in detail (Schaudy, Hölz, Lietard, & Somoza, 2022 ) and shown in Figure 4 SÜSS Model 1000 UV intensity meter with a 365‐nm probe (alternative: Ushio UIT 201 Digital UV intensity meter with Ushio UVD‐365PD 365‐nm probe) …”

“…High‐power Nichia NVSU333A 365 nm UV LED in a custom‐made setup for photocrosslinking, as previously described in detail (Schaudy, Hölz, Lietard, & Somoza, 2022 ) and shown in Figure 4…”

“…Shorter linkers are more stable toward nuclease degradation, but they also negatively affect the accessibility of the template strand for the polymerase in the primer extension. A detailed investigation of the effect of linker length on the process is described elsewhere (Schaudy et al., 2022 ). In our hands, the highest yield of conversion was achieved with a T 10 linker on DNA microarrays from a commercial source.…”

Dipodal Silanes Greatly Stabilize Glass Surface Functionalization for DNA Microarray Synthesis and High-Throughput Biological Assays

“…Glass surfaces are almost universally used as supports for biomolecules in a wide range of high-throughput analyses, including sequencing flow cells, , DNA arrays for gene expression studies, − spatial transcriptomics analysis, DNA − and RNA − interactome analysis, aptamer-binding surveys, , and epitope mapping using peptide arrays. , Glass (generally borosilicate) is inexpensive and widely available and has physical and chemical properties that are often experimentally important including optical transparency, dimensional stability, inertness, and low autofluorescence. The chemical versatility of the silane chemistries adds synergistically to the desirable properties of glass, providing an accessible approach to efficiently derivatize the native hydroxyl groups with a wide variety of functional groups that can be used to create well-defined surface properties, including providing reactive groups suitable for immobilizing biological macromolecules and reactive groups suitable for in situ chemical peptide or nucleic acid synthesis. , In parallel to immobilization chemistries, silanes can also be used to tune the hydrophobicity of the surface, e.g., for promoting tissue-to-surface coupling in spatial transcriptomics, or to provide antifouling properties that can reduce nonspecific adhesion of biomolecules to the surface. − …”