Quantitative Characterization of RNA-Splinted Ligation as a Multipurpose Post-Synthetic Strategy for High-Density DNA Microarrays Synthesized by Nucleic Acid Photolithography

Complex arrays of surface-immobilized DNA oligonucleotides are widely used across diverse fields of bioanalytics, particularly for their applications in massively parallel genomic assays ranging from spatial transcriptomics to protein–DNA recognition studies. DNA microarrays produced by photolithography enable the introduction of versatile chemistries and complexities to surfaces with complete spatial control, but are partially constrained by synthetic error rates for longer probe architectures and by incompatibility with photosensitive chemical building blocks commonly used in phosphoramidite chemistry. Here, we present RNA-splinted ligation as a multipurpose post-synthetic microarray fabrication strategy for extending the complexity of DNA on microarray surfaces. We systematically characterize the parameters governing on-array DNA ligation and demonstrate its applications in labeling, reverse transcription, and the generation of highly complex patterns by introducing an otherwise incompatible photochemistry.