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Ribonuclease HII (RNase HII) is an essential endoribonuclease that binds to double-stranded DNA with RNA nucleotide incorporations and cleaves 5' of the ribonucleotide at RNA-DNA junctions. Thought to be present in all domains of life, RNase HII protects genomic integrity by initiating excision repair pathways that protect the encoded information from rapid degradation. There is sparse evidence that the enzyme cleaves some substrates better than others, but a large-scale study is missing. Such large-scale studies can be carried out on microarrays, and we employ chemical photolithography to synthesize very large combinatorial libraries of fluorescently labeled DNA/RNA chimeric sequences that self-anneal to form hairpin structures that are substrates for Escherichia coli RNase HII. The relative activity is determined by the loss of fluorescence upon cleavage. Each substrate includes a double-stranded 5 bp variable region with one to five consecutive ribonucleotide substitutions. We also examined the effect of all possible single and double mismatches, for a total of &gt;9500 unique structures. Differences in cleavage efficiency indicate some level of substrate preference, and we identified the 5'-dC/rC-rA-dX-3' motif in well-cleaved substrates. The results significantly extend known patterns of RNase HII sequence specificity and serve as a template using large-scale photolithographic synthesis to comprehensively map landscapes of substrate specificity of nucleic acid-processing enzymes.

Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage