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

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 >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.