Fabricating RNA Microarrays with RNA−DNA Surface Ligation Chemistry

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Abstract
A novel surface attachment strategy that utilizes RNA−DNA surface ligation chemistry to create renewable RNA microarrays from single-stranded DNA (ssDNA) microarrays on gold surfaces is demonstrated. The enzyme T4 DNA ligase was used to catalyze the formation of a phosphodiester bond between 5‘-phosphate-modified ssDNA attached to the surface and the 3‘-hydroxyl group of unlabeled RNA molecules from solution in the presence of a complementary template DNA strand. Surface plasmon resonance imaging (SPRI) measurements were performed to characterize the ligation process as well as to verify the bioactivity of the ssRNA microarray in terms of (i) the hybridization adsorption of complementary DNA onto the RNA array to form a surface RNA−DNA heteroduplex and (ii) the hydrolysis of the RNA microarrays with either ribonuclease S or ribonuclease H (RNase H). The hydrolysis of the surface-bound RNA with RNase H required the presence of a surface heteroduplex and, upon completion, regenerated the original 5‘-phosphate-terminated ssDNA array elements. These ssDNA array elements could be ligated again to create a new RNA microarray. These RNA microarrays can be used in the study of RNA−protein/RNA/aptamer bioaffinity interactions and for the enzymatically amplified SPRI detection of DNA in the presence of RNase H.