Abstract

The DNA nanoball (DNB) nanoarray is one of the key components in DNBSEQ sequencing technology. After sequencing, DNB nanoarray loaded with billions of DNBs with their sequences be determined and ready to be desposed of.
Here, we report a sensitive DocMF (DNB-based on-chip motif finding ) system that uses DNB nanoarray to profile protein-DNA interactions. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer adjacent motif (PAM) sequences of different CRISPR systems. DocMF can simultaneously screen both 5′ and 3′ PAMs with high coverage. For SpCas9, we found noncanonical 5′-NAG-3′ (~5%) and 5′-NGA-3′ (~1.6%), in addition to its common PAMs, 5′-NGG-3′ (~89.9%). More relaxed PAM sequences of two uncharacterized Cas endonucleases, VeCas9 and BvCas12a, were extensively characterized using DocMF. Moreover, we observed that dCas9, a DNA binding protein lacking endonuclease activity, preferably bound to the previously reported 5′-NGG-3′ sequence. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA binding proteins. We also describe a sensitive exonuclease III detection method using a “used” DNB nanoarray and demonstrate a detection limit as low as 0.001 U/mL. The 3’-end recessed dsDNA structure formed by sequencing was shown to be a perfect substrate for exonuclease III, but not for other nucleases such as exonuclease I, RecJf and nuclease P1. We developed an exonuclease III assay using the DNB nanoarray, together with other reagents within the BGISEQ-500 sequencing kit, which only required one additional cycle of sequencing. The DNB nanoarray can be reused for the exonuclease III assay at least five times. This method demonstrated superior sensitivity, selectivity, and reusability compared with other assay methods and is accompanied by low cost and simple setup.

Biography

Dr. Xu is a senior director of research in Complete Genomics, Inc/MGI tech. His research interests include DNA nanoball (DNB) based massively parallel DNA sequencing, WGS/WES, DNB based applications, protein engineering, enzymology. He did postdoctoral training in University of Minnesota, University of Pittsburgh and University of Texas at Austin. He earned his Ph.D. in biochemistry in 1989 from Shanghai Institute of Biochemistry, Chinese Academy of Science. He received his B.S. in chemistry from Zhejiang University, China.

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