Affinity Isolation of Defined Genomic Fragments Cleaved by Nuclease S1‐based Artificial Restriction DNA Cutter

Abstract

The human genome is highly susceptible to various modifications, lesions, and damage. To analyze lesions and proteins bound to a defined region of the human genome, the genome should be fragmented at desired sites and the region of interest should be isolated. The few available methods for isolating a desired region of the human genome have serious drawbacks and can only be applied to specific sequences or require tedious experimental procedures. We have recently developed a novel method to isolate a desired fragment of the genome released by site‐specific scission of DNA using a pair of pseudo‐complementary peptide nucleic acids (pcPNAs) and S1 nuclease. When conjugated to biotin, one of the pcPNAs can be used to affinity purify the cleavage product. Here we report a detailed protocol to isolate defined kilobase‐length DNA fragments that can be applied to plasmid or genomic DNA and is not limited by sequence. © 2019 by John Wiley & Sons, Inc.

Ce-based solid-phase catalysts for phosphate hydrolysis as new tools for next-generation nanoarchitectonics

“…Ce 4+ complex can be replaced by nuclease S1. By extending this strategy, a useful tool to clip out a desired double-stranded fragment from huge DNA was developed [ 197 , 198 ]. The key is to attach a biotin to one of the PNA pair which is used to recognize the target scission site.…”

Artificial Restriction DNA Cutter Using Nuclease S1 for Site‐Selective Scission of Genomic DNA

“…The optimal length in terms of synthetic yield, formation of stable invasion complex, and site‐specific DNA scission is 15 bases. Design the pcPNA pair in such a way that they are 5 bases laterally shifted from each other when they invade the DNA (see Fig. A). To isolate a particular fragment of target DNA, synthesize one pcPNA with biotin attached to the C‐terminal lysine via a PEG2 linker (Rajendran et al, ).…”

“… Use the commercially available Boc monomers of G and C that are used for conventional PNA synthesis. To attach lysine residues to the termini of pcPNA, use commercially available Boc‐protected lysine monomer that is used for routine peptide synthesis. Use commercially available Boc‐protected phosphoserine. Synthesize the Boc monomers of D and U s , as well as Rapoport's capping reagent, using previously described methods (Komiyama et al., ). Alternatively, these reagents can be purchased from custom synthesis companies. To isolate the target fragment (Rajendran et al, ), use commercially available N‐Fmoc‐Amido‐dPEG2‐Acid (Quanta BioDesign) and Fmoc‐Lys(biotin)‐OH (from Merck Millipore). Synthesize the pcPNAs as described by Komiyama et al. (). Purify the products by reversed‐phase HPLC.…”

“…The isolation process can be easily carried out by the affinity pulldown method simply by modifying one of the pcPNAs with biotin. See Rajendran et al () for the protocol to isolate defined ARCUT scission products.…”

“…Finally, we show that pcPNA/S1 nuclease‐assisted DNA cleavage can be used for site‐selective scission of the human genome (see Basic Protocol 2 and Support Protocol). We describe how to purify ARCUT scission products in a companion article (Rajendran, Shigi, Sumaoka, & Komiyama, ).…”