Applications of PNA-Based Artificial Restriction DNA Cutters

Abstract

More than ten years ago, artificial restriction DNA cutters were developed by combining two pseudo-complementary peptide nucleic acid (pcPNA) strands with either Ce(IV)/EDTA or S1 nuclease. They have remarkably high site-specificity and can cut only one predetermined site in the human genome. In this article, recent progress of these man-made tools have been reviewed. By cutting the human genome site-selectively, desired fragments can be clipped from either the termini of chromosomes (telomeres) or from the middle of genome. These fragments should provide important information on the biological functions of complicated genome system. DNA/RNA hybrid duplexes, which are formed in living cells, are also site-selectively hydrolyzed by these cutters. In order to further facilitate the applications of the artificial DNA cutters, various chemical modifications have been attempted. One of the most important successes is preparation of PNA derivatives which can form double-duplex invasion complex even under high salt conditions. This is important for in vivo applications, since the inside of living cells is abundant of metal ions. Furthermore, site-selective DNA cutters which require only one PNA strand, in place of a pair of pcPNA strands, are developed. This progress has opened a way to new fields of PNA-based biochemistry and biotechnology.

Keywords: DNA/RNA hybrid; PNA invasion; site-selective DNA cutter; site-selective genome scission.

Figure 1

Clipping of individual telomere from each of the chromosomes in human cells. ARCUT is designed to cut the site where the telomeric repeats (TTAGGG)n terminate and the sequence is specific to each telomere (Xp/Yp here).

Figure 2

Clipping of internal fragments from human genome using the S1 nuclease–pcPNA combination. The genome was first treated with artificial DNA cutter (the combination of two pcPNA strands and S1 nuclease), and then digested by a restriction enzyme NsiI. The products were analyzed by Southern blotting. (a) The sequences of pcPNAs targeting the BFP gene which is stably integrated into the human genome; and (b) Southern blotting. Lane 1, treatment of the genome with S1 nuclease in the absence of the pcPNAs; lane 2, incubation of the genome with the pcPNAs in the absence of S1 nuclease; lane 3, the scission by using the S1 nuclease–pcPNA combination at 37 °C for 1 h; lane 4, the scission by the combination at 50 °C for 30 min; lane M, 1 kbp markers. As presented in (c), the dual scission by the artificial DNA cutter and NsiI should provide two fragments of 0.8 and 1.5 kbp, which were detected in the lower parts of the gels in lanes 3 and 4 in (b).

Figure 3

Site-selective scission of DNA/RNA hybrids by ARCUT. The DNA strand in the upper gel is a part of the epidermal growth factor receptor (EGFR; from human chromosome 7), whereas the DNA in the lower gel is from the proto-oncogene tyrosine-protein kinase (Src; from human chromosome 20). S, DNA/RNA hybrid; I, invasion complex; F, the scission fragments. Scission conditions: [substrate DNA/RNA duplex] = 10 nM, [pcPNA] = 50 nM, [S1 nuclease] = 9 units/μL, [NaCl] = 280 mM, and [ZnSO₄] = 1 mM at pH 4.6 and 25 °C for 3 min.

Figure 4

(a) Recognition of G-rich sequence by one strand of PNA and (b) its site-selective scission by Ce(IV)/EDTA. In (a), one PNA strand forms a G-quadruplex with the upper G-rich DNA strand, and another PNA strand forms Watson-Crick duplex with the lower C-rich DNA strand.

Figure 5

Gel-shift assay for binding of the pcPNA-polyamide conjugate and complementary pcPNA to dsDNA. The pcPNA (blue) binds to 5′-TCATCAGTAA-3′ and the polyamide (orange and green balls) bind to 5′-AGTCCT-3′ in the DNA1. Lane 1, DNA only; lane 2, DNA with two pcPNA strands without the polyamide; lane 3, DNA with the combination of the pcPNA-polyamide conjugate and complementary pcPNA; lane 4, the conjugate only; M, 100 bp ladder. Invasion conditions: [DNA] = 10 nM, [Conjugate 1] = 100 nM, and [pcPNA] = 100 nM at pH 7.0 and 50 °C for 24 h.

Figure 6

Structure of ^(S)−Meγ-PNA which shows unique invasion properties. For the purpose of comparison, the parent PNA is also shown.

Figure 7

Structures of nanomechanical DNA origami devices used to visually detect PNA invasion. (a) Schematic illustration of the system; and (b) structures of the pre-closing zipper elements and PNA.

Figure 7

Structures of nanomechanical DNA origami devices used to visually detect PNA invasion. (a) Schematic illustration of the system; and (b) structures of the pre-closing zipper elements and PNA.