In vivo cloning of artificial DNA nanostructures

Abstract

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.

Fig. 1.

Schematic drawing showing the in vivo replication of a DNA nanostructure. A single-stranded DNA nanostructure (four-arm junction or paranemic cross-over DNA) is inserted into phagemid, transformed into XL1-Blue cells, and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques.

Fig. 2

In vivo replication of a cloverleaf four-arm junction. (A) A 10% denaturing PAGE showing the final replication product. Lane M, 10-nt ssDNA ladder; lane 1, 10-pmol original J1 junction; lane 2, replication product yield from 10-ml saturated cell culture. (B) A 20% nondenaturing PAGE analysis of replicated J1. Lane M, 10-nt ssDNA ladder; lane 1, annealed original J1; lane 2, annealed replicated J1; lane 3, annealed random-sequence 79-mer ssDNA. (C) Endo VII cleavage of the J1 cloverleaf. The two images, Left (L) and Right (R) contain the results of the same experiment, but R has been run further than L to resolve cleavage positions nearer the 3′ end of the strand. Lanes L1 and R3 are untreated controls. Lanes L2 and R1 contain A–G sequencing ladders. The positions of possible cross-overs are indicated by “J” symbols. Lanes L3 and R2 contain the products of Endo VII cleavage experiments. The sites of bands are indicated. (D) Quantitative scans of hydroxyl radical autofootprinting gels. Two sets of traces are indicated: DS, when the molecule is paired with its Watson–Crick complement, and J1, when it is folded into its junction structure. Nucleotides are numbered from the 5′ end, which is labeled. The positions of expected junction-flanking nucleotides are indicated by arrows. In agreement with previous studies of this junction, protections of the J1 strand relative to the DS strand are seen at positions 26 and 27 and at positions 64 and 65 (see E for numbering). In addition, a small amount of protection is seen at position 50 as well, possibly indicating a small amount of cross-over isomerization. (E) Schematic summarizing the results of autofootprinting and Endo VII cleavage experiments. Autofootprinting protection positions are indicted by black triangles and endo VII cleavage positions are indicated by magenta triangles. The main sites of protection or cleavage are in agreement with previous studies of J1.

Fig. 3

In vivo replication of a PX DNA molecule. (A) A 10% denaturing PAGE showing the final replication product. Lane M, 10-pmol original sense PX; lanes 1–3, each lane contains final replication product yield from ≈40-ml saturated cell culture. (B) A 12% nondenaturing PAGE analysis of replicated PX. Lane M, 25-bp dsDNA ladder; lane 1, annealed 146-mer ssDNA with random sequence; lane 2, annealed original sense PX; lane 3, annealed replicated PX. (C) Ferguson analysis of PX molecules and a 125-bp dsDNA (duplex). PX molecules show very similar Ferguson slopes that are significantly different from that of the duplex. (D) Densitometer scans of autofootprinting gels. The DS lanes correspond to the cloned molecule paired with its Watson–Crick complement, and the PX lanes are the PX molecule itself. Downward pointing arrows indicate cross-over positions, and upward pointing arrows are present at loop positions. Protection is seen to flank each of the cross-overs. In addition, a small amount of protection is seen at sites 33 and 105, which might occlude each other. The single loop at position 72 does not seem to be protected significantly. (E) Schematic summarizing the autofootprinting results. Nucleotide positions are numbered, and positions of protection are shown as triangles in rough proportion to the extent of protection for the junction regions. Colored nucleotides indicate the sites of restriction.

Fig. 4

Psoralen cross-linking of the replicated junction (J1-O) structure in vivo and in vitro. (A) A 14% denaturing PAGE analysis of cross-linking products from J1-O. In vivo and in vitro cross-linking products are compared side by side and can be seen to be exactly the same. Black, red, and green lines were labeled beside bands representing un-cross-linked, mono-adducted, and cross-linked J1-O, respectively (see B). A 10-base DNA ladder is loaded in the far left lane as reference. (B) Ferguson analysis of the cross-linking products using denaturing PAGE. Colors of datasets correspond to the labels on the side of the cross-linking product bands in A. The product represented by the upper band shows a significantly different friction constant from the other two DNA species, implying its unique topology. (C) Schematic drawing of the mechanism in which psoralen reacts with the J1-O molecule. As illustrated, the psoralen molecule first induces the balance shift between the two cross-over isomers, and then cross-links (represented by a red curve) the two pyrimidines at the junction site in the presence of UV, altering the topology of the J1-O molecule.