In vivo of artificial DNA nanostructures

Chenxiang Lin*†, Sherri Rinker*†, Xing Wang†‡, Yan Liu*, Nadrian C. Seeman‡§, and Hao Yan*§

*Department of Chemistry and Biochemistry and The Biodesign Institute, Arizona State University, Tempe, AZ 85287; and ‡Department of Chemistry, New York University, New York, NY 10003

Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved September 5, 2008 (received for review June 3, 2008) Mimicking nature is both a key goal and a difficult challenge for the rolling-circle mechanism is also involved in the replication of scientific enterprise. DNA, well known as the genetic-information and bacterial in vivo,sothis carrier in nature, can be replicated efficiently in living cells. Today, result suggested that it might be possible to replicate DNA despite the dramatic evolution of DNA nanotechnology, a versatile nanostructures by using and . In fact, such a method that replicates artificial DNA nanostructures with complex possibility was proposed previously (22–23). Moreover, DNA secondary structures remains an appealing target. Previous success containing a designed hairpin structure has been incorporated in replicating DNA nanostructures enzymatically in vitro suggests into a vector as the substrate for a rolling-circle transcript to that a possible solution could be cloning these nanostructures by produce RNA in Escherichia coli cells (24). However, using viruses. Here, we report a system where a single-stranded to our knowledge, in vivo replication of artificial DNA nano- DNA nanostructure (Holliday junction or paranemic cross-over structures has not been achieved to date. DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy Results and Discussion numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is ver- Design of the Replication Process. We present a biological method to ified by a number of assays including nondenaturing PAGE, Fer- replicate single-stranded DNA nanostructures. The design is illus- guson analysis, endonuclease VII digestion, and hydroxyl radical trated schematically in Fig. 1. A single-stranded Holliday junction autofootprinting. The simplicity, efficiency, and fidelity of nature and PX DNA are chosen as model molecules, because they have are fully reflected in this system. UV-induced psoralen cross-linking been thoroughly characterized and successfully replicated by using is used to probe the secondary structure of the inserted junction in RCA in vitro (21–22). The in vivo replication starts by inserting the CHEMISTRY infected cells. Our data suggest the possible formation of the desired nanostructure into double-stranded phagemid vector Lit- immobile four-arm junction in vivo. mus 28i (Ϸ2.8-kb) with an ampicillin resistance and a single- stranded (M13) DNA replication origin. To do this, the ssDNA that ͉ ͉ ͉ DNA nanotechnology immobile DNA junction self-replication folds into the designed nanostructure is extended at both the 5Ј and synthetic biology 3Ј ends, hybridized to its Watson–Crick complement with end extensions to form a double-stranded (DS) insert with proper sticky he notion that DNA is merely the gene encoder of living ends, and ligated with correspondingly restricted Litmus 28i. The Tsystems has been eclipsed by the successful development of vector with the DS insert is then transformed into E. coli cells BIOCHEMISTRY DNA nanotechnology (1–2). Using branched DNA as the main (XL1-Blue), which are later incubated in an ampicillin-containing building block (also known as a ‘‘tile’’) and cohesive single- culture to ensure the selective amplification of Litmus 28i- stranded DNA (ssDNA) ends to designate the pairing strategy transformed cells. At the next stage, the transformed cells in a single for tile–tile recognition, one can rationally design and assemble colony are infected by helper phage M13KO7 that carries a complicated nanoarchitectures from specifically designed DNA kanamycin resistance gene. M13KO7 has the special property that . Objects in both two and three dimensions with rather than self-replicating, it preferentially packages single- a large variety of geometries and topologies have been built from stranded phagemid bearing the M13 origin and secretes it into the DNA with excellent yield (3–9); this development enables the culture medium (25). The infected cells are then amplified in a construction of DNA-based nanodevices (10–11) and DNA medium containing kanamycin. Finally, the phages are precipi- template directed organization of other molecular species (12– tated, and their single-stranded DNA molecules are extracted and 19). The construction of such nanoscale objects constitutes the restricted. A high copy number of the DNA nanostructure is thus basis of DNA nanotechnology. However, the synthetic scale of obtained as a result of the exponential replication of phagemid oligonucleotides limits the potential applications of DNA nano- vectors in bacteria cells. technology, especially when the nanostructure is designed to be Ͼ made from long ssDNA ( 100 bases). Replicable DNA nano- Replication of a Four-Arm DNA Nanojunction. As a pilot experiment, structures are thus a highly desirable goal to help overcome this we tested the in vivo replication of a four-arm DNA nanojunction barrier. (J1) that is 79 nt long. First, the correct DS insert was verified To this end, several possible solutions have been explored. For by assaying the restricted double-stranded phagemid extracted example, a three-point star motif has been replicated by chemical from the amplified single colony of transformed cells with methods in which the parental nanomotif serves as the template to direct the chemical ligation of nonidentical DNA strands to form the next generation of the nanomotif (20). Another in- Author contributions: C.L., S.R., X.W., N.C.S., and H.Y. designed research; C.L., S.R., and X.W. stance involves a 1.7-kb ssDNA that was used to assemble a DNA performed research; C.L., S.R., X.W., Y.L., N.C.S., and H.Y. analyzed data; and C.L., S.R., X.W., octahedron (together with five short strands); this molecule was Y.L., N.C.S., and H.Y. wrote the paper. cloned in a in bacteria (9). A nicking endonuclease was The authors declare no conflict of interest. used to digest the amplified double-stranded plasmids to obtain This article is a PNAS Direct Submission. the single-stranded heavy chain. This is an inspiring accomplish- †C.L., S.R., and X.W. contributed equally to the work. ment, although the long strand did not form a complete nano- §To whom correspondence may be addressed. E-mail: [email protected] or structure itself without the aid of the short strands. We recently [email protected]. reported the replication of a four-arm DNA nanojunction and a This article contains supporting information online at www.pnas.org/cgi/content/full/ paranemic cross-over (PX) DNA molecule, using rolling-circle 0805416105/DCSupplemental. amplification (RCA)-based enzymatic methods (21–22). The © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805416105 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 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 Fig. 1. Schematic drawing showing the in vivo replication of a DNA nano- ladder; lane 1, 10-pmol original J1 junction; lane 2, replication product yield structure. A single-stranded DNA nanostructure (four-arm junction or parane- from 10-ml saturated culture. (B) A 20% nondenaturing PAGE analysis of mic cross-over DNA) is inserted into phagemid, transformed into XL1-Blue replicated J1. Lane M, 10-nt ssDNA ladder; lane 1, annealed original J1; lane 2, cells, and amplified in vivo in the presence of helper phages. High copy annealed replicated J1; lane 3, annealed random-sequence 79-mer ssDNA. (C) numbers of cloned nanostructures can be obtained readily by using standard Endo VII cleavage of the J1 cloverleaf. The two images, Left (L) and Right (R) molecular biology techniques. contain the results of the same experiment, but R has been run further than L to resolve cleavage positions nearer the 3Ј end of the strand. Lanes L1 and R3 are untreated controls. Lanes L2 and R1 contain A–G sequencing ladders. denaturing PAGE [supporting information (SI) Fig. S1]. Among The positions of possible cross-overs are indicated by ‘‘J’’ symbols. Lanes L3 and four randomly chosen colonies, three appeared to have the R2 contain the products of Endo VII cleavage experiments. The sites of bands correct insert. The error was most likely due to self-ligation of are indicated. (D) Quantitative scans of hydroxyl radical autofootprinting gels. the initially digested phagemid. Glycerol stock of cells containing Two sets of traces are indicated: DS, when the molecule is paired with its the correctly inserted vector was then prepared for long-term Watson–Crick complement, and J1, when it is folded into its junction structure. storage and for later amplification use. The in vivo replication are numbered from the 5Ј end, which is labeled. The positions of efficiency was evaluated by using a denaturing PAGE assay (Fig. expected junction-flanking nucleotides are indicated by arrows. In agreement 2A). It is clear that intact J1 strand is the only replication product 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 visible from the gel. (The top bands represent digested and E for numbering). In addition, a small amount of protection is seen at position undigested phagemid vectors, along with helper phage genomic 50 as well, possibly indicating a small amount of cross-over isomerization. (E) DNA.) Approximately 6 pmol of J1 was produced (estimated Schematic summarizing the results of autofootprinting and Endo VII cleavage from the band intensities in the gel image) from 10 ml of experiments. Autofootprinting protection positions are indicted by black saturated cell culture (OD600 ϭ 1.0). It is very important to point triangles and endo VII cleavage positions are indicated by magenta triangles. out that this amplification is fully scalable. The final yield of The main sites of protection or cleavage are in agreement with previous nanostructure is proportional to the volume of the culture studies of J1. medium used. For example, 150 pmol of J1 is expected to be produced from 250 ml of culture medium; this estimate is tion study on the same molecule, the position of cleavage is not consistent with our final yield, considering the material lost in the final PAGE purification step. The overall yield might be the strongest evidence for the proper cross-over structure, because improved further by optimizing restriction conditions. endo VII has a requirement for minimum-length arms, a require- Denaturing PAGE provides information only on the length of ment that the short horizontal arms may not satisfy (27). DNA strands. Therefore, the purified J1 strand was subjected to We have reported previously that enzymatically generated a number of other assays to verify the correct replication of the unusual DNA motifs can by characterized by hydroxyl radical junction structure. Fig. 2B shows the nondenaturing analysis of autofootprinting experiments; RCA-generated PX molecules the replicated J1 strand. Original sense J1 and replicated J1 were characterized in this fashion (22). Whereas the technique strand exhibited exactly the same mobility on a nondenaturing was originally developed by Churchill et al. (28) to analyze DNA gel, whereas a 79-mer ssDNA with a random sequence migrated branched junctions, and J1 in particular, it is natural to charac- much faster, implying the proper folding of the replicated J1 terize the cloned J1 cloverleaf structure by this method. In this strand. A further test of the structure of the J1 cloverleaf is technique, hydroxyl radicals are generated by Fenton chemistry Ϫ provided by T4 endonuclease VII (endo VII). The cleavage involving Fe(II)EDTA2 (29–30). This technique gives single- pattern is shown in Fig. 2C, and it is summarized by the magenta resolution of nucleotide susceptibility to attack and triangles in Fig. 2E. The cleavage occurs at the expected site, 2 produces a quantitative pattern of backbone cleavage at all or3nt3Ј to the junction on the cross-over segment (26). The possible sites. cleavage is somewhat stronger on the lower left segment than on The strategy of autofootprinting experiments is to compare the upper right one. Although consistent with a previous diges- the chemical attack pattern of each strand when it is part of an

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805416105 Lin et al. Downloaded by guest on September 26, 2021 CHEMISTRY

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 Ϸ40-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 BIOCHEMISTRY protection are shown as triangles in rough proportion to the extent of protection for the junction regions. Colored nucleotides indicate the sites of restriction.

unconventional species with the pattern obtained when the tion results, the data suggest that the replicated J1 cloverleaf has strand is hybridized with its normal Watson–Crick complement adopted the designed structure. (31). Experiments with four-stranded four-arm junctions indi- cate that the patterns of two noncontiguous strands are the same Replication of a PX DNA Molecule. PX DNA, which has a cross-over in both environments, whereas the other two strands, crossing at every possible position between its two parallel helical do- over at the branch point, exhibit protections at the site of the mains (33), is structurally more complicated than the J1 clover- junction. From those experiments, it was concluded that strands leaf. Thus, we challenged our system by replicating a PX DNA with the same patterns in both pairing environments probably molecule, which is folded from a 146-nt ssDNA. Following the have continuous double-helical conformations near the junction, same amplification protocol applied to J1, PX DNA was repli- whereas the other strands (showing protection near the branch cated in vivo with equally high efficiency (Fig. 3A). From Ϸ point) form cross-over structures (28). denaturing PAGE analysis, it can be estimated that a 50-pmol The results of the hydroxyl radical autofootprinting experi- PX strand was replicated from 120 ml of saturated cell culture. It is notable that DNA species shorter than the full-length PX ments on the J1 cloverleaf molecule are shown in Fig. 2D, and strand were present in the restriction digestion product. These they are shown schematically in Fig. 2E by black arrowheads. It byproducts are truncated PX molecules that were also observed is clear that the most salient points of protection are those in the in vitro RCA replication of the same PX motif. However, flanking the branch point in the strand segments on the lower left the byproducts represent a much lower percentage here (Ϸ20%) and on the upper right, with no protection at the equivalent than that in the in vitro RCA replication products (Ϸ50%). This positions on the upper left or lower right segments. It is known is not unexpected, because the cellular machinery provides a 2ϩ that in the presence of Mg , the four-arm branched junction series of enzymes and factors besides polymerase that facilitate assorts pairs of adjacent arms into two stacking domains (28). It the replication process. The in vivo replication efficiency is thus is possible for either pair of junction-flanking corners to form the much higher than that seen in vitro (22). cross-over strands, with the other two being virtually undistorted Three experiments were performed to confirm that the in double-helix-like strands. It is known that the cross-over strands vivo-replicated PX strand can still form the designed nanostruc- are determined by the junction-flanking nucleotides (32), so one ture. First, nondenaturing PAGE (Fig. 3B) demonstrated that would expect that replicated J1 cloverleaf should demonstrate the annealed in vivo-replicated PX molecule migrated at the the same pattern as J1. Indeed, that is the case: The nucleotides same rate as the original PX molecule. As a negative control, a that are protected are the same ones seen in the original 146-mer ssDNA with random sequence ran much slower than the four-stranded J1 molecule. Combined with the endo VII diges- PX molecules because of its relatively loose secondary structure.

Lin et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 26, 2021 Second, Ferguson analysis (Fig. 3C) was used to characterize the friction constant exhibited by the DNA molecules within the nondenaturing polyacrylamide gel; the friction constant is di- rectly related to the surface area and shape of DNA nanostruc- tures. The data points of the PX molecule replicated in vivo and the original PX molecule overlap each other. Furthermore, they both display a very similar slope to that of a previously reported PX motif composed of the same core structure but lacking dT4 loops or ssDNA extensions at the ends of its helical domains (PX without loops) (33). In contrast, the plot for a 125-bp dsDNA exhibits a significantly different slope. This observation strongly suggests the correct folding of the replicated PX structure. Finally, the strongest evidence comes from the hydroxyl radical autofootprinting experiment. The results of autofootprinting experiments are shown in Fig. 3D, and they are summarized in a schematic in Fig. 3E. The results are identical to the previous studies on this same PX nanostructure (22, 33), indicating cross-overs exactly where predicted by the design of the PX molecule. There is some minor protection in the two loops at the top of the diagram, possibly indicating occlusion of each other’s structures. Thus, all three assays confirm that we have replicated the PX nanostructure correctly.

Fig. 4. Psoralen cross-linking of the replicated junction (J1-O) structure in Searching for the Immobile Nanojunction in Vivo. Now that we have vivo and in vitro.(A) A 14% denaturing PAGE analysis of cross-linking products confirmed that the in vivo-replicated DNA strands can fold into from J1-O. In vivo and in vitro cross-linking products are compared side by side the designed nanostructures in vitro, it is interesting to ask and can be seen to be exactly the same. Black, red, and green lines were whether these nanostructures also retain their native conforma- labeled beside bands representing un-cross-linked, mono-adducted, and tion in the cellular environment. The mobile Holliday junction cross-linked J1-O, respectively (see B). A 10-base DNA ladder is loaded in the is a central intermediate in DNA recombination (34–36), but the far left lane as reference. (B) Ferguson analysis of the cross-linking products unnatural immobile DNA nanojunction has not been found in using denaturing PAGE. Colors of datasets correspond to the labels on the side vivo. Here, we intended to use psoralen cross-linking (37) to of the cross-linking product bands in A. The product represented by the upper probe the in vivo secondary structure of the single-stranded J1 band shows a significantly different friction constant from the other two DNA Ј species, implying its unique topology. (C) Schematic drawing of the mechanism in insert. It is known that 4,5 ,8-trimethylpsoralen (TMP) interca- which psoralen reacts with the J1-O molecule. As illustrated, the psoralen mole- lates between DNA base pairs and preferentially cross-links two cule first induces the balance shift between the two cross-over isomers, and then adjacent thymine bases (38) on opposite strands of a DNA cross-links (represented by a red curve) the two pyrimidines at the junction site in double helix (i.e., 5Ј-TA or AT, which is also considered as a the presence of UV, altering the topology of the J1-O molecule. ‘‘hot-spot’’ for TMP to cross-link) when excited by 365-nm UV irradiation. Cross-linking between neighboring thymine and cytosine (e.g., 5Ј-TG) is also possible but is much less likely (38). over, because J1-O lacks ‘‘5Ј-TA’’ or ‘‘5Ј-AT’’ sequences, double- This technique has been used widely to study the secondary stranded vectors in the cell are unlikely to be cross-linked within structure of DNA and RNA in vitro and in vivo. It is especially the J1-O DS insert domain (Fig. S4), strengthening the inter- useful for in vivo structural studies, because psor- pretation of the in vivo cross-linking result. alen, once cross-linked with thymine (or uracil) bases, ‘‘freezes’’ We cloned J1-O in vivo in the same way that we cloned J1 and the in vivo secondary structure of DNA (or RNA) by altering PX. The transformed cells were infected by helper phage, their topologies, which can be readily detected by subsequent in allowed to grow until saturation, and harvested by gentle cen- vitro assays, such as PAGE and electron microscopy. trifugation. The harvested cells were permeabilized by a solution To validate this method for our purpose, we first performed containing EDTA, followed by UV-induced TMP cross-linking in vitro cross-linking on the J1 cloverleaf and a series of its treatment. The phagemid vectors extracted from the UV-treated derivatives and characterized them using denaturing PAGE (Fig. cells were then restricted and run side by side on a 14% S2). The J1 derivatives were designed so that they all keep the denaturing polyacrylamide gel with the in vitro cross-linking same bases flanking the junction point, but each has only zero or products of J1-O (Fig. 4A). It is clear that the product generated one hot-spot on the long arms. On another derivative molecule, by in vivo cross-linking matches that produced by in vitro dT5 loops were substituted by dA5 loops to rule out their cross-linking. Note that on the far right lane, the smearing at the influence on the cross-linking products. It is clear that two starting position of electrophoresis represents digested or undi- common products were generated from all J1 derivatives after gested vectors. Another observation here is that the in vivo psoralen and UV treatment, regardless of their different arm, cross-linking products are relatively fewer than those seen in loop, and end extension sequences. Those two products are vitro. This is because most single-stranded vectors that contain therefore directly related to the pyrimidines flanking the junc- J1-O as cross-linking substrates are packed and released into the tion site. This is not surprising, given the fact that the junction culture medium, leaving the infected cells rich in replicative form structure has been shown to be a preferred substrate for inter- (double-stranded) phagemids, whose DS insert cannot be cross- calating drugs (39). Among various derivative molecules, J1-O, linked. Nevertheless, the same in vivo and in vitro cross-linking which has no regular hot-spot on any arm, was selected for an in products from J1-O can be observed here with no ambiguity. vivo cross-linking study, because we expected TMP to photoreact To understand the topology of the cross-linked products, preferentially with bases at its junction point and thus directly Ferguson analysis was performed by using denaturing PAGE indicate the formation of a junction structure. Indeed, when (Fig. 4B). The un-cross-linked J1-O and the product represented J1-O was denatured by 40% (vol/vol) formamide, it became by the lower band (marked by a red line) on Fig. 4A exhibited immune to TMP (Fig. S3), suggesting that the success of similar friction constants. In contrast, the friction constant of the cross-linking depends strongly on its native conformation. More- other product (marked by a green line on Fig. 4A) was signifi-

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805416105 Lin et al. Downloaded by guest on September 26, 2021 cantly different, suggesting a different topology. It is well known replication system is not significantly hindered by the secondary that besides covalently bridging two pyrimidines, psoralen can structure of the inserted DNA. Third, another also react with a single pyrimidine at the susceptible point, (M13mp18) is also capable of replicating J1 but with much lower forming a monoadduct (37–38). Therefore, the product with efficiency and reproducibility (Fig. S9), possibly owing to the similar topology to the bare J1-O is considered likely to be the conflicts between the native M13 structure (44) and the inserted mono-TMP adduct and the other product is considered to be the artificial nanostructure. Thus, the construction of the vector is cross-linked J1-O, which resembles a circle bearing two antennae crucial to achieve efficient replication. Finally, the psoralen under denaturing conditions (see molecular models in Fig. 4C). cross-linking data suggest not only that nanostructures can be A conundrum arises when one examines the details of the replicated by viruses and bacteria cells, but also that some cross-linking pattern: The pyrimidines disposed to be cross- artificial nanostructures might possibly survive in vivo. Although linked appear to be on opposite helices if the junction adopts the extreme care needs to be taken when interpreting the cross- usual cross-over isomer (stacking pairs) seen for J1 (28). How- linking data, the existence of immobile Holliday junctions in E. ever, Kallenbach and his colleagues (40) have shown that the coli cells has been strongly implied by our data. More concrete presence of intercalating drugs can reverse the favored cross- evidence may be obtained from in vivo autofootprinting (42–43). over isomer. We have ascertained that this occurs when J1-O is In conclusion, we expect this work to open more opportunities in the presence of psoralen (Fig. S5). The cross-over reversal is to scale up structural DNA nanotechnology and to provide shown in the schematic of Fig. 4C. Thus, the cross-linking is not helpful insight on the possible existence and impact of artificial aberrant, and the pattern is consistent with the data. Therefore, nanostructures in living systems. the same cross-linking pattern found in vivo as in vitro can be attributed to the proper of folding of J1-O in the context of a Materials and Methods vector within the cellular environment. Materials. Detailed information for materials used in this study, including DNA oligoonucleotides, restriction enzymes, ligase, phagemid vectors, competent Conclusions cell, helper phage, and chemicals, can be found in SI Text. We have demonstrated a system that utilizes viruses and bacte- rial cells to replicate complete artificial DNA nanostructures. All In Vivo Replication of DNA Nanostructures. Equal-molar sense and antisense steps involved in the replication process are based on well nanostructure (J1, J1-O, or PX; see SI Text for sequences) strands (90 nM) were ϫ established molecular cloning techniques (41), making the annealed from 94°C to room temperature over 45 min in 1 TAE-Mg buffer [to CHEMISTRY method easy to master and reproduce. However, applying those mM Tris-acetic acid (pH 8.0), 12.5 mM magnesium acetate, and 1 mM EDTA] to yield a DS insert. Two micrograms of Litmus 28i (500 ␮g/ml) were double techniques to DNA bearing complicated secondary structures is restriction digested by 20 units of PstI and 15 units of SacI in 50 ␮lof1ϫ NE not necessarily trivial. Instead, the work reported here is signif- Buffer 1 [10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.0)] at icant, both for applications and on a fundamental basis. Com- 37°C for 3 h. The reaction was stopped by the addition of denaturing buffer, pared with the in vitro enzymatic amplification method, in vivo and the digested vector was purified via agarose gel electrophoresis. Digested replication provides higher efficiency by taking advantage of the vector (100 ng) was ligated with 0.16 pmol of preannealed DS insert (Ϸ3-fold naturally existing cellular machinery. Such improvement be- excess) in 20 ␮lof1ϫ T4 ligase buffer [50 mM Tris⅐HCl, 10 mM MgCl2, 1 mM ATP, comes obvious when more complicated nanostructures (e.g., PX and 10 mM DTT (pH 7.5)] at 4°C overnight. Ligated vector (50 ng) was DNA) are encountered. It is also worth pointing out that the transformed into competent XL1-Blue cells, plated on LB-ampicillin (LB-Amp) BIOCHEMISTRY replication is fully scalable. Only a small amount of DNA agar plates, and incubated at 37°C overnight. Double-stranded phagemid was (subpicomole quantities) is needed to start the replication. Once extracted from cells in 5 ml of saturated cultures that were amplified from a single colony by using the plasmid miniprep kit. The correct insertion was successfully transformed by the correctly inserted vector, the verified by restriction digestion, followed by denaturing PAGE. One milliliter cells can be stored for later amplification. The amplification can of glycerol stock of XL1-Blue cells (OD600 ϭ 0.5) with correctly inserted phage- be scaled up readily by increasing the volume of the culture mid were then infected by 50 ␮lof1ϫ 1011 M13KO7 helper phage and medium. Moreover, by replicating two different nanostructures incubated overnight at 37°C in 250 ml of LB-Amp culture containing 25 ␮g/ml (Holliday junction and PX) and the same nanostructure formed kanamycin A. The cells were harvested for later cross-linking studies. The by different DNA sequences (J1 and J1-O), we expect this bacteriophage particles that contain single-stranded vector were precipitated strategy to be universally useful for replicating nanostructures from the supernatant by addition of 10 g of PEG and 7.5 g of NaCl, followed formed by a single DNA strand. by centrifugation at 10,000 ϫ g. Protein shells were removed from the We have used commercialized kits (competent cells, phage- single-stranded vector by phenol/chloroform extraction. DNA was recovered mid vectors, restriction enzymes, etc.) throughout the study. In by ethanol precipitation, redissolved in 0.9 ml of water, and restricted by 500 units of PstI and 360 units of SacI in the presence of 1 nmol of restriction the future, the conditions can be optimized (e.g., engineering the helpers in 1 ml of 1ϫ NE Buffer 1. The digested single-stranded vector was vector) to improve the replication yield further. The cloning of resolved on a 10% denaturing polyacrylamide gel, and the correctly replicated DNA nanostructures with knotted topologies has yet to be insert (J1, J1-O, or PX) was excised from the gel and eluted. Typically, 50–100 tested. Nevertheless, the method reported here could potentially pmol of ssDNA can be recovered. become a routine laboratory method to prepare moderate amounts of long ssDNA with designated sequences. An exciting Characterization of Replicated Nanostructure. Nondenaturing PAGE, Ferguson possibility is that this technique could find application in the analysis, hydroxyl radical autofootprinting, and endonuclease VII cleavage evolution and in vitro selection of DNA nanostructures. In this experiments were used to characterize the replicated DNA nanostructures. case, it can serve to amplify enriched DNA nanostructures after Detailed protocols can be found in SI Text. each selection cycle. The tolerance of natural systems for artificial objects is of great Psoralen Cross-Linking Study. Detailed information for protocols used in importance in synthetic biology. An interesting yet open ques- psoralen cross-linking experiments can be found in SI Text. tion is how the cellular replication machinery is affected by a ACKNOWLEDGMENTS. We thank Profs. Jiunn-Liang Chen and Thomas D. foreign DNA insert. Although it is beyond the scope of this work Tullius for helpful discussions. This work was supported by grants from the to address this question, several clues can be obtained here. First, National Science Foundation (NSF), the Office of Naval Research, the Air Force we found that J1 and an unstructured 79-mer ssDNA were Office of Scientific Research, the Army Research Office (ARO), and the Na- replicated equally well by using the same in vivo cloning proce- tional Institutes of Health and by funding from Arizona State University (to H.Y.), by grants from the NSF, ARO, and the U.S. Department of Energy dure (Fig. S6). Second, both J1 and PX retained sequence (Figs. (subcontract from the Research Foundation of State University of New York) S7 and S8) and structure integrity after replication. These (to N.C.S.), and by grants from the W. M. Keck Foundation and the National findings suggest that the efficiency and fidelity of the natural Center for Research Resources to New York University.

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