Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures

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Article Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures

1,2, 3,4, 1, Saminathan Ramakrishnan †, Sivaraman Subramaniam †, Charlotte Kielar ‡, Guido Grundmeier 1 , A. Francis Stewart 3,4 and Adrian Keller 1,*

1 Technical and Macromolecular Chemistry, Paderborn University, Warburger Str. 100, 33098 Paderborn, Germany; [email protected] (S.R.); [email protected] (C.K.); [email protected] (G.G.) 2 Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA 3 Biotechnology Center, Department of Genomics, Technische Universität Dresden, Tatzberg 47-51, 01307 Dresden, Germany; [email protected] (S.S.); [email protected] (A.F.S.) 4 Cluster of Excellence Physics of Life, Technische Universität Dresden, 01062 Dresden, Germany * Correspondence: [email protected] These authors contributed equally to this work. † Present address: Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf, ‡ Bautzner Landstraße 400, 01328 Dresden, Germany.

Academic Editor: Ramon Eritja Received: 29 September 2020; Accepted: 30 October 2020; Published: 3 November 2020

Abstract: Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA–protein nanostructures may find applications in the crystallization of DNA–protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

Keywords: DNA nanotechnology; Holliday junctions; atomic force microscopy; single-strand annealing proteins; Redβ

1. Introduction The Holliday junction (HJ) is an intermediate DNA structure involved in genetic recombination and DNA repair. The biological signiﬁcance of HJs includes facilitating the strong binding of regulatory proteins and target sites of recognition signals during replication [1]. The folding or emergence of double helical junctions and their stability strongly rely on three critical factors: base stacking [2], sequence composition [3], and ionic strength [4]. However, innate branch migration in HJs is a distinct phenomenon that makes the HJ structures highly unstable [5]. Active pair exchange between bases in homologous strands and in particular at the branch points migrates the

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sequences along the structure until the HJ eventually disappears [6]. For previous structural and functional analyses, the in vitro production of HJs was required. Thereby, it became possible to understand how junction-resolving enzymes such as resolvases bind and split the duplexes in the HJ structures, which is an essential event in recombination [7]. Furthermore, the synthesis of immobile HJs is a cornerstone of structural DNA nanotechnology [8]. Initially, Seeman designed immobile HJ structures by reevaluating the concept of branch point migration and its influence on junction instability with the aim to arrange them in three-dimensional crystal lattices. [9]. Seeman et al. also studied the orientation-specific cleavage function of resolvase enzymes in synthetic HJ structures [10] and other critical biophysical characteristics [11–15]. The immediate applications of such synthetic HJs also included the understanding of functional proteins [16] and epigenetic markers [17], as well as applications in biomolecular engineering [18]. Importantly, in DNA nanotechnology, the concept of immobile HJ synthesis linked to a peculiar sticky-end strategy was employed to assemble highly ordered crystal-like lattices in two and three dimensions. Unfortunately, this strategy usually requires complex and time-consuming thermal annealing protocols, which in extreme cases may extend over several days [19], while typically resulting in only moderate synthesis yields. Consequently, there is a need for fast and low-temperature strategies for DNA nanostructure synthesis with high assembly yields [20–26]. Many phages encode their own homologous recombination systems often as 50-30 exonucleases paired with single-strand annealing proteins (SSAPs). In the lambda phage, the red recombination system includes lambda exonuclease (Redα) paired with the SSAP Redβ [27]. Genome integrity is maintained by repair of double-strand breaks (DSB) through homology directed repair (HDR) [28]. One of the reaction pathways of HDR involves single-strand annealing (SSA) mediated by SSAPs. SSAPs have been divided into two categories, namely ATP-dependent, e.g., RecA and Rad51, and ATP-independent, which in particular includes Redβ [29]. Redβ is a 30 kDa protein and a central player in the recombinant DNA engineering method called recombineering [30–32]. In the absence of DNA, Redβ polymerizes to form a shallow right-handed helix. Upon annealing two DNA strands, Redβ forms a very stable left-handed helical nucleoprotein filament (Figure1a). These filaments have a curved form without rigid edges [29]. Optical tweezer single-molecule studies revealed that the Redβ nucleoprotein filament is nucleated by a remarkably stable DNA clamp to initiate HDR [33], while biochemical studies have established a complete model of HDR [34]. In this work, we utilized the annealing efficiency of Redβ to coat single-stranded (ss) DNA with Redβ monomers and then anneal them to complementary sequences to form DNA nanostructures based on rigid HJ motifs. The aim of this work was to (i) assess the potential of Redβ in the in vitro assembly of complex DNA structures, (ii) investigate whether the protein-coated DNA nanostructures provide a viable route for crystallizing the DNA–protein complexes, and (iii) produce extended lattices of rigid DNA–protein nanostructures at room temperature for applications in nanoelectronics [35,36], plasmonics [37,38], and molecular Molecules 2019, 24, x FOR PEER REVIEW 3 of 15 lithography [39,40].

Figure 1. Cont.

Figure 1. Experimental strategy for the Redβ-mediated assembly of Holliday junction (HJ) structures. (a) Characteristic features of Redβ. Models of the Redβ quaternary structure are shown for the protein alone (20 × 20 nm2), in complex with ssDNA, and in an annealed complex in the presence of two complementary ssDNA (20 × 20 nm2). The models were adapted from [29]. (b) Design of HJ structures. Different HJ structures composed of four ssDNA strands (S1–S4) were designed using UNIQUEMER 3D [41] and employed as substrates in this study. (c) Optimization of DNA/Redβ ratios by gel electrophoresis. The arrowhead pointing downwards indicates the free ssDNA, while the asterisks indicate the assembled HJ structures. (d) HJ assembly by Redβ. (i) Schematic view and atomic force microscopy (AFM) image of a single HJ structure (50 × 50 nm2). (ii) Schematic view, size exclusion chromatography (SEC) purification, and AFM image of a hierarchical HJ assembly (100 × 100 nm2).

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Figure 1. Experimental strategy for the Redβ-mediated assembly of Holliday junction (HJ) structures. Figure 1. Experimental strategy for the Redβ-mediated assembly of Holliday junction (HJ) structures. (a) Characteristic features of Redβ. Models of the Redβ quaternary structure are shown for the (a)protein Characteristic alone (20 features20 nm of2), Redβ. in complex Models with of ssDNA,the Redβ and quaternary in an annealed structure complex are show in thenpresence for the protein 2× aloneof two (20 complementary × 20 nm ), in ssDNAcomplex (20 with20 ssDNA, nm2). The and models in an were annealed adapted complex from [29 ].in ( bthe) Design presence of HJ of two × complementarystructures. Diff ssDNAerent HJ (20 structures × 20 nm composed2). The mode ofls four were ssDNA adapted strands from (S1–S4) [29]. ( wereb) Design designed of HJ using structures. DifferentUNIQUEMER HJ structures 3D [41] andcomposed employed of asfour substrates ssDNA in strands this study. (S1– (cS4)) Optimization were designed of DNA using/Red UNIQUEMERβ ratios 3Dby [41] gel and electrophoresis. employed asThe substrates arrowhead in pointing this study. downwards (c) Optimization indicates the of free DNA ssDNA,/Redβ while ratios the by gel electrophoresis.asterisks indicate The the arrowhead assembled HJ pointing structures. downwards (d) HJ assembly indicate by Reds theβ.( ifree) Schematic ssDNA, view while and atomicthe asterisks force microscopy (AFM) image of a single HJ structure (50 50 nm2). (ii) Schematic view, size exclusion indicate the assembled HJ structures. (d) HJ assembly× by Redβ. (i) Schematic view and atomic force chromatography (SEC) purification, and AFM image of a hierarchical HJ assembly (100 100 nm2). microscopy (AFM) image of a single HJ structure (50 × 50 nm2). (ii) Schematic view× , size exclusion chromatographyFew previous (SEC) attempts purification, to employ and proteins AFM image in the of construction a hierarchical of HJ DNA assembly nanostructures (100 × 100 nm have2). been reported. For instance, Praetorius et al. made efforts to synthesize a complete DNA origami

nanostructure through stitching complementary sequences by DNA-binding TAL proteins [42]. The TAL proteins not only annealed but also provided rigidity to the DNA origami structures. Similarly, Schiffels et al., demonstrated the stiffening of flexible dsDNA structures using the HDR protein RecA [43]. In contrast to these proteins, however, Redβ does not form linear protein–DNA filaments but rather Molecules 2020, 25, 5099 4 of 15 ring-like structures in the presence and absence of complementary ssDNA (see Figure1a). While this intrinsic curvature may impose some limitations to nanostructure assembly, it may also present a possibility to synthesize more complex shapes and structures. Upon ssDNA binding, Redβ breaks into different nucleoprotein filaments depending on the length of the ssDNA (see Figure1a) [ 29]. When a complementary ssDNA sequence is added, a highly stable dsDNA–protein complex is formed. Since it does not require ATP to anneal, this Redβ-assisted duplex formation occurs already at room temperature. During annealing, every Redβ protein monomer occupies approximately 11 bases in the ssDNA sequence, and efficient DNA annealing requires at least 15–20 base pairs [29]. Therefore, we have designed different four-arm HJ structures from 44 mer, 66 mer, and 88 mer oligonucleotides (Figure1b). After determining the optimal ssDNA/Redβ ratio for Redβ-assisted annealing (Figure1c), the HJ structures were assembled at room temperature and characterized by atomic force microscopy (AFM, Figure1d). Furthermore, we also have synthesized hierarchical assemblies of HJ structures (Figure1d) in order to assess the ability of Redβ to form complex and extended DNA nanostructures and pave the way for its inclusion in the DNA nanotechnology toolkit.

2. Results and Discussion

2.1. Assembly of Blunt-End HJ Structures from 44 mer, 66 mer, and 88 mer Oligonucleotides at 37 ◦C In a 44 mer HJ structure, every arm has a 22 mer dsDNA sequence. In order to estimate the optimal ssDNA/Redβ ratio during Redβ-assisted assembly, different ratios ranging from 1:1 to 1:4 were tested. For this, the individual strands S1–S4 were first coated with Redβ at 37 ◦C, subsequently mixed together, and incubated at 37 ◦C in order to anneal the HJ structure. Interestingly, even for the DNA mixture without Redβ, a significant shift of the corresponding band is observed in the gel image in Figure2. This indicates that at this temperature, there is already some hybridization of the four partially complementary ssDNAs occurring. In the presence of Redβ, however, a much stronger shift is observed. In particular, at an ssDNA/Redβ ratio of 1:1, the agarose gel analysis shown in Figure2 reveals a broad band corresponding to the ssDNA completely coated with Red β monomers with some fast-migrating bands corresponding to residual ssDNA sequences with incomplete Redβ coating. Increasing the Redβ concentration resulted in all ssDNA sequences being coated with no residual ssDNA detected. The gel bands have a smeared look due to the weak interaction of ssDNA with Redβ. From these initial tests, we have decided to use 1:1 and 1:2 ratios of ssDNA/Redβ to achieve complete coating of the oligonucleotides and promote HJ assembly. After incubating the individual ssDNA strands with Redβ, all four sequences were mixed to form the HJ structure at room temperature. However, the AFM image of the assembled HJ structures in Figure2 shows plenty of full rings and few C-shaped structures corresponding to free Redβ and incomplete HJ structures even at an ssDNA/Redβ ratio of 1:2. This mixture of structures further supports the weak binding of Redβ to short ssDNA sequences. Nevertheless, a few four-arm HJ structures can be identified in the AFM image in Figure2 and are in fair agreement with the design (see the schematic structure in Figure2). Since each arm can accommodate about two monomers, each of them exhibits a tiny protrusion. Typically, the ring-like structure formed by the Redβ monomers has a height of approximately 3 nm [29], which is in good agreement with height of the HJ arms as observed in the height profile in Figure2. Furthermore, patches of increased height are visible in the center of the four-arm HJ structure due to the crossover of the protein–DNA filaments. The 66 mer ssDNA sequence was tested with ssDNA/Redβ ratios of 1:1 and 1:2. The broader bands of the Redβ–ssDNA complex observed in Figure3 may indicate a slightly increased a ffinity of Redβ to the longer ssDNA sequence. Unlike the case of the 44 mer oligonucleotide, the gel analysis in Figure3 shows that the 66 mer sequence has been completely coated with Red β already at an ssDNA/protein ratio of 1:1. The AFM image in Figure3 shows the 66 mer HJ structures assembled at this ratio. No full ring structures corresponding to Redβ monomers assembled without DNA are observed in this sample, which is thus indicative of the complete saturation of the ssDNA sequences. Molecules 2019, 24, x FOR PEER REVIEW 5 of 15

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FigureThe 662. merThe HJsch structuresematic structures are composed show of the two 44 half-ring mer HJ shapes junction connected design atassembled the central by crossover thermal point. (i) and RedSinceβ-mediated each 33 bp(ii) arm annealing can accommodate. The sodium about borate three Red agaroseβ monomers, gel analys a totalis of shows six monomers the saturation are concentrationpresent on one ratio half of of Red theβ HJ to structure. ssDNA. ItThe is knowndownward from- thefacing literature arrowheads [29] that (black) a complete in lanes Redβ S1,ring S2, is S3, andcomposed S4 of the of gel 11–12 image monomers. indicate Inthe agreement free ssDNA, with while that, the the AFM asterisks image i inndicate Figure the3 shows HJ structures. that the six The monomers on each side of the crossover point of the HJ form a half-ring, resulting from the natural black downward-facing arrowhead in the first lane 1:0 indicates self-annealed HJ structures without curvature of the Redβ ﬁlament, which bends the dsDNA arms of the HJ structures upon addition of a Moleculesprotein 2019. ,Note 24, x FORthat PEERthe apparent REVIEW differences in HJ band intensity for the different ssDNA/Redβ ratios5 of 15 third monomer. are not due to any loss of DNA but rather due to a smearing of the bands (indicated by further asterisks), which results from the formation of a variety of different DNA–protein complexes. The AFM image depicts the resulting Redβ-mediated HJ assemblies. The height profiles were taken along the lines indicated in the zoom. The AFM image has a size of 300 × 300 nm² and a height scale of 4 nm.

The 66 mer ssDNA sequence was tested with ssDNA/Redβ ratios of 1:1 and 1:2. The broader bands of the Redβ–ssDNA complex observed in Figure 3 may indicate a slightly increased affinity of Redβ to the longer ssDNA sequence. Unlike the case of the 44 mer oligonucleotide, the gel analysis in Figure 3 shows that the 66 mer sequence has been completely coated with Redβ already at an ssDNA/protein ratio of 1:1. The AFM image in Figure 3 shows the 66 mer HJ structures assembled at this ratio. No full ring structures corresponding to Redβ monomers assembled without DNA are Figure 2. The schematic structures show the 44 mer HJ junction design assembled by thermal (i) observedFigure in 2.this The sample, schematic which structures is thus show indicative the 44 mer of theHJ junction complete design saturation assembled of theby thermal ssDNA (i )sequences and . and Redβ-mediated (ii) annealing. The sodium borate agarose gel analysis shows the saturation The 66 mer HJ structures are composed of two half-ring shapes connected at the central crossover Redβ-mediatedconcentration (ii ratio) annealing of Redβ .to The ssDNA. sodium The downward-facing borate agarose arrowheads gel analys (black)is shows in lanes S1,the S2, saturation S3, point.concentration Sinceand each S4 of33 ratio the bp gel of arm image Red canβ indicate to accommodatessDNA. the free The ssDNA, downward about while the three-facing asterisks Redβ arrowheads indicate monomers, the HJ (black) structures. a intotal lanes The of black S1, six S2, monomers S3, are presentand S4 downward-facingon of theone gel half image of arrowheadthe indicate HJ structure. in the the free first lanessDNA, It is 1:0 known indicates while thef self-annealedrom asterisks the literature HJ indicate structures [29]the without HJ that structures. protein.a complete The Redβ ring isblack composed downwardNote that of the 11-facing apparent–12 monomers arrowhead differences .in In in the HJag bandfirstreement lane intensity 1:0 with indicates for that, the di fft selfheerent -AFMannealed ssDNA image/Red HJβ structureratiosin Figure ares not without 3 show s that due to any loss of DNA but rather due to a smearing of the bands (indicated by further asterisks), the sixprotein monomers. Note thaton eachthe apparent side of differences the crossover in HJ pointband intensityof the HJ for form the different a half- ring,ssDNA resulting/Redβ ratios from the which results from the formation of a variety of different DNA–protein complexes. The AFM image are not due to any loss of DNA but rather due to a smearing of the bands (indicated by further natural curvdepictsature the of resultingthe Redβ Red βfilament,-mediated HJwhich assemblies. bends The the height dsDNA profiles werearms taken of the along HJ the structures lines upon asterisks), which results from the formation of a variety of different DNA–protein complexes. The addition of indicateda third monomer in the zoom.. The AFM image has a size of 300 300 nm2 and a height scale of 4 nm. AFM image depicts the resulting Redβ-mediated HJ assemblies.× The height profiles were taken along the lines indicated in the zoom. The AFM image has a size of 300 × 300 nm² and a height scale of 4 nm.

point. Since each 33 bp arm can accommodate about three Redβ monomers, a total of six monomers are presentFigure on one 3. Thehalf schematic of the HJ structures structure. show It the is 66known mer HJ from junction the design literature assembled [29] bythat thermal a complete (i) Redβ Figure 3.and The Red schβ-mediatedematic structures (ii) annealing. show The the sodium 66 mer borate HJ junction agarose geldesign analysis assembled shows the by saturation thermal (i) and ring is composed of 11–12 monomers. In agreement with that, the AFM image in Figure 3 shows that Redβ-mediatedconcentration (ii) ratioannealing of Redβ. toThe ssDNA. sodium The downward-facing borate agarose arrowheads gel analys (black)is shows in the gel the image saturation the six monomers on each side of the crossover point of the HJ form a half-ring, resulting from the concentrationindicate ratio the free of ssDNA, Redβ whileto ssDNA the white. The asterisks downward indicate-facing the HJ structures.arrowhead Thes AFM(black) image in depictsthe gel image natural curvtheature resulting of Redtheβ -mediatedRedβ filament, HJ assemblies. which The heightbends proﬁles the dsDNA were taken arms along theof linesthe indicatedHJ structures in upon the zoom. The AFM image has a size of 300 300 nm2 and a height scale of 4 nm. addition of a third monomer. ×

Figure 3. The schematic structures show the 66 mer HJ junction design assembled by thermal (i) and Redβ-mediated (ii) annealing. The sodium borate agarose gel analysis shows the saturation concentration ratio of Redβ to ssDNA. The downward-facing arrowheads (black) in the gel image

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Moleculesindicate2020, 25 ,the 5099 free ssDNA, while the white asterisks indicate the HJ structures. The AFM image depicts 6 of 15 the resulting Redβ-mediated HJ assemblies. The height profiles were taken along the lines indicated in the zoom. The AFM image has a size of 300 × 300 nm² and a height scale of 4 nm. The 88 mer oligonucleotides were also incubated with ssDNA/Redβ ratios of 1:1 and 1:2. As can be seenThe in the 88 mer gel analysisoligonucleotides in Figure were4, the also ssDNA incubated sequences with ssDNA are not/Redβ completely ratios of coated 1:1 and at 1:2. the As 1:1 can ratio, whichbe seen can in be the attributed gel analysis to the in larger Figure number 4, the ssDNA of available sequences Redβ are-binding not completely sites in the coa longerted at ssDNA the 1:1 ratio strands., Atwhich this ratio, can be a largeattributed fraction to the of freelarger ssDNA number is observedof available in Redβ the form-binding of fast-migrating sites in the longer bands. ssDNA At an ssDNAstrands/Red. Atβ thisratio ratio, of 1:2, a large however, fraction complete of free ssDNA saturation is observed of ssDNA in the with form Red ofβ fastmonomers-migrating is observedbands. withAt thean ssDNA corresponding/Redβ ratio band of 1:2 migrating, however, much complete slower. saturation Moreover, of ssDNA the completely with Redβ saturated monomers band is at theobserved 1:2 ratio with looks the very corresponding faint compared band to migrating the case ofmuch the slower 66 mer. oligonucleotideMoreover, the completely (see Figures saturated3 and4 ). Theseband observations at the 1:2 ratio indicate looks very that thefaint interaction compared atoﬃ thenity case of Red of theβ and 66 mer ssDNA oligonucleotide weakens once (see the Figure ssDNAs 3 and 4). These observations indicate that the interaction affinity of Redβ and ssDNA weakens once sequence exceeds a certain length. In the AFM image of the assembled 88 mer HJ structures in Figure4, the ssDNA sequence exceeds a certain length. In the AFM image of the assembled 88 mer HJ there are many full rings and plenty of half-rings observed at an ssDNA/Redβ ratio of 1:1. Due to their structures in Figure 4, there are many full rings and plenty of half-rings observed at an ssDNA/Redβ long sequence length, it may be possible for the 88 mer HJ structures to show partially and completely ratio of 1:1. Due to their long sequence length, it may be possible for the 88 mer HJ structures to show closed rings on both sides of the crossover point. There are about four monomers bound on each 44 bp partially and completely closed rings on both sides of the crossover point. There are about four arm,monomers corresponding bound on to each 1/3 of 44 the bp completearm, corresponding ring shape. to 1 When/3 of the the complete four monomers ring shape. in W eachhen ofthe the four four armsmonomers come together, in each theirof the intrinsic four arms curvature come together, yields a their HJ structure intrinsic composedcurvature ofyields two a almost HJ structure ring-like shapescomposed with aof prominent two almost bulge ring-like at the shape crossovers with a point. prominent bulge at the crossover point.

FigureFigure 4. 4.The The schematicschematic structures structures show show the the 88 88mer mer HJ HJjunction junction design design assembled assembled by thermal by thermal (i) and (i) andRed Redβ-mediatedβ-mediated (ii) (iiannealing) annealing.. The The sodium sodium borate borate agarose agarose gel gel analys analysisis shows shows the the saturation saturation concentrationconcentration ratio ratio of of RedRedββ to ssDNA. ssDNA. The The downward downward-facing-facing black black and andwhite white arrow arrowheadsheads in the ingel the gelimage image indicate indicate the the free free ssDNA ssDNA and andself-annealed self-annealed HJ structure HJ structuress without without protein, protein,respectively respectively,, while whilethe asterisk the asteriskss indicate indicate the protein the protein-annealed-annealed HJ structures HJ structures.. The AFM The image AFM depict images the depicts resulting theresulting Redβ- Redmediatedβ-mediated HJ assemblies. HJ assemblies. The height The height profiles profiles were taken were takenalong alongthe lines the indicated lines indicated in the zoom in the. The zoom. TheAFM AFM image image has has a size a size of 300 of 300 × 300 300nm² nmand2 aand height a height scale scaleof 4 nm. of4 nm. × LilleyLilley described described two two broad broad classes classes of of HJHJ conformations,conformations, namely namely open open and and stacked stacked [44] [44. ].The The open open conformation has a characteristic feature of four arms being at a 90° angle to each other with a lack conformation has a characteristic feature of four arms being at a 90◦ angle to each other with a lack of centralof central base pairing.base pairing. The stacked The stacked form hasform a characteristichas a characteristic feature feature of slow of branch slow branch migration migration as shown as by severalshown studies by several [5,6,45 studies] and is [5,6,45] characteristic and is forcharacteristic the HJ in the for absence the HJ of in proteins. the absence Red βof, however,proteins. adoptsRedβ, a however, adopts a characteristic right-handed helix in the absence of DNA and changes its helicity characteristic right-handed helix in the absence of DNA and changes its helicity to being left-handed to being left-handed upon ssDNA annealing (see Figure 1) [29], with the characteristic C shapes of upon ssDNA annealing (see Figure1)[ 29], with the characteristic C shapes of the DNA–protein the DNA–protein filaments indicating strong DNA bending. Based on our current understanding, filaments indicating strong DNA bending. Based on our current understanding, this Redβ-induced this Redβ-induced bending of the DNA strands will probably affect the conformation of the bending of the DNA strands will probably affect the conformation of the assembled HJ, which may assembled HJ, which may thus deviate from the known isomers described above. However, further thus deviate from the known isomers described above. However, further crystallographic or numerical studies will be required to elucidate the detailed DNA conformations of the different Redβ-mediated HJ structures. Molecules 2019, 24, x FOR PEER REVIEW 7 of 15

crystallographic or numerical studies will be required to elucidate the detailed DNA conformations Molecules 2020 25 of the different, , 5099Redβ-mediated HJ structures. 7 of 15

2.2.2.2. Hierarchical AssemblyAssembly of HJ StructuresStructures withwith StickySticky EndsEnds WeWe have have further further designed designed HJ structures HJ structures with sticky with ends sticky for hierarchical ends for hierarchical assembly. The assembly. experimental The strategyexperimental for hierarchical strategy for self-assembly hierarchical is outlinedself-assembly in Figure is outlined5. For the in purification Figure 5. F ofor the the single purification HJ structures of the andsingle hierarchical HJ structures assembly, and hierarchical the amount ofassembly, Redβ has the to beamount scaled of up Red in order has to to meet be scaled the detection up in order limit ofto themeet SEC the purification detection methodology. limit of the For SEC this, purificatio we haven chosen methodology. an ssDNA /ForRed β this,ratio we of 1:25 have based chosen on ouran experimentalssDNA/Red results ratio of when 1:25 88based mer on were our purified experimental by SEC results (see Supplementary when 88 merMaterials). were purified by SEC (see Supplementary Materials).

FigureFigure 5. 5.Experimental Experimental strategy strategy for speciesfor species enrichment enrichment of S1S4 of and S1S4 S5S8 and HJ structuresS5S8 HJ and structures puriﬁcation and ofpurification ﬁnal hierarchical of final assemblieshierarchical using assemblies SEC. using SEC.

TheThe schematic schematic structure structure in in Figure Figure6a shows 6a shows that two that arms two (S2S3 arms and (S2S3 S3S4) and of S3 theS4 S1S4) of HJ the structure S1S4 HJ arestructure ds 44 mer,are ds which 44 mer can, which accommodate can accommodate about four about Redβ fourmonomers. Redβ monomers. The other twoThe armsother (S4S1two arms and S1S2)(S4S1 areand each S1S2 composed) are each composed of two domains: of two adomains: ds 22 mer a ds close 22 tomer the close crossover to the pointcrossover and point an ss 22and mer an atss the22 mer distal at the end distal of the end arm, of the which arm, is which acting is asacting a sticky as a sticky end. A end similar. A similar design design is also is also employed employed for thefor the S5S8 S5S8 HJ structureHJ structure (Figure (Figure6b). 6b In). theIn the S1S4 S1S4 HJ HJ structure, structure the, the sticky sticky ends ends of of the the S4S1 S4S1 and and S1S2 S1S2 armsarms areare complementarycomplementary to those of the S6S7S6S7 and S S7S87S8 arms of the S5S8 HJ structure structure,, respectively respectively.. Interestingly,Interestingly, we we observed observed e ffiefficientcient HJ HJ structure structure assembly assembly by Redby Redββ at (23 at (22)3 ± C.2) Therefore,°C . Therefore, from from here ± ◦ on,here we on, performed we performed HJ assembly HJ assembly as well as hierarchical well as hierarchical assembly assembly using Red usingβ at 23 Redβ◦C. The at 23 individual °C. The Redindividualβ-assembled Redβ- S1S4assembled and S5S8 S1S4 HJ and structures S5S8 HJ werestructures subjected were to subjected SEC for enrichmentto SEC for enrichment of the individual of the species (see Figure5 and Materials and Methods). The enriched species from the two peaks obtained in Molecules 2019, 24, x FOR PEER REVIEW 8 of 15 individual species (see Figure 5 and Materials and Methods). The enriched species from the two peaks obtainedMolecules in SEC2020 and, 25, 5099 fractions from each peak’s summit were pooled and analyzed along8 of 15with load and concentrated samples by agarose gel electrophoresis (see Figure 6). For the S1S4 and S5S8 designs,SEC the and SEC fractions loads from show each the peak’s presence summit of were HJ pooled species and ( analyzedsee asterisks along with in the load corresponding and concentrated lanes in Figure 6samples). Pooled by and agarose concentrated gel electrophoresis Peak I (see fractions Figure6 ).show For the the S1S4 presence and S5S8 of designs, free DNA the SEC substrates loads (black arrowheadshows), the while presence the P ofeak HJ II species samples (see asterisksshow complete in the corresponding saturation lanes (asterisks in Figure). The6). Pooled AFM andimage of the S1S4 HJconcentrated structures Peakin Figure I fractions 6a show thes highly presence concentrated of free DNA substrates single species (black arrowheads), with sticky while ends the at two of Peak II samples show complete saturation (asterisks). The AFM image of the S1S4 HJ structures in the arms. Since the S2S3 and S3S4 arms have lengths of 44 nucleotides, many S1S4 HJ structures have Figure6a shows highly concentrated single species with sticky ends at two of the arms. Since the S2S3 an almostand closed S3S4 arms ring have at one lengths side of and 44 nucleotides, a half-ring many at the S1S4 other HJ structures. Nevertheless, have an almost a certain closed variability ring at of HJ structuresone is side observed and a half-ring and can at thebe attributed other. Nevertheless, to the presence a certain variabilityof different of HJ number structuress of is Redβ observed monomers. Upon bindingand can be with attributed ssDNA, to thethe presence Redβ monomers of different numbers adhere of and Red β growmonomers. filaments Upon of binding multiple with lengths. Therefore,ssDNA, an 88 the mer Redβ ssDNAmonomers does adhere not and necessarily grow filaments have of multiple to be occupied lengths. Therefore, by precisely an 88 mer eight Redβ ssDNA does not necessarily have to be occupied by precisely eight Redβ monomers. If there are only monomers. If there are only seven monomers on an 88 mer ssDNA instead of eight, the HJ structure seven monomers on an 88 mer ssDNA instead of eight, the HJ structure will exhibit a slightly different will exhibitlength a andslightly curvature. different For instance, length there and arecurvature. HJ structures For observed instanc withe, there one armare being HJ structures shorter than observed with onethe arm others being or even shorter missing than completely the others (highlighted or even bymissing green arrows completely in Figure (highlighted6), which demonstrates by green arrows in Figurethe 6), necessity which of demonstrates enriching the individual the necessity species. of Even enriching though the the individual individual species species. of S1S4 Even and S5S8 though the individualwere species enriched of by S1S4 SEC, thereand areS5S8 still were plenty enriched of incomplete by HJ SEC, structures there observed are still in plenty the AFM of images incomplete in HJ Figure6 that have only one, two, or three arms. structures observed in the AFM images in Figure 6 that have only one, two, or three arms.

Figure 6. The schematic structures show the (a) S1S4 and (b) S5S8 HJ junction designs assembled by Figure 6.thermal The sch andematic Redβ-mediated structures annealing. show the The (a) correspondingS1S4 and (b) sodiumS5S8 HJ borate junction agarose designs gel analyses assembled by thermal wereand Red conductedβ-mediated with the annealing input and. The the corresponding pooled fractions sodium after SEC, borate as explained agarose in gel Figure analyses5. were conductedThe with downward-facing the input and arrowheads the pooled in fractions the gel image after indicate SEC, as the explained free ssDNA, in Figure while the 5. asterisksThe downward- facing arrowindicatehead the HJs in structures. the gel The image AFM imagesindicate of Peak the I (seefree Figure ssDNA,5) were while recorded the after asterisk SEC puriﬁcation.s indicate the HJ The AFM images show completely formed HJ structures with the green arrows indicating HJ structures structures. The AFM images of Peak I (see Figure 5) were recorded after SEC purification. The AFM with one arm missing. The AFM images have a size of 300 300 nm2 and a height scale of 4 nm. images show completely formed HJ structures with the ×green arrows indicating HJ structures with one arm missing. The AFM images have a size of 300 × 300 nm² and a height scale of 4 nm.

Molecules 2020, 25, 5099 9 of 15

For hierarchical assembly, Peak I and Peak II from the S1S4 assembly were mixed with the corresponding Peak I and Peak II from the S5S8 assembly, respectively. Four discrete bands can be seen in the lane corresponding to the concentrated Peak I of S1S4 and S5S8 HJ structures after SEC (gel image in Figure7, white asterisks). Surprisingly, the Peak II combination did not show any Molecules 2019, 24, x FOR PEER REVIEW 9 of 15 hierarchical assemblies (see Figure7). Therefore, the Peak I sample was used for AFM imaging. Several different structures were observed by AFM (see Figure S2), a selection of which is shown in Figure7. Several different structures were observed by AFM (see Figure S2), a selection of which is shown in Both the S1S4 and S5S8 HJ structures have shapes consisting of a closed ring connected to a half-ring Figure 7. Both the S1S4 and S5S8 HJ structures have shapes consisting of a closed ring connected to a due to the differences in the dsDNA domains in the different arms. When one S1S4 and one S5S8 half-ring due to the differences in the dsDNA domains in the different arms. When one S1S4 and one anneal with each other, however, they form structure (ii), which resembles a chain of three closed S5S8 anneal with each other, however, they form structure (ii), which resembles a chain of three rings (see Figure7). There are plenty of such chain-like structures observed in the AFM images in closed rings (see Figure 7). There are plenty of such chain-like structures observed in the AFM images Figure S2. In some structures, however, individual arms are missing, or additional arms are present in Figure S2. In some structures, however, individual arms are missing, or additional arms are present (see Figure7, structures (iii) and (iv)). Interestingly, if one of the sticky arms of the S1S4–S5S8 dimer (see Figure 7, structures (iii) and (iv)). Interestingly, if one of the sticky arms of the S1S4-S5S8 dimer is free and anneals with a complementary ssDNA–Redβ complex, it will form a 2D square lattice is free and anneals with a complementary ssDNA–Redβ complex, it will form a 2D square lattice structure (i) as seen in the corresponding AFM image in Figure7. Formation of multiple-ring structures structure (i) as seen in the corresponding AFM image in Figure 7. Formation of multiple-ring is also possible due to a cross-linking of free sticky arms with hierarchical structures. In summary, structures is also possible due to a cross-linking of free sticky arms with hierarchical structures. In any free S1S4 and S5S8 structure can anneal with any complementary sticky end, as observed after summary, any free S1S4 and S5S8 structure can anneal with any complementary sticky end, as SEC purification in the AFM images in Figure7. Therefore, we next attempted to assemble individual observed after SEC purification in the AFM images in Figure 7. Therefore, we next attempted to 66 mer HJ structures into extended hierarchical networks. assemble individual 66 mer HJ structures into extended hierarchical networks.

Figure 7. TheThe sch schematicematic structures structures show show possible possible hierarchical assemblies composed of S1S4 and S5S8 β HJ junction junction structures structures assembled assembled by thermal by thermal and Redand Red-mediatedβ-mediated annealing. annealing The corresponding. The corresponding sodium sodiumborate agarose borate gelagarose analysis gel was analysis conducted was conducted with the input with and the theinput pooled and fractionsthe pooled after fractions SEC, as after explained SEC, asin Figureexplained5. The in asterisksFigure 5 indicate. The asterisk the hierarchicals indicate HJ the assemblies. hierarchical The HJ AFM assemblies images of. The Peak AFM I (see images Figure 5of) werePeak recorded I (see Figure after SEC 5) were purification recorded and after show SEC hierarchical purification HJ assemblies and show corresponding hierarchical to HJ the assemblies schematic structures (i–iv). The AFM images have a size of 125 125 nm2 and a height scale of 4 nm. corresponding to the schematic structures (i–iv). The× AFM images have a size of 125 × 125 nm² and a height scale of 4 nm.

2.3. Assembly of Hierarchical Networks Composed of 66 mer HJ Structures We aimed at a two-dimensional hierarchical structure assembly by designing a 66 mer HJ structure with self-complementary arms in its structure. In this HJ structure, each arm is composed of a 22 mer ds domain and a 22 mer sticky end. The sticky end of arm S12S9 is complementary to that

Molecules 2020, 25, 5099 10 of 15

2.3. Assembly of Hierarchical Networks Composed of 66 mer HJ Structures We aimed at a two-dimensional hierarchical structure assembly by designing a 66 mer HJ structure withMolecules self-complementary 2019, 24, x FOR PEER arms REVIEW in its structure. In this HJ structure, each arm is composed of a10 22 of 15 mer ds domain and a 22 mer sticky end. The sticky end of arm S12S9 is complementary to that of arm S10S11,of arm whereas S10S11, the whereas sticky the end sticky of arm end S9S10 of arm is complementaryS9S10 is complementary to that of to arm that S11S12 of arm (see S11S the12 schematic(see the structuresschematic in Figurestructure8).s Individualin Figure 8 sequences). Individual were sequences first treated were with first Redtreatedβ for with annealing Redβ for and annealing subjected to SECand subje (seected Materials to SEC and (see Methods) Materials followedand Methods) by agarose followed gel by electrophoresis agarose gel electrophoresis to analyze the to analyze two peak fractions.the two However, peak fractions. we found However, that the we concentrated found that Peakthe concentrated I and Peak II P fractionseak I and contained Peak II fractions structures thatcontained were too structures large to enterthat were the geltoo evenlarge afterto enter a long the rungel even (see after gel analysis a long run in Figure(see gel8, analysis arrowhead). in TheFigure rapid 8 Red, arrowheadβ-mediated). The assembly rapid Redβ of large-mediated complex assembly structures of large is also complex evident structure from thes is AFMalso evident image in Figurefrom8 .the The AFM four image sticky in arms Figure of the8. The HJ structuresfour sticky werearms annealedof the HJ withstructures self-complementary were annealed with arms self in- all thecomplementary directions, thus arms forming in all a the huge directio clump.ns, thus When forming analyzed a huge carefully, clump. the When clump analyzed has numerous carefully, highly the branchedclump microstructures has numerous (see highly AFM branched zoom in microstructuresFigure8). Since the (see dsDNA–Red AFM zoomβ complex in Figure can 8) exhibit. Since highly the dsDNA–Redβ complex can exhibit highly flexible structural conformations in all directions, the flexible structural conformations in all directions, the polymerization reaction extended in all directions polymerization reaction extended in all directions until the free sticky ends were depleted. The sizes until the free sticky ends were depleted. The sizes of the clumps observed in the AFM images were thus of the clumps observed in the AFM images were thus of the order of several microns. Such mesh-like of the order of several microns. Such mesh-like structures may be interesting for the crystallographic structures may be interesting for the crystallographic investigation of Redβ–dsDNA complexes and investigation of Redβ–dsDNA complexes and thereby provide a better biophysical understanding of thereby provide a better biophysical understanding of recombineering. Moreover, this extraordinary β recombineering.ability of Redβ Moreover, to anneal thiscomplex extraordinary dsDNA assemblies ability of Redat roomto annealtemperature complex may dsDNA open up assemblies possible at β roomapplications temperature of Redβ may openand other up possible SSAPs in applications DNA nanote ofc Redhnologyand. other SSAPs in DNA nanotechnology.

FigureFigure 8. 8.The The schematic schematic structures structures show show the the design design and and hierarchical hierarchical assembly assembly of of a a66 66 mermer HJHJ structure in whichin which each armeachhas arm complementary has complementary sticky sticky end sequencesend sequences for hierarchical for hierarchical assembly. assembly The. sodiumThe sodium borate agaroseborate gel agarose analysis gel shows analysis that shows the assembled that the structures assembled are structures too large are to enter too largethe gel to (indicated enter the by gel the black(indicated arrowhead). by the The black AFM arrowhead) image of the. The concentrated AFM image Peak of Ithe shows concentrated the efficient Peak Red I βshows-assisted the hierarchical efficient assemblyRedβ-assisted of the66 hierarchical mer HJ structures. assembly Aof zoomedthe 66 mer section HJ structure in the AFMs. A zoomed image depictssection in the the microstructures AFM image presentdepicts inside the themicrostructures hierarchical structure. present inside The AFM the hierarchical image has a structure. size of 5 The5 µ mAFM2 and image a height has scalea size of of 20 5 nm.× 5 µm2 and a height scale of 20 nm. ×

3. Materials and Methods

Molecules 2020, 25, 5099 11 of 15

3. Materials and Methods

3.1. Protein Expression, Affinity, and Preparative Gel Filtration Chromatography For in vitro studies, Redβ was purified using one-step Strep-tag II/Strep-Tactin affinity chromatography which allows mild protein purification under physiological conditions as described previously [29,34,46]. Quality and quantities of the protein from the two steps above were monitored by analytical SDS-PAGE analysis. Purified Strep-tag II-tagged protein was >95% pure at a concentration range of 0.5–10.0 mg/mL when quantified on a Nanodrop 1000 (Thermo Fisher Scientific GmbH, Bremen, Germany).

3.2. Establishment of a Holliday Junction Assembly Protocol Using Redβ Different immobile HJ structures with different lengths were designed using UNIQUEMER 3D software version 1.0 [41]. Oligonucleotides were purchased from Metabion international AG (Planegg/Steinkirchen, Germany) (HPLC-purified). To optimize HJ assembly, 10 pmol of each ssDNA was incubated with 10, 20, 40, or 80 pmol of Redβ at 37 ◦C for 20 min in four sterile 1.5 mL Eppendorf tubes. After this step, all reaction contents from respective ratios were mixed in one sterile 1.5 mL Eppendorf tube (final volume 20 µL) and incubated at 37 ◦C for 20 min. The reaction was stopped by adding 4 µL of 6 using 1 sodium borate buffer DNA loading dye and 3 µL of 50% glycerol. As controls, × each individual ssDNA was also loaded. The reaction mixtures were loaded and electrophoresed on 2% or 3% sodium borate agarose gels using 1 sodium borate buffer. After electrophoresis, gels were × stained with 1 sybrGold (Thermo Fisher Scientific GmbH, Bremen, Germany) in 1 sodium borate × × buffer for 15 min at 25 ◦C. Visualization of DNA was carried out using the Gel DocTM XR+ imaging system (Bio-Rad laboratories GmbH, Feldkirchen, Germany). All the sequences are listed in Table S1. For AFM imaging of 44 mer, 66 mer, and 88 mer HJ structures assembled at room temperature (see Figures1–3), 1:4 ratios (in µM) of individual ssDNA sequences and Redβ were mixed in separate Eppendorf tubes and incubated at 37 ◦C for 20 min. Then, the complete volume of all the ssDNA–protein complexes was added to a sterile Eppendorf tube, mixed well, and incubated at 37 ◦C for 20 min to facilitate HJ structure formation. The same protocol was used for the hierarchical structure assembly without SEC purification and AFM analysis at 5 and 24 h time intervals (see Figure S3). The AFM imaging of the HJ structures and hierarchical assembly are described in Section 3.5.

3.3. Redβ-Mediated Annealing and Purification of Hierarchical HJ Assemblies with Sticky Ends (S1S4 and S5S8) For S1S4 + Redβ, 12.5 µL of 66 mer ssDNA S1, S2 and 88 mer ssDNA S3, S4 (1 µM) was pipetted into four individual sterile 1.5 mL Eppendorf tubes. For S5S8 + Redβ, 12.5 µL of 88 mer ssDNA S5, S6 and 66 mer ssDNA S7, S8 (1 µM) was pipetted into four individual sterile 1.5 mL Eppendorf tubes. An amount of 370 µL of 87 µM Redβ (pre-incubated at 37 ◦C for 20 min) was pipetted into each of these 8 tubes and the final volumes were adjusted to 1250 µL by addition of 870 µL of sterile B1 buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1 mM MgCl2). The reaction contents were mixed well by gentle pipetting and incubated at 25 ◦C for 20 min. Afterwards, the contents of all four tubes, namely S1, S2, S3, and S4, were mixed in a 15 mL Falcon tube and incubated overnight at 25 ◦C. Similarly, the contents of the other four tubes, namely S5, S6, S7, and S8, were mixed in another 15 mL Falcon tube and incubated overnight at 25 ◦C. Following overnight incubation, the reaction mixture S1S4 + Redβ was loaded using a 5 mL sample loop onto the preparative superdex 200 column (16/600) (GE Lifesciences, Chicago, IL, USA) connected to an Äkta purifier (GE Healthcare, Chicago, IL, USA). The column was pre-equilibrated with B2 buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1 mM MgCl2). Preparative size exclusion chromatography (SEC) was carried out at 1 mL/min flowrate. The eluting fractions were collected and based on the profile of the peaks, and only the summit fractions from the peaks were analyzed and pooled. A similar protocol was followed for the reaction mixture S5S8 + Redβ as well. The eluting fractions were collected and pooled as described above. For the purpose of quality control, we performed purification of S1S4 + Redβ species as well Molecules 2020, 25, 5099 12 of 15 as S5S8+Redβ species separately as the first step. Once we confirmed the purification of the desired species, we went further and combined S1S4 + Redβ with the S5S8 + Redβ species. Peak I pools and Peak II pools of S1S4 + Redβ with S5S8 + Redβ runs were combined appropriately and purified further by another preparative SEC run. The resulting peak fractions were grouped as Peaks I and II. Purified complexes were analyzed by agarose gel electrophoresis in order to visualize DNA and on 8% native PAGE or 4–16% gradient BlueNative PAGE gels followed by colloidal coomassie staining to visualize the protein. Afterwards, the desired species were used for AFM imaging.

3.4. Redβ-Mediated Assembly of Two-Dimensional Hierarchical HJ Networks (S9S12) For the two-dimensional hierarchical network assembly, 4 sticky-end ssDNAs (66 nucleotides long), namely S9, S10, S11, and S12, which could assemble in a left–right and up–down manner (see Figure8) were used along with Red β. We set up an optimization protocol to check the Holliday junction network formation by Redβ similar to the S1S4 + Redβ and S5S8 + Redβ work above. SEC puriﬁcation (see Figure5) was used for enriching the S9S12 + Redβ species. Following SEC, peak fractions were pooled and analyzed on 2% sodium borate agarose gel to visualize the S9S12 DNA and S9S12 + Redβ species. We used 8% native PAGE gel followed by colloidal coomassie staining to visualize the protein present in the S9S12 + Redβ complex. Afterwards, the desired species from Peak I was used for AFM imaging.

3.5. AFM Imaging An amount of 5 µL of the Redβ-annealed HJ assemblies was deposited onto freshly cleaved mica surfaces. An amount of 100 µL of fresh B2 buﬀer was added and the samples were allowed to adsorb for 15 min. The samples were washed with 5 mL of HPLC-grade water and dried with a stream of ultrapure air. The AFM images were recorded in intermittent contact mode using an Agilent 5100 AFM (Agilent Technologies, Inc., Santa Clara, California, CA, USA) and HQ:NSC18/Al BS cantilevers (MikroMasch Europe, Wetzlar, Germany).

4. Conclusions In this study, we have successfully optimized the conditions for the assembly of blunt- or sticky-ended HJ structures using Redβ. We have shown that these assembled structures are stable and hence could be purified by SEC. Our experiments with increasing lengths of HJ arms using three different lengths of ssDNA substrates support previous findings concerning the monomer spacing on DNA [29]. Most importantly, however, we have successfully demonstrated the ability of Redβ to assist in the concerted annealing of HJ structures and their hierarchical assembly into complex networks. As we have employed the full-length Redβ in this study, we observed left-handed helical filaments with curved ends. Future studies employing purified DNA binding domains or shorter DNA binding peptides of Redβ could aim at having more rigid-ended complex HJ assemblies. This could open up new avenues in the fields of macromolecular patterning and DNA nanotechnology.

Supplementary Materials: The following are available online, Redβ-mediated annealing and purification of 88 mer HJ structures, gel analysis of the purified and enriched 88 mer HJ structures and corresponding AFM images, AFM images of hierarchical structures with SEC purification, AFM images of individual HJ structures and hierarchical structures without purification, oligonucleotide sequences. Author Contributions: Conceptualization, S.R., S.S. and A.K.; methodology, S.R., S.S. and A.K.; validation, S.R., S.S. and A.K.; formal analysis, S.R. and S.S.; investigation, S.R., S.S. and C.K.; resources, A.F.S., G.G. and A.K.; data curation, S.R., S.S. and C.K.; writing—original draft preparation, S.R. and S.S.; writing—review and editing, S.R., S.S., C.K., G.G., A.F.S. and A.K.; visualization, S.R. and S.S.; supervision, G.G., A.F.S. and A.K.; project administration, A.K.; funding acquisition, A.F.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by funding from the Deutsche Forschungsgemeinschaft grant STE 903/6-1 “Initiation of homologous re-combination by Red beta and other single strand annealing proteins” to A.F.S. S.S. Molecules 2020, 25, 5099 13 of 15 was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) STE903/6-1 and by Germany’s Excellence Strategy–EXC-2068–390729961–Cluster of Excellence Physics of Life of TU Dresden. Conflicts of Interest: The authors declare no conflict of interest.

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