Spontaneous Assembly of an Organic–Inorganic Nucleic Acid Z‐DNA Double‐Helix Structure

Abstract Herein, we report a hybrid polyoxometalate organic–inorganic compound, Na2[(HGMP)2Mo5O15]⋅7 H2O (1; where GMP=guanosine monophosphate), which spontaneously assembles into a structure with dimensions that are strikingly similar to those of the naturally occurring left‐handed Z‐form of DNA. The helical parameters in the crystal structure of the new compound, such as rise per turn and helical twist per dimer, are nearly identical to this DNA conformation, allowing a close comparison of the two structures. Solution circular dichroism studies show that compound 1 also forms extended secondary structures in solution. Gel electrophoresis studies demonstrate the formation of non‐covalent adducts with natural plasmids. Thus we show a route by which simple hybrid inorganic–organic monomers, such as compound 1, can spontaneously assemble into a double helix without the need for a covalently connected linear sequence of nucleic acid base pairs.

The central dogma of molecular biology is built upon the DNAd uplex. [1] Perfectly aligned so that its two linear information polymer strands can be non-covalently joined to their mutual complements,the unwinding of the two DNA strands and their subsequent replication provides the mechanism for the perpetuation of genetic information in biol-ogy. [2] However,a se legant and profoundly simple as this system is,the means by which the first DNAduplexes formed remain unclear. [3] One possibility could be that self-replicating minimal "inorganic" materials were able to spontaneously form as ystem capable of bridging this gap,l eading to the emergence of living systems. [4] Thus the very basic evolutionary information could have been encoded in naturally occurring periodic systems in the form of the directionality of crystalline layers,lattice defects,orchirality,for example.This "information" could then be transferred to molecules adsorbed on the surface of the material but this theory has lacked evidence or an experimental framework for development since it was proposed by Cairns-Smith in 1966. [4] We hypothesized that the formation of simple hybrid inorganicorganic units provides am odel of an intermediate class that electrostatically assemble into structures possessing characteristics similar to those of the information copying motifs found in biology. [5] To explore this idea, we set about synthesizing av ery simple prototype nucleobase-metal oxide hybrid:g uanosine monophosphate with a{Mo 5 O 15 }-based polyoxometalate.This compound, Na 2 [(HGMP) 2 Mo 5 O 15 ]·7 H 2 O( 1), was formed by the condensation reaction of guanosine monophosphate (GMP, 2)a nd sodium molybdate upon acidification. [6] To our surprise,w ef ound that compound 1 reproducibly forms an exceptionally intricate crystalline structure with similar dimensions to those of Z-DNA, but without hydrogen-bond base pairing (see Figure 1a nd Table 1; for ac omprehensive version, see the Supporting Information, Table S2). The compound crystallizes in the space group P6 5 22 containing as ixfold screw axis with al eft-handed twist and two orthogonal twofold rotational axes as symmetry elements. Within the structure,t he anions consisting of an inorganic POM core and two ligands protruding from opposing sides are situated along the resulting helix, and are interconnected by arow of Na + cations.
Theinorganic moiety consists of aring of five condensed molybdate(VI) anions capped by phosphate groups above and below the ring plane in the so-called Strandberg geometry. [7] Theg uanosine is connected to this moiety via the oxygen atom on the ribose ring at position 5' (Scheme S1). Theo rganic ligands are connected to the neighboring polyoxoanions through hydrogen bonds donated by the amino group,f orming ad imer (Figure 1b). Stacking interactions between the guanine rings and chelate coordination of Na + cations by the hydroxy groups of the ribose ring reinforce the linkage.T he sixfold symmetry axis runs through the center of the dimers,c reating al eft-handed double helix ( Figure 1c,d and Movie S1).
Thed ifferent major conformations of DNA, namely A-, B-, and Z-DNA( Figure S1), can be differentiated by their geometrical parameters (see Table 1a nd Table S2). [8] The symmetry and geometrical parameters of 1 are closely related to the Z-form of DNA. Thetendency of aDNA sequence to form the Z-conformation increases with abundance of guanosine. [9] Further,t he rise per turn and incline towards the central symmetry axis are very similar in these structures. Both compound 1 and Z-DNAform dimers that wind around the sixfold symmetry axis with left-handed helicity;however, these are different in their nature. [10] Thed imers of the guanosine Strandberg anion result from direct hydrogen bonding between the ligand and the POM (Figure 1b), whereas dimers of Z-DNAc onsist of two purine-pyrimidine base pairs,w here only the organic moieties are hydrogenbonded. Furthermore,in1,the guanosine ligands are stacked in an antiparallel orientation. Theg eometry of the ribose rings in 1 and Z-DNAd etermines the overall shape of the macromolecule and thus plays ad ecisive role in the refinement of the data obtained for different forms of DNAb yf iber X-ray diffraction. [8a, 11] TheC 2 '-endo conformation is assumed by the ribose rings of 1,a sc oordination of aNa + ion by two oxygen atoms is only possible in this conformation. It is important to note that the Z-form of DNAi ss tabilized by high salt concentrations as cations effectively shield the negative charges of opposing strands from one another (i.e., the phosphate-phosphate distance decreases from 12 in B-DNAt o8 in Z-DNA), [2] and as imilar effect can be observed in the structure of 1.
Thee xceptional aspect of structure 1 is the close resemblance to the iconic helical DNAstructure.Inprevious work involving helical structures,t hey were also compared with that of DNA: [12] this includes studies that show the ability of nucleotide-like organic molecules to arrange themselves into DNAd ouble-helix dimensions without the need for ab ackbone. [13] However,n one of the previously reported organic-inorganic structures display helical parameters that approach those of any of the major DNAc onformations. Compound 1,o nt he other hand, can be overlaid in ah elical sense as the number of dimers per turn, helical pitch, and rise per turn all map almost perfectly onto the Z-form of DNA, marking structure 1 as particularly unusual (see Table 1a nd Table S2). [14] Upon amore detailed structural analysis,wefound that of the two major components of the hybrid anion, only guanosine displays chiral centers and hence the ability of building homochiral structures.The "simple" compounds like hydrated Na 2 GMP (3)and H 2 GMP (4)crystallize in the chiral space group P2 1 2 1 2 1 ,b ut do not form helical structures like 1. [15] Theguanine rings are hydrogen-bonded to the ribose or water molecules instead. According to aCCDC search, there are 46 monomeric structures of GMP and derivatives thereof, 24 of which are transition-metal complexes of GMP.Some of them crystallize with polar or chiral symmetries,b ut no base pairing with stacking interactions has been observed. As such, compound 1 is the first non-oligomeric GMP derivative crystallizing with sixfold symmetry to form ad ouble helix similar to the DNAd uplex, and the helical parameters are almost identical to those of Z-DNA. In contrast, many other DNAm odels with or without nucleotides display different helicity,s ymmetry,a nd parameters,s uch as rise and number of dimers per turn, or have no DNAbackbone substitute. [12a,16] It is worth noting that the adenosine analogue of 1, Themost intriguing question posed by the above structure is whether compound 1 remains intact in aqueous solution, and whether the helical structure is preserved. To investigate Table 1: Structural featureso fideal B-and Z-DNA as well as 1.

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Communications 1142 www.angewandte.org this,w ee mployed several analytical techniques to probe the nature of compound 1 in solution (maintained acidic as hydrolysis is observed at neutral pH). As polyoxometalate anions can easily speciate in aqueous solution, we explored the solution stability of the hybrid anions in water by 31 P NMR spectroscopy ( Figure S5). The 31 Presonance appears as amultiplet in the NMR spectrum, confirming the persistence of the PÀOÀRb onds of 1 in water. Furthermore,I MS-MS analysis revealed as eries of oligomeric peaks,w hich may be assigned to a[ (GMP) 2 (Mo 5 O 15 ) 1 (K) W (Na) X (H) Y (H 2 O) Z ] m series (GMP = C 10 H 13 N 5 O 8 P), further corroborating the persistence of 1 in solution and its tendencytoself-associate (see Figure S7).
To examine the conformation of the extended structure of 1 in solution, we employed circular dichroism, at echnique that is widely used to investigate the structure of DNAa nd other biomolecule assemblies in solution (Figure 2). At room temperature,o nly aw eak signal is observed;h owever, upon cooling, ad istinctive pattern emerges with maxima around 210 and 260 nm, showing the formation of amore structured framework as the solution becomes less dynamic.U pon reheating,the original structure does not reform. Instead, the character of the 5 8 8Cs pectrum remains,where the intensities of the differential absorption bands are reduced but their pattern remains unchanged. This is indicative of aw elldefined secondary structure.H owever,t his pattern does not include features consistent with the structure of Z-DNA (negative minimum at 290 nm and positive maximum at 260 nm). This is perhaps unsurprising as the structural similarity of 1 and Z-DNAresults from different interactions, and neither confirms nor negates the persistence of the helical structural motif in solution. [17] As 1 is ad erivative of guanosine monophosphate (2), we compared its CD spectrum to that of 2 (also measured in an acidic medium), which showed that they are clearly different. Fore xample,a t 210 nm, am aximum is observed in the spectrum of 1 while am inimum is observed for 2,a nd am aximum is observed around 260 nm for 1,whereas no such feature is observed for 2. [18] Inferences from CD on the secondary structure of 1 proved elusive,p erhaps as the acidic pH value prevents many of the hydrogen-bonding interactions that define more well-known solution structures.
AFM data was obtained after drop-casting as olution of 1 on af reshly cleaved mica surface (see the Supporting Information for the exact conditions). Ther esulting fibers display an oticeable helical twist (Figure 3a). However,t he features seen on the surface by AFM depend strongly on the local concentration. In some areas,atight and systematic network of fibers with aheight of around 3.5 nm is observed ( Figure S11). Guanosine self-assembly has already been studied by AFM and is known to result in fiber-like structures on mica. [19] In most reports,t he height of these fibers is between 1.5-2.0 nm. Theh eight of 3.5 nm measured in this study is aconsequence of the inorganic core,and is consistent with the helicoidal diameter measured in the crystal structure  (3.20 nm). It is expected that the interactions between 1 and the surface are mediated by Na + cations as freshly cleaved mica surfaces are negatively charged.
Thea bility of 2 to act as al ow-molecular-weight gelator (LMWG) is well documented and due to its tendency to build macromolecular aggregates. [20] Amore pronounced version of this behavior might be expected for 1 as other Strandbergtype inorganic-organic hybrids have also shown gelator properties. [21] To test this,w ec hose the most pragmatic test to characterize the LMWG properties of 1-the tube inversion method (Figure 3b). Thec ritical gelation concentration (CGC) of 1 at room temperature is 0.009 m at pH 1.2 (the right-most vial in Figure 3b). This value corresponds to 1.28 wt %, which is striking given the high molecular weight of 1 compared to other LMWGs. [22] Other POM hybrids have also been shown to act as gelators, [21] but none formed hydrogels.Given the fact that pure 2 does not form hydrogels even at 0.3 m (50 times the CGC of 1 at pH 1.2) at neutral pH, one could assume that synergistic supramolecular interactions between the organic and inorganic moieties of 1 are responsible for gel formation.
Thei nteractions between polyoxoanions and biomolecules are not only of general interest, but could also be useful for the development of POM-based drugs and chemical biology tools. [23] To evaluate the ability of 1 to interact with afunctional biopolymer,the hybrid compound was incubated with double-stranded (ds) and single-stranded (ss) plasmid DNA( pGLO). No effect on the DNAm igration during electrophoresis was detected for ds-DNA, but anew band was observed for ss-DNA ( Figure 3c,l anes Ca nd D). No interaction with either ss or ds plasmid DNAw as detected after incubation of the inorganic Strandberg anion Na 6 Mo 5 P 2 O 23 (5;F igure 3c,l anes Ea nd F). [7] Thep resence of the new band after incubation of the ss-DNAwith 1 and its absence following the incubation with the inorganic core anion 5 suggests an interaction between the free guanosine ligands on 1 and single-stranded DNA.
In conclusion, the most intriguing feature of compound 1 is its structural similarity to Z-DNA. [17] We have shown that neither 1) ac ovalent backbone nor 2) hydrogen-bond-mediated base pairing is necessary for the build-up of homochiral helical structures,a nd that 3) guanosine seems to strongly influence the rotational direction of the helix and the overall structural geometry.T his is interesting as it has often been said that at some point in evolutionary history,a ni nformation-carrying biopolymer "must have arisen based on purely chemical means". [25] Herein, we have described anew class of compounds that spontaneously forms an analogue of anucleic acid double helix. This takes place without the need for apreprogrammed or biochemically written linear sequence of nucleic acid base pairs.T he similarities of the extended structure of 1 and Z-DNAa re unprecedented, with no other simple nucleobase monomer or hybrid showing such as tructure out of over 750 000 entries in the Cambridge Structural Database.F urthermore,t his work shows that biochemical machinery is not required to produce adouble helix with this degree of similarity to Z-DNA. Given the current gap between the inorganic and biological world, we would like to suggest that hybrids such as 1 might offer abridge between the inorganic and biological worlds as depicted in Figure 4. Forinstance,Benner has shown that the formation of ribose is promoted by molybdate under acidic conditions similar to those required to form 1. [26] We postulate that the key to understanding the "evolution of evolution" is exploring avenues that reduce the information required such that as pontaneously assembled double helix can become programmable,t hat is,b ecome an evolvable polymer sequence. This is currently under investigation in our laboratory along with the search for the simplest synthetic conditions that yield the monomers. . The increase in complexity from inorganicm aterials to DNA, RNA, and proteins (from left to right). We therefore suggest that the inorganic-organic hybrid 1 can be seen as an example system that can be placed between the spontaneously forming "inorganic" world and the evolvable "biological" world. [24]