The Resurgence of Acyclic Nucleic Acids

REVIEW

The Resurgence of Acyclic Nucleic Acids

by Su Zhanga), Christopher Switzer*b), and John C. Chaput*a)

a) Center for Evolutionary Functional Genomics, The Biodesign Institute, and Department of Chemistryand Biochemistry at Arizona State University, Tempe, Arizona 85287-5301, USA

(phone: (480)727-0392, e-mail: [email protected])b) Department of Chemistry, University of California, Riverside, California 92521, USA

(phone: (951)827-7266, e-mail: [email protected])

This review examines acyclic nucleoside analogs as therapeutic agents, potential progenitorcandidates to RNA, and novel building blocks for nucleic-acid nanotechnology. Together, these areas ofresearch provide new insights into the structural and functional properties of nucleic acids and suggestnew paradigms for nucleic acid self-assembly.

Introduction. – The structural simplicity of acyclic nucleic acids has captured theattention of chemists and biologists alike for nearly three decades. Early work in thefield of nucleoside mimetics led to the discovery that certain acyclic nucleic acidspossess significant therapeutic activity [1– 7]. Acyclic nucleosides have also attractedthe attention of researchers interested in studying the origin and early evolution of life.Research in this area examined the potential of acyclic nucleic acids as templates andsubstrates for nonenzymatic oligomerization experiments [8– 14]. This work providedsupport for the idea that acyclic nucleotides might have served as progenitors to RNA[15], and initiated a line of research that is helping to unravel the age-old mystery ofhow primitive biopolymer systems evolved into a modern genetic system. Morerecently, acyclic nucleic acids have been studied as substrates for polymerase-mediatedprimer extension reactions in hopes of identifying a set of conditions that will lead tothe selection and amplification of completely unnatural nucleic acid aptamers andenzymes [16 –18]. If successful, such studies will facilitate a direct comparison of thefunctional properties of RNA with simpler genetic materials like FNA (FlexibleNucleic Acid) and GNA (Glycerol Nucleic Acid, also known as Glycol Nucleic Acid;Fig. 1) [19 – 21]. Because some acyclic nucleic acids adopt very stable structures [22 –24], these molecules are also being examined as possible building blocks for unnaturalnucleic-acid nanotechnology [25 – 27]. In the fullest sense, acyclic nucleic-acid systemsare leading to an expanded understanding of the structural basis of genetic polymers.This review highlights recent findings in the area of acyclic nucleic-acid chemistry andfocuses entirely on nucleic-acids composed of sugar-phosphate backbones.

Acyclic Nucleoside Derivatives as Small-Molecule Therapeutics. – Early researchin the field of nucleoside mimetics led to the unexpected discovery that certain acyclic

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� 2010 Verlag Helvetica Chimica Acta AG, Z�rich

nucleic-acid analogs exhibit remarkable antiviral properties when compared with theircyclic-sugar counterparts. The first synthesis and antiviral examination of this type ofcompound produced a molecule called acyclovir (ACV) [1], which to this day remains afirst-line agent in the treatment of many herpes infections. Further development of thisclass of antiviral compounds produced a second generation of molecules that showimproved bioavailability and efficacy. Among these newer acyclic nucleosides areganciclovir, penciclovir, adefovir, and cidofovir (Fig. 2) [2– 7].

In-depth studies into the mechanism of acyclovir�s activity in cells revealed thatACV is transformed by herpes-specific thymidine kinase (TK) into acyclovir mono-phosphate (ACV-MP), which is then phosphorylated by cellular guanylate kinase tothe nucleoside diphosphate (ACV-DP). Several cellular kinases promote the trans-formation of ACV-DP to the triphosphate form (ACV-TP), which is the active drug.ACV-TP was found to be a potent inhibitor of the herpes virus due to its ability tofunction as a chain terminator during DNA synthesis. This drug is highly selective, andendogenous TK enzymes found in normal healthy cells exhibit only limited activitywith the ACV nucleoside [28].

Acyclic Oligonucleotide Analogs and Nucleic-Acid Evolution. – The notion thatthere was an ancestral period in the Earth�s history in which RNA served both asgenetic information carrier and catalyst is commonly referred to as the �RNA world�hypothesis [29 – 33]. Naturally occurring catalytic RNA molecules, such as self-splicingGroup I introns, RNase P, and, most critically, the ribosome could be the evolutionaryremnants of an earlier time when life�s biology was based entirely on RNA [34 – 38].

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)246

Fig. 1. Chemical structures of FNA, GNA, TNA, and RNA

Whether RNA was the first genetic material or simply an intermediate in the evolutionto the modern DNA/RNA genetic system is unclear. Several lines of evidence suggestthat RNA is an unlikely candidate as the first genetic material, and many now favor thepossibility that life emerged from a simpler genetic system during a period of life�shistory referred to as the �pre-RNA world� [15]. Early experiments into the synthesisand chemical stability of ribose demonstrate that ribose is difficult to synthesize underconditions that are considered relevant to the prebiotic world [39], and, once made,ribose shows only limited stability [40]. This drawback is somewhat mitigated by therecent finding that borate ions favor the synthesis of ribose over other aldose sugars andstabilize sugars relative to decomposition [41]. Despite this recent advance, competi-tion between d- and l-stereoisomers of activated ribose nucleotides leads to severeinhibition of nonenzymatic template-directed oligomerization. This phenomenon,referred to as enantiomeric cross-inhibition, would limit the amount of RNA productobtained from a racemic mixture of activated mononucleotides [42].

Considering such obstacles, many nucleotides have been examined as progenitorcandidates to RNA [19 –21] [43 – 47]. Systematic analysis of the chemical etiology ofRNA led to the finding that several nucleotide analogs are capable of adopting stableduplex structures by Watson –Crick base pairing [46] [47]. The acyclic sugar nucleo-sides, GNA and FNA represent two very interesting candidates, as these molecules arestructurally much simpler than natural DNA or RNA [19] [20]. Indeed, both acyclicnucleosides can be obtained under primitive reaction conditions using prebioticallyplausible molecules [15]. The incorporation of prochiral acyclonucleosides intooligonucleotides causes the central C-atom of the monomer to become stereogenic,with each monomer adopting either a d-like or l-like orientation (Fig. 3) in the chain.However, the sugar backbone might, in the case of FNA or related acyclonucleotides,


Fig. 2. Acyclic nucleoside analogs with antiviral activity

be sufficiently flexible to allow racemic mixtures of d- and l-like monomers to adoptsimilar conformations such that replication could take place without being affected bythe absolute configuration of the stereogenic center [15]. As a class of promising RNAprogenitors, acyclic nucleic acid systems have been extensively investigated, andexperimental evidence has reinforced the hypothesis that structurally simplifiedanalogs might have played a transitional role, which eventually led to the emergence ofcatalytic RNA molecules.

Nonenzymatic Synthesis of Acyclic Oligonucleotides. Nonenzymatic, template-directed oligomerization would have been important in the prebiotic evolution offunctional RNA-like molecules, as it would have enabled the replication of geneticinformation from parent to daughter strands. These molecules would have then beenexposed to the pressures of natural selection, which would have allowed certainmolecules with favorable traits to evolve over time. The hypothetical evolution of theseearly genetic systems would have proceeded in three distinct stages of polymerization(Fig. 4) [48]. In the first stage, monomers would have combined to form a templatestrand. Once formed, the template would have been copied in the second stage togenerate a complementary strand. In the third and final stage, the complementarystrand is itself copied, leading to the synthesis of a new strand that is identical insequence to the original template. While the second stage in this process, in isolation, istermed copying, the second and third stages, in combination, lead to formal replication.

Fig. 3. Enantiomers of FNA and GNA


The earliest examples of template-copying reactions with acyclic nucleotidesinvolved the oligomerization of activated FNA diphosphate analogs on conventionalpolynucleotide templates [8]. In these cases, successful oligomerization resulted in theformation of a product strand composed of pyrophosphate linkages (Fig. 5) rather thanthe more common phosphodiester linkages found in natural DNA and RNA. ActivatedFNA-G monomers were shown to rapidly oligomerize on poly-C templates. Controlreactions performed in the absence of the template yielded only small amounts of theelongated product. Template-directed oligomerization efficiency of the FNA-Gdiphosphate analog was found to be lower than that of the dG diphosphate analog.Nevertheless, the observed results suggest that atactic products derived from prochiralFNA-G diphosphate monomers are successfully extended during template-directedsynthesis. The latter finding is important, because it demonstrates that this systemavoids the problem of enantiomeric cross-inhibition seen with RNA monomers.

In contrast to the above studies, Chaput and Switzer examined a series of atactictemplates with FNA-C bearing a natural phosphodiester backbone to determine ifthese molecules might direct the synthesis of activated GMP monomers [14]. Analysisof the template-copying reactions indicated that atactic FNA-C oligonucleotidessupport template-directed synthesis, albeit with reduced efficiency when compared todC templates. A key finding was that both single and multiple FNA-C residues in atemplate are capable of directing the synthesis of the complementary strand. RNasedigestion showed that regioselective control of 3’,5’-phosphodiester bond-formation inthe product strand was gradually lost as more FNA-C residues were introduced into thetemplate. The main conclusion was that atactic FNA templates with naturalphosphodiester linkages support template-directed synthesis, and although thetemplate efficiency was less than with natural DNA, it may be presumed to be muchhigher than what would be obtained from a template bearing a mixture of all 24 (16)possible ribonucleoside isomers.

Watson –Crick Base-Pairing Properties of Acyclic Nucleotide Analogs. Shortly afterJoyce et al. first proposed that acyclic nucleotide analogs might have existed as potentialRNA predecessors [15], Schneider and Benner prepared a series of oligonucleotidesthat contained FNA-Tresidues at one or more positions [19]. Analysis of the stability of

Fig. 4. Replication of an early genetic system


these molecules revealed that a single FNA-Tresidue lowered the melting temperatureby 9 – 158 when compared to the all-natural DNA duplex. The destabilization is similarto that caused by a GT mismatch. Although poly(dA) failed to hybridize with isotacticstrands of FNA-T, Merle et al. reported that poly(T) associates with atactic FNA-A12,resulting in a modest Tm value of 248 [45]. In the latter case, circular dichroism (CD)traces also exhibited characteristic differences between natural and hybrid DNAduplexes. These observations indicate that, while FNA oligonucleotides can associatewith complementary DNA, such association is sequence-dependent. Accordingly, thesefindings do not exclude the possibility of FNA as a pre-RNA molecule. Indeed, it maybe na�ve to suppose that the first genetic material was comprised of completely uniformbackbones [15].

Despite the inability of FNA to form stable duplex structures with DNA, Heubergerand Switzer recently demonstrated that both (S)- and (R)-fNTP antipodes aresubstrates for several DNA polymerases [18]. Several polymerases accepted racemic,(R)- and (S)-fNTPs as substrates (Fig. 6) in primer extension reactions, leading tomultiple incorporations of flexible nucleotides. Although the efficiency of nucleotideincorporation was affected by such factors as the incubation time and temperature,polymerase type, and specific nucleotide bases, the overall results demonstrated that,on average, antipodal fNTPs are not distinguished. Mismatched fNTP – templatecombinations led to only very limited primer extension. These results show that


Fig. 5. Bis-activated acyclic monomers. a) Comparison of the chemical structure of bis-activated FNA-Grelative to bis-activated riboG. b) Oligonucleotide templates with pyrophoshate and phosphodiester

linkages.

functional molecular-recognition properties, including Watson– Crick base pairing, areretained by the structurally simple FNA system even though it has additional degrees offreedom in its backbone relative to DNA. The above findings with FNA haveimplications for the pre-RNA world and molecular evolution. These polymerasestudies highlight the observation that acyclic structures, in general, and FNA, inparticular, might be a means to preserving the informational properties of a polymer,while avoiding enantiomeric cross-inhibition during replication.

Overall, the above studies on FNA lend support to the feasibility of �genetictakeover�, the circumstance where one informational polymer (or, in the general case,genetic system) is superseded by another, on the basis of fitness [49]. While the degreeof duplex stability required to sustain a primitive self-replicating system, as well as thestructural features necessary to maintain such stability, remain unclear [19], GNAprovides an intriguing counterpoint to FNA, as detailed in the section to follow.

Glycerol Nucleic Acid (GNA). – Systematic evaluation of RNA etiology led to thediscovery of threose nucleic acid (TNA; Fig. 1) as a simple nucleic-acid system with a

Fig. 6. DNA polymerase-mediated FNA synthesis. a) d- and l-flexible nucleoside triphosphates. b)Lanes 2–9: primer extension on four different DNA heptanucleotide templates of the four bases with(S)- and (R)-fNTPs as indicated. Template composition: lanes 2 and 6: G7; lanes 3 and 7: A7; lanes 4 and8: T7; lanes 5 and 9: C7. In addition to polymerase, all reactions included 1U Tth pyrophosphatase. Lanes1 and 10 contain radiolabeled primer only. c) MALDI-TOF-MS of polymerization products formedunder conditions shown in inset i, with (S)-fATP. Calculated mass for [(Mþ3)þHþ ]¼8519, [(Mþ4)þ

Hþ ]¼8821. Figure taken from [18].


repeating five-atom backbone unit [46] [47]. Inspired by this work, Meggers and co-workers discovered another alternative nucleic acid system called GNA (Fig. 1), whosestructure derives directly from TNA [20]. GNA has an acyclic backbone composed of aC3 sugar connected by phosphodiester linkages. The repeating backbone unit containsone stereogenic center, which results in two enantiomers, namely (S)- and (R)-GNA.Both (S)- and (R)-GNA form antiparallel duplex structures with complementary GNAstrands of the same stereoconfiguration, and GNA duplexes are generally more stablethan natural DNA and RNA. For this reason, GNA represents the most economicalsolution to a stable nucleic acid structure.

Watson –Crick Base-Pairing Properties of GNA. Since the original description byMeggers and co-workers [20], the thermal and thermodynamic properties of GNA havebeen studied in detail using temperature-dependent UV absorption and CD spectro-scopy. In general, GNA duplexes were found to be thermally superior to natural DNAand RNA duplexes of the same sequence (Fig. 7) [21– 24]. However, GNA duplexesare not always thermodynamically more stable than natural duplexes, and GNA oftenexhibits higher melting temperatures with lower thermodynamic stability [24]. GNA ishighly sensitive to nucleotide mismatches, with one study reporting a decrease in themelting transition of 6– 188 depending on the sequence. This effect becomes even morepronounced, when a second mismatch is incorporated into the GNA duplex. Cross-pairing between GNA and RNA homopolymers is observed, but results in adramatically weaker helix than pure GNA or pure RNA. No cross-pairing is observedbetween homopolymers of GNA and DNA, suggesting that the GNA helix is


Fig. 7. A standard temperature-dependent UV melting curve for GNA, TNA, RNA, and DNA of theduplex A4T3AT3AT2A2/T4A3TA3TA2T2. In the case of RNA, the sequences contained uridine in place of

thymidine. Figure adapted from [24].

incompatible with the standard B-form DNA helix. Cross-pairing between (R)- and(S)-GNA is highly destabilizing, and only complementary A15 and T15 strands havebeen shown to associate, albeit with very weak thermal stability [22].

Determining the structures and conditions that may have given rise to a primitiveRNA world is fundamental to understanding the origins and early evolution of life. Oneapproach to this problem is to compare the capacity for independent genetic systems totransfer genetic information in the form of Watson – Crick base pairing, betweenthemselves and RNA [46]. Since nucleic acid base pairing is a chemical propertyconsidered fundamental to biology, genetic systems that are capable of adopting stablehelical structures with themselves and RNA are considered potentially naturalprogenitor candidates of RNA. While many nucleic acid systems have been comparedto RNA [46], very few pre-RNA candidates have been compared to one another. Inone example, Chaput and co-workers examined the ability of TNA to undergo base-pairing with opposite complementary strands of GNA [24]. TNA and GNA areinteresting molecules for this type of comparison, since polymers of both systems arecapable of forming stable Watson – Crick duplex structures with themselves and RNA,thereby providing a plausible mechanism for the transfer of genetic informationbetween successive genetic systems. Moreover, because the chemical structure of GNAderives directly from TNA, it is interesting to examine whether GNA and TNA mightrepresent common intermediates in the same evolutionary pathway to RNA.Unfortunately, detailed analysis of the molecular-recognition properties of GNA andTNA revealed that TNA and GNA are unable to undergo base-pairing by intersystemcross-pairing [24]. This observation suggests that GNA and TNA might represent twoindependent paths to a hypothetical RNA world.

GNA Structural Analysis. The X-ray crystal structure of a self-complementary (S)-GNA double helix (Fig. 8) was recently solved to a resolution limit of 1.3 � [50]. Theincorporation of two GNA hydroxypyridone (H) nucleotides into the sequence (3’-CGHATHCG-2’) facilitated the introduction of two heavy atoms (CuII) that were usedto phase the crystallographic data. The three-dimensional structure revealed that (S)-GNA adopts a right-handed helix that is significantly different from the standard A-and B-form helix observed for natural RNA and DNA, respectively. The GNA helixhas a pitch of 60 � with 16 base pairs per helical turn. This results in a helix with a largehollow core and one large minor groove. The major groove is replaced by a convexsurface.

A characteristic feature of the GNA duplex is the large backbone-base inclinationof the duplex from 42 to 508, a situation that is similar to unnatural hexose nucleic acids[51] [52], but very different from the 08 found in standard B-form DNA. This results ina large average slide (3.4 �) between adjacent base pairs, and extensive interstrandbase stacking. In contrast, intrastrand base stacking dominates base – base interactionsin A- and B-form helical structures. This special interaction suggests that base-stackingcontributions are different along each of the two GNA strands – a prediction that waslater tested using single-nucleotide overhangs [53]. In this case, 2’-nucleotide over-hangs dramatically enhance the thermal and thermodynamic properties of the GNAhelix, while 3’-nucleotide overhangs have no effect on duplex stability. Theseobservations indicate that base stacking is thermodynamically more favorable forGNA duplex formation than for DNA duplex formation. Another unusual character-


istic of GNA duplex structure is the involvement of the 1’-methylene group inhydrophobic interactions with the p-system of neighboring nucleobases on the samestrand. These unique backbone interactions are not found in natural duplexes, and mayaccount for the high stability of GNA duplexes.

Polymerase-Mediated Primer Extension of GNA. Many nucleic acid systems withstructures that are prebiotically more accessible than RNA have been examined assubstrates for polymerase-mediated template-copying reactions [54– 58]. One goal ofthese experiments is to identify a set of conditions that will lead to the evolution ofunnatural nucleic acid aptamers and enzymes. Szostak and co-workers recentlyexamined the ability of GNA triphosphates (gNTPs) to function as substrates fornatural DNA polymerases [16]. Using a standard primer-template assay, many DNApolymerases were screened for the ability to extend a DNA primer with GNA. Whilemost DNA polymerases readily incorporated one GNA residue, further primerextension beyond a single nucleotide was not observed. The difficulty in synthesizinglonger GNA strands may be due to an incompatibility between gNTPs and natural


Fig. 8. X-Ray crystal structure of the GNA octamer duplex (PDB code 2JJA). Coloring by atom type withCu ions shown in orange. The figure was obtained using Pymol.

polymerases. The situation was very different when the enzymatic synthesis of DNAwas examined on GNA templates [17]. In this case, a variety of DNA polymerasesshowed GNA-dependent DNA-polymerase activity, with Bst DNA polymerasecapable of synthesizing the full-length DNA product on a GNA template. The authorsnoted that, in addition to the presence of manganese ions in the reaction buffer, thesubstitution of adenosine for diaminopurine in the template and DNA monomersresulted in higher reaction efficiency. This example demonstrates that a stable duplexstructure between the product and template strands is not required for enzyme-mediated polymerization, consistent with the FNA results noted above.


Fig. 9. Characterization of two GNA 4-helix junctions. a) The design used to assemble the GNA 4 HJ(left). Nondenaturing gel-electrophoresis mobility-shift assays showing the monomer, dimer, trimer, andtetramer forms of the 4 HJ (right). b) CD Analysis of the 4 HJs derived from (R)- and (S)-GNA. Figure

taken from [25].

GNA Applications. DNA Nanotechnology relies on sequence minimization rules toself-assemble DNA tiles into nanostructures with well-defined shapes and lattices [59–63]. While a significant effort has been made to expand the programmability of naturalB-form DNA, very little attention has been given to the use of unnatural nucleic acidsas building blocks for nanotechnology. By expanding DNA nanotechnology to includealternative polymers, it should be possible to create nanostructures with chemical andphysical properties not found in natural DNA. Toward this goal, Chaput and co-workers recently described the synthesis of a nanostructure composed entirely of GNA[25]. The GNA nanostructure, a 4-helix junction (4 HJ) motif (Fig. 9) mimics an earlierstructure composed of DNA [64]. Because the GNA backbone contains only onestereogenic center per repeating unit, it was possible to synthesize two mirror-imagenanostructures using (S)- and (R)-GNA. A major finding was that the GNA 4 HJ wassignificantly more stable than the earlier 4 HJ composed entirely of DNA (Tm 738 forGNA vs. 378 for DNA) [64]. This feature coupled with the ability to construct left- andright-handed nanostructures provides new opportunities for building highly stablenanostructures with unique topologies that are not readily available to DNA.

Conclusions. – In summary, acyclic nucleic acids have been studied in a variety offormats for reasons that are both applied and fundamental. These studies have led tothe discovery of nucleic acid analogs with unusual chemical and physical properties asdetailed here. While many early studies on the stability and function of acyclic nucleicacids concluded that they have limited fitness, the recent finding that molecules such asGNA (and FNA) are functional is leading to a resurgence in acyclic nucleic-acidchemistry. While the full implications of these molecules are not yet known, recentsuccesses suggest that acyclic nucleic acids could find their way into many newtechnologies and possibly help solve old conundrums.

We gratefully acknowledge members of the Chaput laboratory for helpful comments and discussions,and Mr. Chad Simmons for assistance with the figures. This work was supported by grants from the NewFrontier�s Program at the Biodesign Institute to J. C. C. and the NASA Exobiology Program to C. S.

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Received September 8, 2009


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The Resurgence of Acyclic Nucleic Acids