METHODS 23, 141–148 (2001)doi:10.1006/meth.2000.1115, available online at http://www.idealibrary.com on

Probing Structure and Function with Alternative NucleicAcids Bearing 28,58-Linked, Zwitterionic, andIsocytosine?Isoguanine Components

Christopher Switzer1 and John C. Chaput

Department of Chemistry, University of California, Riverside, Californ

The incorporation of alternative functional components into nucleic acidscan provide insight into what molecular features are necessary for aninformational macromolecule to be successful. It can also provide a meansto improve particular physical characteristics of nucleic acids for diagnosticand therapeutic purposes, or probe mechanisms. By testing the fitness of

nucleic acid-like molecules derived by structural permutations of RNA, itmay also prove possible to trace a path from simple prebiotic precursorsto biotic molecules. This article describes the applications of 28,58-phospho-diester linked, zwitterionic, and base-permuted nucleic acid deriva-

acid mimic is whether its strands stably associate by

tives. q 2001 Academic Press

The availability of efficient methods for the solid-phase synthesis of DNA and RNA has provided a step-ping off point for constructing structurally modified al-ternatives to natural nucleic acids in the laboratory.This article focuses on the chemical synthesis and appli-cations of several kinds of alternatives to natural nu-cleic acids. One reason for investigating the propertiesof nonstandard nucleic acids is to identify general mo-lecular features required for a polymer to be a suitablegenetic material. Another is simply to learn more aboutthe genetic material nature has selected for us. Fea-tures found in natural DNA and RNA whose necessity

or purpose may be tested include: (i) the carbohydrate-backbone unit, (ii) the repeating negative charge, and(iii) the Watson–Crick hydrogen bonding. What followsis a brief survey of some of the structures that have beenexamined thus far and their properties. The methods

1 To whom correspondence should be addressed. Fax: 909-787-4713.E-mail: switzer@citrus.ucr.edu.

1046-2023/01 $35.00Copyright q 2001 by Academic PressAll rights of reproduction in any form reserved.

ia 92521

reported here focus on one aspect of each of the catego-ries noted, specifically 28,58-phosphodiester linked nu-cleic acids, zwitterionic DNA, and DNA incorporatingthe isomeric bases isoguanine and isocytosine.

Approaches to the alteration of nucleic acid structurecan be grouped into the broad categories of alternativesystems based on natural motifs and “synthetic” sys-tems. Clearly, a prime property of a genetic material isthe capacity for specific self-recognition. In DNA andRNA this occurs by means of Watson–Crick base pair-ing and stacking interactions in a double helix. It fol-lows that an initial screen often employed for a nucleic

way of specific pairing interactions. Once recognitioncan be established, other important properties to testinclude a capacity for nonenzymatic and enzymatic rep-lication, transcription, translation and recombination.

CARBOHYDRATE-BACKBONE ALTEREDNUCLEIC ACIDS

A variety of naturally occuring structures have beenused to systematically examine the implications of re-placing the 38,58-linked ribofuranosyl group in nucleicacids with other moieties. Pyranosyl-RNA (p-RNA) andhexose RNA are structurally related alternative nucleicacid analogs in which the furanose ring has been re-placed by a pyranose ring (1). These modifications re-

sult in biopolymer systems with linear rather than heli-cal strand arrangements in which complementaryWatson–Crick pairing is observed (1, 2). This enablesthe conclusion that a five-membered ring as found inribofuranose is not essential. All p-RNA stereoisomers

141

SWITZER AN142

yield stronger Watson–Crick base pairs than RNA (2).However, p-RNA is an autonomous genetic system inthe sense that no tendency to pair with natural RNAhas been observed. While homo-DNA shares with p-RNA an ability to form unusually stable Watson–Crickbase paired double strands, fully hydroxylated hexose-RNA variants have highly diminished Watson–Crickpairing abilities (1). The fact that p-RNA can also repli-cate in the absence of enzyme assistance makes it apossible evolutionary alternative to RNA (3). Pentosealternatives to ribofuranose include arabinose, xylose,and lyxose. Arabinonucleic acids have been shown toform stable double helices with natural DNA and RNA(4). The recognition properties of xylose DNA have alsobeen investigated, and it has been shown to give a du-plex of stability similar to that of natural DNA (5). Ithas been proposed that xylofuranosyl and lyxofuranosylnucleotides are unlikely candidates for incorporation

into primitive macromolecules owing to the ready cycli-

FIG. 1. Examples of alternative nucleic acids. Applications of the hi

D CHAPUT

a conformationally restricted nucleotide in which the28-oxygen and 48-carbon are linked by a methano bridge(7). LNAs constructed from b-D-ribo, b-D-xylo, and a-D-ribo subunits exhibit strong binding affinities towardnatural oligonucleotides via complementary base pair-ing (7). Bicyclo-DNA (8) and tricyclo-DNA (9) constituteanother class of conformationally restricted oligonucle-otides wherein the core bicyclic motif involves creatingan ethano bridge between the 38 and 58-carbons of deox-yribose. This results in systems that bind strongly toDNA and RNA, and that selectively bind using theHoogsteen face of purine bases (8, 9). The unusual pair-ing selectivities found in this series are a direct resultof alterations to backbone torsion angles relative tonatural nucleic acids. Hexitol nucleic acids (HNAs) areanother class of carbohydrate backbone-modified nu-cleic acid derivatives. HNA is constructed from 1,5-D-arabino-2,3-dideoxyanhydrohexitol, where the nucleo-

base prefers to adopt an equatorial position and the

. HNAs havelect for onephoroimida-ected oligo-

poly(HNA) prefers an A-type geometry (10)zation reaction they undergo when their 58-phosphatesare activated (6). the unusual property of being able to se

enantiomer of an activated nucleoside phosA large number of “synthetic” oligonucleotides withaltered carbohydrate backbones have also been exam- zolide during nonenzymatic template-dir

merization (11).ined. Locked nucleic acids (LNAs) are one example of

ghlighted derivatives are discussed in this article.

B

DNA tethers an amino group to the phosphorus atom

28,58-LINKED, ZWITTERIONIC, AND

CHARGE-ALTERED NUCLEIC ACIDS

Synthetic alternatives to nucleic acids systems bear-ing altered electrostatic properties have proved capableof natural DNA and RNA recognition. Methylphospho-nate DNA (12), peptide nucleic acids (PNAs) (13), sul-fone DNA (14), cationic DNA (15), and ribonucleic gua-nidine (16) are representatives of this type of alteration.In methylphosphonate DNA, one of the phosphodiesteroxygens is replaced by a methyl group and the resultingstructure is charge neutral. Methylphosphonate DNAbinds to DNA, but has diminished affinity for RNA (17).PNA (13) is composed of a neutral repeating polyamide-linked ethylaminoglycine unit incorporating a nucleo-base, and bears a stronger affinity for complementaryDNA than DNA has for itself, and also binds well toRNA (19). The case has been made for PNA as a prebi-

otic reagent (20), and, in addition, PNA has been shown

FIG. 2. Representative phosphoramidites for solid phase synthesisof oligonucleotides whose applications are the subject of this article.

ASE-PERMITTED NA DERIVATIVES 143

been shown to have a distorted backbone conformation(22) that mimics the transition state during restrictionenzyme cleavage of DNA (23). All of the aforementionedstructures eliminate charge entirely. A related ap-proach has been to replace the negatively charged phos-phodiester with a positively charged entity. Cationic

efficiently copies under the direction of a DNA poly-

to enter into nonenzymatic template-directed syntheticreactions with RNA (21). Sulfone DNA (14) is an isost-eric variant in which the phosphodiester groups havebeen replaced by dimethylenesulfone linkages, andforms highly stable self-structures. Sulfone DNA has

(15). The result is a structure that binds to DNA, butnot RNA. Ribonucleic guanidine replaces the entirephosphodiester unit with a guanidino group. Here thereis found very strong affinity between the guanidino-DNA and natural nucleic acids (16).

BASED-ALTERED NUCLEIC ACIDS

Nucleic acids bearing nonstandard base pairs havebeen used to explore the principles by which nucleicacids interact with themselves and enzymes. The ex-trapolation (24) of four nonstandard base pairs consis-tent with a Watson–Crick pairing geometry in additionto A?T and G?C pairs has led to a number of chemicaland biochemical studies of their properties. Apart fromthe iC?iG base pair to be discussed below, two otherbase pairs capable of Watson–Crick hydrogen bondingthat have been scrutinized include k–p (25) and V–J(26), and involve donor?acceptor?donor and donor?

donor?acceptor hydrogen-bonding patterns, respec-tively. It has been found from these studies that thethermodynamic discrimination of matched versus mis-matched pairs in a double helix attainable with bothsets of additional bases is quite good, and that they canserve as ready substrates for DNA and RNA polymer-ases, although their acceptance by polymerases hasbeen shown to be idiosyncratic (26). An expanded setof encodable nucleotides have been proposed to improvethe catalytic fitness of nucleic acids in comparson toproteins (24). Hydrogen bonding has been shown notto be an absolute requirement for base-pair replication.A hydrophobic analog of an A?T base pair, F?Z (27),

merase.Uses of alternative nucleic acids incorporating 2858-

linked DNA and RNA, zwitterionic DNA, and thenucleobases iC and iG are presented below, after discus-sion of their preparation in the laboratory.

DESCRIPTION OF METHODS

Methodology for the preparation of 28,58-linked DNAhas been reported for the five bases U (28), T (29, 30),

N

cleotides to concentrated ammonium hydroxide not

SWITZER A144

C (31–33), A (28–30), and G (31–33). Synthesis pro-ceeds via the phosphite–triester approach and solid-phase synthesis with base-protected phosphoramiditesin exact analogy with the synthesis of standard 38,58-linked DNA (Fig. 2). 28,58-Linked RNA may also beprepared using the same general methodology devel-oped for 38,58-linked RNA (34, 35). 38-Deoxynucleosidephosphoramidites are now available commercially al-though their cost may be prohibitive for some applica-tions. Phosphoramidites for the preparation of 28,58-linked RNA are available commercially as well.

Zwitterionic DNA may also be prepared via appro-priate phosphoramidites (Fig. 2) using the standardmethodology for DNA synthesis (36, 37). However, caremust be taken when deprotecting and purifying zwitter-ionic DNA owing to the fact that the amino functiontends to react with common by-products and reagentsused in the purification of oligonucleotides. For exam-ple, acrylonitrile released during phosphate deprotec-tion can form adducts with primary amines. We havefound that pure oligonucleotides are obtained throughreversed-phase HPLC purification. Zwitterionic DNAsare freely soluble in aqueous media.

Oligodeoxynucleotides incorporating methylisocytos-ine (MiC) and isoguanine (iG) may be prepared fromappropriately protected phosphoramidite derivatives.The MiC (38–40) and iG (38, 39, 41) phosphoramiditesshown in Fig. 2 can be used to prepare oligodeoxynucleo-tides. 5-Methylisocytosine is preferred over isocytosineowing to the enhanced stability of the 5-methyl deriva-tive to basic conditions encountered during oligonucleo-tide deprotection (38, 42). The 5-methyl group of MiCleads to increased sensitivity to acid depyrimidination,however (38, 39). 2-N,N-Dialkylformamidine-protectedMiC phosphoramidite derivatives are sufficiently sta-ble to the acidic detritylation conditions used during

oligonucleotide synthesis to enable efficient coupling

FIG. 3. Schematic illustration of a 28,58-linked oligocytidylate templazolide (64).

D CHAPUT

N,N-dibutylformamidine (40, 43)-protected MiC phos-phoramidites. Care must be taken in the handling ofMiC containing DNA or RNA owing to its tendencytoward hydrolytic deamination under alkaline condi-tions, such as ammonium hydroxide oligonucleotide de-protection. Thus, it is essential that exposure of oligonu-

exceed 16 h at 558C. MiC bearing DNA is sensitiveto acid, and exposure to acidic conditions results indepyrimidination. Even pH values in the realm of ,5–6can lead to degradation depending on time of exposureand temperature.

APPLICATIONS

28,58-Linked DNA and RNA: Recognition Properties

28,58-Linked DNA and RNA can self-associate to formdouble and triple helices that have less thermodynamicstability than the corresponding natural structures(28,30,31,34,35,45,46). In addition, 28,58-linked DNAand RNA associate with natural RNA to make hetero-duplexes and triplexes with stabilities similar to thoseof their natural counterparts, but, in contrast, interactonly weakly with natural DNA (35, 47–52). The solutionstructure of a purely 28,58-linked DNA duplex (45) indi-cates an A-type structure with alternating 28-endo, 38-endo sugar puckers, an outcome that was predictedon purely theoretical grounds (53). The fact that 28,58-linked oligonucleotides selectively bind to natural RNAmakes them candidates for diagnostic or antisense ap-plications. Several modified 28,58-linked nucleosides

have been prepared including formacetal and bicyclic analogs (54, 55). 28,58-Linked RNA/38,58-linked RNA(39, 40, 43). N,N-Dibutylformamidine-protected MiCduplexes are reported not to be RNase H substratesphosphoramidite is available commercially, as is N,N-(56, 57). Stems I, II, and III within a hammerheaddibutylformamidine diphenylcarbamate-protected iGribozyme have been replaced by 28,58-linkages withoutphosphoramidite. However, work has been conducted

with both the N,N-dimethylformamidine (39, 44)- and impairing catalytic activity (58).

te-directed oligomerization of guanosine 2-methyl-58-phosphoroimida-

28,58-LINKED, ZWITTERIONIC, AND B

28,58-Linked DNA and RNA: Capacity for InformationTransfer

38,58-Phosphodiester linkages are possibly preferredevolutionarily not only because they form more stablehelical structures, but also because of their relativeinertness within such structures. 28,58-Linked RNA isstereoelectronically disposed toward hydrolytic cleav-age in right-handed helices (59). Despite this, if ever28,58-linkages were formed on the primitive earth theywould likely have persisted for a finite period. In thisregard, it is interesting to note that 28,58-linked RNAappears to be an unavoidable outcome of nonenzymaticoligomerization reactions in the absence of oligonucleo-tide templates (61–63). Accordingly the properties of28,58-linked RNA as templates in non-enzymatic reac-tions have been explored (60, 63–65). In particular, ithas been shown that purely 28,58-linked oligocytidylatetemplates (and mixed linkage templates) are competentat directing the synthesis of complementary oligogua-nylates (Fig. 3) (60, 63, 64). Additionally, 28,58-linkedoligouridylate serves as a template for the ligation ofactivated 28,58-(pA)2 (65). A reverse-transcriptase hasalso been found to read through a 28,58-linkage defectin an RNA template (66).

Zwitterionic DNA

Fully zwitterionic DNA strands bind to complemen-tary DNA as well as natural DNA at modest ionicstrengths (50 mM NaCl) (36, 37). However, no effect on

duplex stability is observed on increasing the solution

FIG. 4. Illustrations of the two iG?T geometries observed by X-raycystrallographic analysis of a dodecamer duplex (75).

ASE-PERMITTED NA DERIVATIVES 145

neutralization as a mechanism for DNA bending byproteins via a “phantom protein” model (67, 68). Theo-retical predictions indicate an asymmetric distributionof charge about a helix would lead to a force that bendsDNA (69, 70). In practice, a laterally asymmetric distri-bution of tethered cations leads to DNA bending consis-tent with theoretical models (67,68).

iC and iG DNA and RNA: Recognition Properties

iC and iG form a base pair in DNA duplexes that isas stable or more stable than G?C pairs (38, 39, 43, 71).However, the discrimination of mispaired bases by iCand iG appears to be somewhat less than that achievedby natural bases in the case of iG?T mispairs (38). Thereis also the added complexity that iG adopts at leastthree tautomeric forms (38, 72–74). Of particular rele-vance is that high resolution crystal structural analysisof a dodecamer duplex incorporating two iG?T mispairsshows two distinct pairing geometries—wobble andWatson–Crick—that are presumed to incorporate N1–H and O2–H iG tautomers, respectively (Fig. 4) (75).The same study also gave evidence of multiple pairinggeometries in solution through NMR spectroscopy. Oli-gonucleotides bearing the four bases iG, iC, T(U), andA are capable of recognizing complementary naturalDNA or RNA by forming parallel-stranded helices (39,41). The structure of parallel DNA has been probed byboth NMR (76) and fluorescence (39) spectroscopies.Homo-DNA and p-RNA give stable iG?G Watson–Crickpairs via the iG N3–H tautomer (74, 77). iG also exhib-its monovalent cation mediated self-pairing similar toG. Parallel-stranded tetraplexes are induced in iG-DNAby potassium ions (78–80), and iG and G have beenshown to form mixed quartet structures under the sameconditions (78, 80). A new type of DNA helix, a parallel-stranded pentaplex, derives from iG rich DNA strandsand cesium ions (81). The capacity for iG to form pen-taplexes appears to originate from the 678 angle (nearlythe quintet optimum angle of 728 or 3608/5) made byits van der Waals surface along the hydrogen-bondingfaces (Fig. 5) (81). iG mononucleosides in organic sol-vent have been shown to form pentamer complexes un-der the influence of cesium ions by NMR spectros-copy (82).

ionic strength, consistent with approximately equiva-lent charge densities for the double- and single-stranded states. This observation is also consistent withnet charge neutrality for the zwitterionic strand. Zwit-terionic nucleotides have been used to evaluate charge

iC and iG DNA and RNA: Replication, Transcription,Translation, and Recombination

The iC?iG base pair is recognized by DNA (83–85)and RNA (42, 83, 84) polymerases and reverse tran-scriptases (84, 85). The major N1–H tautomeric formof iG is presumed to pair with iC in the polymerase

SWITZER AN146

active site via Watson–Crick base pairing. However, Talso incorporates iG under the direction of polymerases(42, 83, 84), presumably via the minor O2–H iG tauto-mer and a Watson–Crick geometry as well (75). Theability of an A triphosphate to outcompete an iG tri-phosphate for T in a template is polymerase dependent,where both the ability and inability of A to successfullycompete have been noted (42, 85). Translation of amRNA incorporating the codon iCAG by a tRNA bear-ing the anticodon CUiG proved successful, but wasplauged, albeit to a limited extent, by a competing rec-ognition of UAG by the iG bearing anticodon (86). AnRNA helix incorporating iC has been used to probeaminoacylation by a tRNA synthetase (87), and theeffect of replacing conserved adenosines with iG in thehammerhead ribozyme has been examined (88). A 70-nt DNA strand and a 40-bp DNA duplex containing 19iG?iC base-pair substitutions throughout are sub-strates for the Rec A protein of E. coli (44). Modeling

studies show that iG and iC are able to form triples consistent with the proposed R-form triplex intermedi-ate for homologous DNA recombination (Fig. 6) (44, 89). FIG. 6. Hypothetical R-form triples that may be formed by iG and iC

iC?iG and 28,58-linked nucleic acids are isomeric alter- during E. coli Rec A protein-mediated strand exchange as determinedfrom PM3 semiempirical and ab initio calculations (44).natives to natural nucleic acids that may well have

been accessible during early biopolymer evolution.

FIG. 5. Comparison of Cs+-iG-quintet and K+-G-quartet structures fthe iG van der Waals surface compares favorably with the expected 72found for G meets the anticipated quartet optimum of 908 (3608/4) (81

D CHAPUT

ound in parallel-stranded DNA complexes. The 678 angle created by8 optimum for quintets (3608/5), whereas the corresponding 908 angle).

28,58-LINKED, ZWITTERIONIC, AND B

Zwitterionic DNA is a formally net charge neutral nu-cleic acid-like polymer. All three of these systems retainsome level of the functionality seen in natural DNAand RNA, and can be used to test issues relating to

hydrogen-bonding, helix, and electrostatic interactions. In the case of iC- and iG-bearing nucleic acids it mayyet prove possible to engineer a fully functioning six-component genetic system in conjunction with the fourgenomic bases using the known strengths and weak- nesses of this isomeric base pair.

ACKNOWLEDGMENT

This work was supported by grants from the NIH and NASA.

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