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Review

Recognition of the unique structure of DNA:RNA hybrids

Nicholas N. Shaw, Dev P. Arya*

Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, SC 29634, USA

Received 7 February 2008; accepted 18 April 2008

Available online 27 April 2008

Abstract

Targeting nucleic acids using small molecules routinely uses the end products in the conversion pathway of ‘‘DNA to RNA, RNA to protein’’.However, the intermediate processes in this path have not always been targeted. The DNAeRNA interaction, specifically DNA:RNA hybridformation, provides a unique target for controlling the transfer of genetic information through binding by small molecules. Not only doDNA:RNA hybrids differ in conformation from widely targeted DNA and RNA, the low occurrence within biological systems further validatestheir therapeutic potential. Surprisingly, a survey of the literature reveals only a handful of ligands that bind DNA:RNA hybrids; in comparison,the number of ligands designed to target DNA is in the thousands. DNA:RNA hybrids, from their scientific inception to current applications inligand targeting, are discussed.� 2008 Published by Elsevier Masson SAS.

Keywords: DNA:RNA hybrids; Ligand targeting; Aminoglycoside; Polyamide; Ethidium bromide

1. History

Six years after the report on the double-helical structure ofDNA by Watson and Crick [1], the DNA:RNA hybrid wasproposed to address the interaction between DNA and RNA[2]; a wonderful retrospective account can be found in thefollowing reference [3]. The first synthetic DNA:RNA hybridstructure was formed in 1960 through the reaction of oligo-deoxythymidylic acid with polyriboadenylic acid [4]. Amere year later, the first DNA:RNA hybrid helix was formedthrough the novel annealing of a RNA strand, with a comple-mentary DNA strand [5,6]. X-ray fiber diffraction studies firstappeared in 1967 [7], and confirmed CD spectroscopy obser-vations that the conformation of the DNA:RNA hybrid wasdifferent from B-form DNA. The crystal structure ofa DNA:RNA hybrid was solved in 1982 [8], while solutionbased conformational analysis, first used to study a DNA:RNAhexamer [9] was followed shortly by polymeric DNA:RNAhybrids in 1985 [10].

In the initial report where the presence of DNA:RNAhybrids was suggested [2], the mechanism of their formation,

* Corresponding author. Tel.: þ1 864 6561106.

E-mail address: [email protected] (D.P. Arya).

0300-9084/$ - see front matter � 2008 Published by Elsevier Masson SAS.

doi:10.1016/j.biochi.2008.04.011

as a product of hybridization between a single-stranded DNAtemplate strand and a newly formed RNA strand, was alsohypothesized. Experimental data supported this hypothesis in1960 [11]. This was 10 years before Temin [12] and Baltimore[13], independently, yet simultaneously, reported the use ofreverse transcriptase in the synthesis of a new DNA strand,via RNA template strand. Originally proposed by Rich [2],this became known as reverse transcription, the secondexample of DNA:RNA hybrid formation. In 1975, the firstexample of DNA:RNA hybrid formation in the replication ofDNA was discovered [14]. Preceded by their biological signif-icance, DNA:RNA hybrids as therapeutic targets logicallyfollowed.

2. Importance

2.1. Transcription [15,16]

The DNA mediated synthesis of RNA is a finite processwhich initiates at promoter sites within the DNA template,proceeds in the 50 to 30 direction and terminates at terminatorsites within the DNA template. RNA polymerase binds to theinitiation site, unwinds a helical turn of duplex DNA and be-gins to incorporate ribonulecotides sequentially to the 30 end

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1027N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039

of the growing RNA strand. Ribonuclotide selection involvesthe interaction of the incoming base with the template DNAstrand and the ability of the base to fit the active site of the en-zyme [17]. Termination is less well understood; the termina-tion process involves (i) cessation of RNA chain growth, (ii)the release of the RNA strand from the DNA template and(iii) the release of RNA polymerase from the DNA [18]. Ithas been suggested that termination concludes with the releaseof the RNA strand from an unstable DNA:RNA hybrid [19].Furthermore, the lability of some hybrid sequences may aidthe dissociation of the RNA strand from the DNA template[20,21].

2.2. Reverse transcription

The RNA mediated synthesis of DNA was originally pro-posed [22] and substantiated [23] as an integral part of the ret-roviral life cycle. Expanded to eukaryotic cells [25], as muchas 10% of the eukaryotic genome is a direct product of reversetranscription [24]. Mechanistically similar to the transcriptionprocess, reverse transcription relies on the dual activity of re-verse transcriptase (RT) [24,26]. DNA:RNA hybrid formationcan be found following RT mediated DNA synthesis and priorto RT mediated RNase H cleavage of the template RNAstrand. Since reverse transcription relies on the dual role ofRT, the intermediate DNA:RNA hybrid form provides an en-ticing target for targeting by small molecules, prior to reattach-ment and subsequent cleavage by RT.

2.3. DNA replication [27]

The discussion of DNA replication may seem unnecessarywhen addressing DNA:RNA hybrid duplexes, howeverDNA:RNA hybrids play an important role in DNA replication.DNA replication begins with the unwinding of duplex DNAby DNA helicase followed by priming of the DNA strandsby proteins and enzymes [18] and proceeds in: (1) a heavy(H)-strand origin (OH), 50 to 30, leading strand synthesis and(2) a light (L)-strand (OL), 30 to 50, which begins replicationupon exposure to the single-stranded region of DNA [28], lag-ging strand synthesis. Lagging strand synthesis utilizes Oka-zaki fragments [29]. This process is repeated, formingDNA:RNA hybrids every 100e200 nucleotides on the DNAlagging strand. The DNA polymerase deletes the RNA primerswhich are then replaced by DNA using DNA ligase.

2.4. Mitochondrial DNA

Human mitochondrial DNA (mtDNA) is present in the mi-tochondria of the cell. Circular in conformation, mtDNA en-codes for the remaining machinery of protein synthesis notencoded for in nuclear DNA [27]. The mechanism of replica-tion is similar to that of nuclear DNA, however, the priming ofmtDNA for DNA replication requires the sole use of RNAprimers [30]. DNA:RNA hybrids are prerequisite to the initia-tion of transcription and serve as primers for DNA replicationof the leading strand [18,31,32]. Similar use of RNA primers

in the replication of DNA can be found in prokaryotes aswell [33].

2.5. DNA:RNA hybrids of therapeutic interest

Despite reports of the existence of DNA:RNA hybrids ofpotential therapeutic importance (reports of the persistent ex-istence of DNA:RNA hybrids in human cytomegalovirushave been made [34]); extensive studies reporting the targetingof these DNA:RNA hybrids as therapeutic strategies have notyet been examined, with the following notable exceptions.

The reverse transcription of HIV-1 has attracted substantialattention [27,35]. HIV-1 contains two identical polypurinetracts (PPTs). The first PPT only contains two pyrimidine ba-ses and extends to 25 bases from the 30 long terminal repeatend of the viral RNA. The second PPT lies further towardsthe 50 end of the viral RNA and closer to the primer bindingsite used by tRNA to initiate minus strand DNA synthesis.Both of these PPTs are identical and 18 nucleotides in length[36]. Reverse transcription (minus strand DNA synthesis) ofboth PPTs occurs after DNA strand transfer by reverse tran-scriptase, providing a unique r(Pu):d(Py) type DNA:RNA hy-brid [26]. This unique PPT DNA:RNA hybrid exists untilcleavage of the RNA template strand (50 to the PPT) and cleav-age of the tRNA and PPT. Targeting of DNA:RNA hybrids arediscussed in subsequent sections of the manuscript.

Telomerase activity, seemingly synonymous with G-quad-raplex formation, is a well understood process, of whichDNA:RNA hybrid targeting is an equally viable approachfor telomerase inhibition. Telomerase activity is associatedwith cellular immortality [37] and its potential as a universalcancer target has been proposed [38,39]. Telomeres exist asa protein coupled DNA complex with long repeats of a simple6 base sequence, d(TTAGGG)n which becomes shortened witheach cellular division [40]. The 30 tail of the parent DNA telo-mere is longer than its compliment and forms a DNA:RNA hy-brid upon binding to the active site of telomerase. Binding ofsmall molecules to the DNA:RNA hybrid can potentially offera therapeutic method for controlling telomerase activity, pre-venting telomere extension, by distorting the substrate/enzymeinteraction, or preventing dissociation of the enzyme from thesubstrate [41].

3. Stability and conformation

3.1. Stability

DNA:RNA hybrid composition can be isolated to homopurineand homopyrimide strands, which gives rise to deoxyribo(pyrimi-dine):ribo(purine), d(Py):r(Pu), and deoxyribo(purine):ribo(pyri-midine), d(Pu):r(Py) type hybrids. The designation of pyrimidine(Py) and purine (Pu) bases is consistent through this manuscript,analogous to (Y) and (R), found in other accounts, respectively.Homopurine:homopyrimidine polymer and oligomeric structuresdecrease in stability in the order of r(Pu):r(Py) > r(Pu):d(Py) > d(Py):d(Pu) > r(Py):d(Pu), as suggested by Hung et al.[42,43]. However, the introduction of long stretches of

Fig. 1. A and B form nucleic acids.

1028 N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039

(A)n:(T or U)n reverses the trend, an observation made by Dubinset al. [44]. The polymeric duplexes containing stretches of(A)n:(Tor U)n and at least one homoribonucleic acid strand followthe general hierarchy for stability DNA:DNA > DNA:RNA typed(Py):r(Pu) > RNA:RNA > DNA:RNA type d(Pu):r(Py).

Oligmeric DNA:RNA hybrids containing homopurine andhomopyrimidine tracts, stabilized by a single GeC base pairat each end, d(CT5G):r(GA5C) and d(CA5G):r(GU5C), demon-strate the same hierarchy [19] as duplexes containing (A)n:(T or U)n. It should be noted that the stability of d(Pu):r(Py)duplexes is low and although triplex formation is possible,the duplex structure does prevail [45]. Furthermore, Hall andMcLaughlin [46] demonstrate that oligomers of mixed basesequences, not isolated to purine or pyrimidine strands, showexceptions to the previously described trends. In these excep-tions, homoduplexes, d(Pu):d(Py) and r(Pu):r(Py), are morestable than heteroduplexes, d(Pu):r(Py) and d(Py):r(Pu).

An extensive survey and review of DNA:RNA hybrid sta-bility while varying (1) the percentage of (A)n:(T or U)n con-tent, (2) oligomeric length and (3) the percentage of d(Py)content in each of the strands, offers the most complete anal-ysis of hybrid stability [47]. Lesnik and Freier demonstratethat in general: (i) an inverse relationship is displayed betweenthe percentages of (A)n:(T or U)n content in the hybrid duplexand the thermal stability. (ii) Hybrid duplexes of equal d(Py)and d(Pu) content are thermally similar when comparing thed(Pu):r(Py) and d(Py):r(Pu) forms. (iii) Decreasing oligomericlength decreases the thermal stability. (iv) Isolation of (A)n:(T or U)n content to continuous tracts results in a lower ther-mally stable duplex than when the same percentage of (A)n:(Tor U)n content is dispersed throughout the sequence. (v) Con-tinuous tract (A)n:(T or U)n content results in d(Py):r(Pu)thermal stability lower than the corresponding d(Pu):r(Py)form, at physiological conditions, as expected.

It has also been suggested that DNA:RNA hybrid stabilityis related to the ability of the hybrid duplex to associatewith a third single strand [43,48]. The d(Py):r(Pu) hybriddoes not associate with a third strand of DNA, whiled(Pu):r(Py) hybrids easily accommodate a third strand toform d(Pu):r(Py):d(Pu) [43]. Furthermore, with the exceptionof long stretches of (A)n:(T or U)n, the stability of duplexescan also be attributed to the relative stability of the ribosechain. Unfavorable van der Waals contacts [49] have been ob-served between the sugar and pyrimidine nucleotides, result-ing in the expectation that within a double helix,a pyrimidine nucleotide is less stable that a purine nucleotide.

3.2. Conformation

For the sake of this discussion, we examine DNA:RNA hy-brid conformation by individually focusing on DNA:RNA hy-brids of polymeric length as well as DNA:RNA hybrids ofoligomeric length, further defined by homo ribose:deoxyriboseoligomers and chimeric oligomers. For comparison purposes,the conformation of various DNA:RNA hybrids will be com-pared to conformationally distinct A and B form helices(Fig. 1).

3.2.1. Polymeric DNA:RNA hybridsThe first DNA:RNA hybrid structure [7] resisted conforma-

tional changes upon relative humidity changes, a techniquecommonly used to drive the A- to B- transition in duplexDNA. The observation, by Milman et al. [7], that DNA:RNAhybrids resembled A-form DNA while differing from theRNA duplex, coupled with solution studies of DNA:RNA hy-brids [50,51], spurned a 20 year debate on the conformationand structure of DNA:RNA hybrids. X-ray fiber diffractionstudies of poly(rA):poly(dT) suggest a structure similar tothat observed for B-form DNA duplexes when the hybridwas in a highly solvated state [7] suggest a structure similarto that observed for B-form DNA duplexes. Parallel circulardichorism (CD) [52] and Raman [53] experiments refutea complete B-form model for the DNA:RNA hybrid. Furtheranalysis by 31P solid state NMR suggests poly(rA):poly(dT)is capable of existing in an A-like conformation at relativelylow humidity [54] while increasing the solvation from 87%to 92% yields a B-like conformation [55]. Equilibrium Ramanspectroscopy further clarifies the conformational differencesare a result of differences in furanose sugar conformation,unique to each respective ribose and deoxyribose strand[56], of which the global B-like conformation is dictated bythe burial of the thymidine methyl [48] in the d(Py) strand[57]. Steely et al. suggest [10] that ‘the d(Py) strand contrib-utes to the overall B-like conformation and concurrently stabi-lizes the duplex’. Currently, it is generally accepted that theconformation of poly(rA):poly(dT) is polymorphic, capableof undergoing an A- to B- type transition upon relative humid-ity changes [57,58].

Polymorphic structures, type d(Pu):r(Py), poly(dA):poly(rU) and poly(dI):poly(rC) further broaden the conforma-tional spectrum in which DNA:RNA hybrid structures exist.The model for poly(dA):poly(rU) suggests a ‘heteromerous’structure [59]. This term was used to describe the overall con-formation of the duplex in which the one strand, the d(Pu)strand, displays B-form characteristics while the other strand,the r(Py) strand, displays A-form characteristics. The globalconformation of poly(dA):poly(rU) is strongly driven by ther(Py) strand and the global conformation of the hybrid is anA-type duplex closer in conformation to the A-form of RNA


[48]. Congruently, the analogous DNA:RNA hybrid, poly(dI):poly(rC) displayed similar ‘heteromerous’ conformation[48,49]. However, the r(Py) has a diminutive effect on theoverall conformation of the poly(rI):poly(dC) duplex, whencompared to poly(dA):poly(rU) [59].

Mixed base polymeric DNA:RNA hybrids poly(dAC):poly(rGU) and poly(dGU):poly(rAC) have also been extensivelystudied by Gray [52]. Poly(dAC):poly(rGU) and poly(dGU):poly(rAC) are more similar to the analogous A-form RNAduplex. Furthermore, conformational differences between theindividual heteroduplexes mirror differences observed in thepolymeric analogs poly(dA):poly(rU) and poly(rA):poly(dT).The authors suggest poly(dGU):poly(rAC) is more A-likethan poly(dAC):poly(rGU). These duplexes are also suscepti-ble to dehydration driving the conformation to complete A-form conformation.

3.2.2. Oligomeric DNA:RNA hybridsPardi et al. used seven-mer oligomeric DNA:RNA hybrids

containing an A5:(T or U)5 stretch, sealed by terminal G-Cbase pairs as the first instance of oligomeric studies ofDNA:RNA hybrids by NMR [60]. The authors suggestDNA:RNA hybrid conformation belonging to the A-form, isdriven by the A-form conformation of the RNA strand, whilethe DNA strand is predominantly B-form. Unfortunately, par-tial triplex formation of d(CA5G):r(GU5C) precludes the as-signment of this hybrid to A-form or B-form.

Oligomeric DNA:RNA hybrids, restricted in base composi-tion to homopurine and homopyrimidine strands, have beenstudied by both solution [61e63] and crystal [64e66] tech-niques. Solution studies suggest a ‘heteromerous’ duplex con-taining A-form conformation in the RNA strand while theDNA strand conformation was closer to B-form [63]. How-ever, crystal structure observations [64e66] seemingly dispelthe notion that the conformation is ‘heteromerous’ by suggest-ing the conformation is homogeneous and closer to A-form.

The most complete analysis of the conformational relation-ships between homopyrimidine and homopurine DNA:RNAhybrids and their corresponding DNA and RNA duplexeswas made comparing duplexes 50-(GAAG)3-30 and 50-(CTTC)3-30 [67]; thymidine bases were replaced by uracil inribose strands. The DNA:RNA hybrid, d(Pu):r(Py) is closerin global conformation to B-form than d(Py):r(Pu); in factthe CD spectra closely resembles the CD spectra ofd(Py):d(Pu). The opposite was true for the d(Py):r(Pu) hybrid;the CD closely resembles the duplex RNA analog r(Py):r(Pu),which further perpetuates the idea that d(Py):r(Pu) oligomerichybrids are closer to A-form than analogous d(Py):r(Pu) du-plexes. In fact, the authors suggest predictions of the globalconformation of duplexes can be made based on whether ornot the hybrid duplex contains a DNA or RNA purine strand.

A survey of mixed base DNA:RNA hybrids suggests theconformation varies depending upon the percentage of purinecontent in the DNA strand. Fedoroff et al. utilized a mixedbase DNA:RNA hybrid and described its conformation as anintermediate between A-form and B-form [68]. The ‘hetero-merous’ nature of DNA:RNA hybrids reprises in the

DNA:RNA hybrid of 33% purine content in the DNA strand[69,70]. Increasing DNA purine content increases the debatein the conformational contributions from the respectiveDNA:RNA strands. One report suggests the DNA strand con-tributes B-form characteristics [71], while other reports sug-gest the DNA strand contributes A-form and B-formcharacteristics, a result of homologous sugar pucker[70,72,73] or distribution of independent sugar pucker in theDNA strand [74].

3.2.3. Chimeric DNA:RNA hybridsChimeric DNA:RNA hybrids are formed when DNA:RNA

hybrid helix is joined to double-helical DNA. Studies ondGi:rCkdCn confirmed the presence of a bend joining two dis-tinctly unique conformations within the polymer [75,76].Crystallized in 1982, the chimera [r(GCG)d(TATACGC)]2

[8] suggests the global conformation adopted by the duplexis close to the A-form RNA double helix, although discrep-ancies exist [8,77]. Subsequent conformational treatments re-vealed that chimeric hybrids are polymorphic withconformationally distinct contributions from each portion[78]. Furthermore, the RNA influence from the ribonucleicstrand in DNA:RNA hybrids is greatly diminished in chimericduplexes [79]. Solution studies on chimeras reveal that regard-less of flanking DNA:RNA hybrids with DNA duplexes or thecontrary flanking of a central core DNA by DNA:RNA hy-brids, the portion of the hybrid retains A-form characteristicsthat lie closer to B-form, while the DNA portion of the duplexexists as predominant B-form [80e82]. Even single junctionhybrids composed of a DNA duplex and DNA:RNA hybridduplex displayed the dual conformation characterized bya DNA:RNA hybrid:DNA bend [83e85].

3.3. Sugar conformation

The ribose sugar pucker is a major contributor to the helicalparameters of nucleic acids. Ribose sugar pucker of ribonu-cleic acids are generally found in the C30-endo (‘north’ orequivalent C20-exo) conformation, an accommodation madeby the sugar to facilitate the 20-hydroxyl [55]. The C30-endo ri-bose drives the conformation of homoribonucleic strands to-wards A-form, affecting most helical parameters, giving riseto the ‘heteromerous’ nature of DNA:RNA hybrids. Deoxyri-bose conformation is much more accommodating to a numberof different conformations and generally assumes a C20-endo(‘south’ or equivalent C30-exo) conformation. This conforma-tion is characteristic of B-form. Another sugar pucker, an in-termediate between the north and south conformations, isC40-endo and arises at various points in the subsequent discus-sion on the effect of sugar pucker on DNA:RNA hybrids heli-cal parameters (see Figs. 2 and 3).

3.3.1. Polymeric DNA:RNA hybridsPolymeric DNA:RNA hybrids generally follow two trends.

Refinement of the secondary structure of poly(rA):poly(dT)suggests the sugar puckers, regardless of strand, are a slightvariant of the C20-endo conformation [10,54]. However, Gupta

Fig. 2. Sugar conformations found in DNA:RNA hybrids. Adapted from ‘Principles of Nucleic Acid Structure’, Wolfram Saenger, p. 18.


and coworkers make the case that ‘in order to facilitate the 20-hydroxyl, a displacement of the RNA bases from the helicalaxis is observed. This displacement results in a concomitantchange in the tilt and twist of these bases as well as a changein a backbone torsion angle [10].’ However, there existsa ‘high energy barrier’ through which the ribose sugar ofRNA must pass in order to convert to C20-endo, a barrier sig-nificantly lower for DNA ribose [86]. Dehydration convertsthe global ribose sugar pucker to C30-endo and results in pa-rameters closer in agreement to A-form [55]. Unlike thecase of d(Py):r(Pu), in which global conformational changescan be accommodated in both strands of the duplex,d(Pu):r(Py) type polymeric duplexes are not as forgiving.Driven strongly by the conformational stubbornness of ther(Py) strand to exist in an A-form state with C30-endo sugarpucker, helical parameters tend to be more A-form in charac-teristic, with deviations arising from the d(Pu) strand[59,62,63].

Fig. 3. Definition of helical parameters pitch (P), axial rise per residue (h),

x-displacement (x-disp.), inclination (inc.) and residues per turn (shown with

n ¼ 6 residues per turn), found in Table 1, adapted from ‘Principles of Nucleic

Acid Structure’, Wolfram Saenger, p. 25.

3.3.2. Oligomeric DNA:RNA hybridsOligomeric DNA:RNA hybrids generally reflect the trends

observed in polymeric hybrids. However, discrepancies arisein the conformation of the sugar pucker. C30-endo sugar puckeris generally observed in the RNA strand, while variation in theinterpretation of the deoxyribose conformation as an equilib-rium between C30-endo/C20-endo or alternatively interpretedas C40-endo [73,74]. Further assessment of sugar pucker sug-gests that the flexible DNA strand exists with a fraction of de-oxyribose sugars in the C30-endo form, increasing in the orderof DNA:DNA duplexes, d(Pu):r(Py) and d(Py):r(Pu), whichmay be attributed to oligomeric d(Py):r(Pu) type hybrids bear-ing more A-form characteristics [61,62]. Mixed purine/pyrim-idine bases tend to assume the same general trends as observedin homopurine/homopyrimidine hybrids. Fedoroff et al. pro-pose ‘DNA:RNA structure parameters (roll and tip) can be at-tributed to DNA strand propensity to roll about the long basepair axis while RNA susceptibility to rotate along the shortbase pair axis affects parameters such as tilt and inclination[74].’ Also, interpretation of DNA:RNA hybrid sugar puckeras global C30-endo has been reported [64,65,87].

3.3.3. Chimeric DNA:RNA hybridsChimeric DNA:RNA hybrids are structurally unique, as al-

ready discussed. The extent of sugar pucker on the overallstructure of the duplex is surprising. In the chimera, hybrid du-plex flanked by DNA duplexes, the hybrid portion of the du-plex adopts C30-endo conformation in the ribose sugars,however with the exception of the junction, deoxyribonucleo-tides (which adopt C40-endo conformation) assume a C20-endoconformation exclusively [81]. DNA duplexes flanked byDNA:RNA hybrids have been suggested to have a global sugarpucker of C30-endo with a conformation characteristic ofA-form [88,89]. However, more recent studies have demon-strated that DNA residues adopt a conformation close in agree-ment to C40-endo [83], as alluded to by Mellema et al. [79].


4. Targeting DNA:RNA hybrids

Although polymorphic in structure, as demonstrated in pre-vious sections, DNA:RNA hybrids offer a unique structure totarget and potentially inhibit biological functions. However,a survey of the literature reveals the existence of less thanten ligands which specifically bind DNA:RNA hybrids. Inthe course of this section, the binding of these ligands toDNA:RNA hybrids is examined. Attention is paid to ligandbinding class as well as targeting biologically significantDNA:RNA hybrids. Structures, targets, binding constantsand buffer conditions can be found in Table 2.

4.1. Ligand classes

An examination of various ligand binding modes toDNA:RNA hybrids is conducted in the subsequent section. In-terestingly enough, DNA:RNA binding by small moleculeshas been achieved via all available modes: intercalation,groove binding as well as dual recognition. It should be notedthat although the binding of antisense oligonucleotides tomRNA forms DNA:RNA hybrids, this therapeutic approachtargets single-stranded RNA templates and is excluded fromthe following discussion, however, the use of antisense oligo-nucleotides is addressed in the section covering the PPT ofHIV-1.

4.1.1. IntercalationUsing a technique developed by Muller and Crothers [90],

Chaires et al. identified the intercalator, ethidium bromide, asa DNA:RNA hybrid, poly(rA):poly(dT), preferential binding

Table 1

Helical parameters displayed by a wide array of DNA:RNA hybrid type duplexes

Helix

type

Pitch

height (A)

Axial

rise/re

B-DNA B 33.7 3.37

Polymeric DNA:RNA hybrids

poly(rA):poly(dT)

Hydrated B 33.7 3.46

79% rel. humidity A 36.0 3.03

poly(rI):poly(dC) A 35.6 2.97

poly(dI):poly(rC) A 31.3 e

poly(dA):poly(rU) A 33.7 e

Oligomeric DNA:RNA hybrids

d(CTCTTCTTC):r(GAAGAAGAG) A 34.2 2.9

d(GTGAACTT):r(AAGUUCAC) A e 3.2

d(CAGTCCTC):r(GAGGACUG)b A e 3.2

d(CGCGCCCCAA):r(UUCGGGCGCC) A e 2.9

Chimeric DNA:RNA hybrids

[d(CGC)r(AAA)-d(TTTGCG)]2 A/B e 3.1

r(GCG)d(TATACGC) A/B 33 2.6

A-DNA A 28.2 2.56

A-RNA A 30.0 2.73

Adapted and expanded from ‘Principles of Nucleic Acid Structure’, Wolfram Saena x-displacement.b Average values.

ligand [91]. An ensuing report assayed 85 ligands against var-ious nucleic acids and focused on binding to poly(rA):po-ly(dT). This assay confirmed the presence of five ligands:ellipticine, ethidium bromide, coralyne, propidium andTAS103, which recognized the poly(rA):poly(dT) structure.Of these ligands, ethidium bromide showed the highest prefer-ence for poly(rA):poly(dT). Complete thermodynamic profilesfor ellipticine, propidium and ethidium bromide binding topoly(rA):poly(dT) yield binding constants for the three ligands(see Table 2). Inasmuch, an inverse relationship between theIC50 (inhibitory concentration giving 50% inhibition of poly(rA):poly(dT) degradation by RNase H) and the observedligand binding constant [92e94] is observed. This assay wasexpanded to simultaneously provide the thermal stabilizationafforded by the ligand on the preferentially targeted nucleicacid structure, while in competition with other nucleic acidstructures [95]. Ethidium bromide competition dialysis find-ings were corroborated in this mixed melting experiment.

Actinomycin D (AMD) has demonstrated the ability to in-hibit reverse transcriptase activity [96,97] through the inhibi-tion of double-stranded DNA synthesis [96]. Comprised ofan intercalating phenoxazome ring, substitution for the carbonat the 8-position of the planar ring system by nitrogen in-creases the selectivity of the compound for DNA:RNA hybrids[98]. Unfortunately, affinity of N8AMD (Table 2) for theDNA:RNA hybrid is slightly lower than the natural product.Of note, the authors suggest that AMD actually bindsDNA:RNA hybrids, structures previously believed to be un-able to bind AMD. However, the preference of AMD forDNA duplexes dominates. Similarly, substitution at the sp2

carbon at position 8, by fluorine increases the preference of

sidue (A)

Residues

per turn

Inclination (�) Dx (A)a Ref.

10 �5.9 e [59,87,88]

9.7 e e [55]

11.9 e e [55]

12 þ5.68 e [49]

10 þ15 e [59]

11 þ13 e [59]

11.8 þ7.9 �4.0 [65]

11 þ17 �4.5 [71]

10.9 þ6.8 �3.7 [68]

10.9 þ13.9 �3.3 [72]

11 �1.3 �1.8 [81]

10.9 þ20 �2.0 [8]

11.0 þ20.0 e [88]

11.0 þ17.0 e [89]

ger, p. 235.

Table 2

Structures, targets, binding constants and buffer conditions for ligands and their respective DNA:RNA hybrid targets

Structure Target Ka Ref.

Ethidium bromide

NH2

H2N

Br- N+ poly(rA):poly(dT): 7 mM Na2HPO4, 2 mM NaH2PO4,

180 mM NaCl, 1 mM EDTA, pH 7.0e7.1

1.1 � 105;

fluorescence

[91]

poly(dA):poly(rU): 10 mM sodium calcodylate,

0.5 mM EDTA, 100 mM NaCl, pH 5.5

9.3 � 106;

calorimetric

[109]

Propidium iodide

NH2H2N

N+

N+2

. I-

poly(rA):poly(dT): 7 mM Na2HPO4, 2 mM

NaH2PO4, 180 mM NaCl, 1 mM EDTA, pH 7.0e7.1

3.3 � 105;

fluorescence

[91]

EllipticineN

N

CH3

CH3

poly(rA):poly(dT): 10 mM sodium calcodylate,

0.5 mM EDTA, 100 mM NaCl

4.4 � 105;

fluorescence

[91]

O

N NH2

O

CH3CH3

NN

X

O

R1R1

R2

R1 = -Thr-D Val-Pro-Sar-MeVal

R1 = C; X = H (AMD)

d(GGAGCAGGAC):r(CUCCUGCUCC): 20 mM

phosphate, 300 mM NaCl, pH 7.0

3.4 � 104;

UV-spectroscopic

[98]

R1 ¼ C; X ¼ H (AMD) 50-AUAUAUGCAUAUTTTTTATATGCATATAT-30:a

50 mM TriseHCl, 10 mM MgCl2, 10 mM KCl, pH 7.5

2.6 � 105;

UV-spectroscopic

[99]

R1 ¼ N (N8AMD) d(GGAGCAGGAC):r(CUCCUGCUCC); 20 mM

phosphate, 300 mM NaCl, pH 7.0

1.6 � 104;

UV-spectroscopic

[98]

10

32

N.N

.Shaw

,D

.P.

Arya

/B

iochimie

90(2008)

1026e1039

R1 ¼ C; X ¼ F (F8AMD) 50-AUAUAUGCAUAUTTTTTATATGCATATAT-30:a

50 mM TriseHCl, 10 mM MgCl2, 10 mM KCl, pH 7.5

1.4 � 105;

UV-spectroscopic

[99]

O

NH

NO

NH

N

NO

NH

N

NH

X

R

O

R

O

R =

X = CH (MT-9)

d(CAAAGATTCCTC):(GTTTCTAAGGAG):a 200 mM

TriseHCl, 50 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, pH 7.6

5.4 � 107;

electrophoretic

[105]

X ¼ N (MT-15) d(CAAAGATTCCTC):(GTTTCTAAGGAG):a 200 mM

TriseHCl, 50 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, pH 7.6

w106;

electrophoretic

[105]

Paramomycin

O

O

O

O

OO

HOHO

HO

OH

NH2

NH2

H2NH2N

H2N

OH

OH

OH

OH

d(GCCACTGC):r(GCAGUGGC): 10 mM sodium calcodylate,

0.1 mM EDTA, total Naþ 60 mM

[104]

(pH 6.0) 4.3 � 106

(pH 7.0) 1.2 � 106;

calorimetric

50-CGGGCGCCACTGCTAGAG-30; 30-

GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS, 0.1 mM

EDTA, total Naþ 100 mM, pH 7.5

4.2 � 105;

calorimetric

[26]




1.8 � 105;

calorimetric

[26]

Ribostamycin

OH

O

O

OO

HOHO

HO

NH2

NH2

H2NH2N

OH

OH




1.1 � 1010;

calorimetric

[26]




1.5 � 104;

calorimetric

[26]

(continued on next page) 10

33

N.N

.Shaw

,D

.P.

Arya

/B

iochimie

90(2008)

1026e1039

Table 2 (continued)

Structure Target Ka Ref.

Neomycin

O

O

O

O

O

O

HOHO

HO

NH2

NH2

NH2

H2NH2N

H2N

OH

OH

OH

OH


GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS,

0.1 mM EDTA, total Naþ 100 mM, pH 7.5

1.1 � 106;

calorimetric

[26]


GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS,

0.1 mM EDTA, total Naþ 100 mM, pH 7.5

1.4 � 105;

calorimetric

[26]



1.7 � 106;

calorimetric/UV

b



9.9 � 106;

calorimetric/UV

[109]

NM

N+

NH2H2N

O

SNH

O

O

O

O

OO

HOHO

NH2

OH

OH

H2N OHOH

NH2

NH2

H2NH2N



1.9 � 108;

calorimetric/UV

b



4.8 � 1010;

calorimetric/UV

[109]

a In all cases, bold denotes RNA nucleotides. Buffer conditions and method of binding constant determination are noted throughout.b Unpublished results.

10

34

N.N

.Shaw

,D

.P.

Arya

/B

iochimie

90(2008)

1026e1039


the ligand for DNA:RNA hybrids, although not notably higherthan N8AMD. The binding constant of F8AMD towards a hair-pin DNA:RNA hybrid, 50-aua uau gca uau-TTT TTA TATGCA TAT AT-30, containing a five thymine loop (RNA basesdenoted in lowercase), is nominally lower than AMD (seeTable 2). However, both conjugates of AMD display a uniqueselectivity for leukemia cells, inhibiting cellular growth by50% at less than 1.0 nM! Although the binding mode ulti-mately leading to retardation of leukemia cell growth is notknown, the authors hypothesize these conjugates bindDNA:RNA hybrids and validate further development [99].

4.1.2. Major groove bindingAminoglycosides have been recognized as A-form nucleic

acid major groove binders [100e103] and shown to signifi-cantly stabilize DNA:RNA hybrids [103]. Among all amino-glycosides studied, neomycin showed the maximumstabilization for DNA:RNA hybrid duplexes and even inducedthe formation of hybrid triplexes [100]. Later, paramomycin,an aminoglycoside, and its complexation with DNA:RNA hy-brids was studied by Barbieri et al. [104]. Thermal stabiliza-tion of a mixed base RNA 8-mer duplex, afforded uponparamomycin complexation, is 6.3 �C, not surprising sinceaminoglycosides traditionally bind RNA. However, thermalstabilization of the DNA:RNA hybrid analog was on parwith the RNA duplex at 6.2 �C. Furthermore, the binding ofparamomycin to the hybrid duplex induces a shift in the hybridduplex to a more A-form like conformation. Additionally, itwas found that paramomycin binds the RNA duplex at higherbinding affinities than the corresponding DNA:RNA hybrid(Table 2). Discrimination between paramomycin binding theRNA duplex and hybrid duplex is enthalpy driven. However,the difference between binding constants between the RNAduplex and DNA:RNA hybrid is diminutive in comparison tothe analogous DNA duplex. Finally, the authors suggest thebinding of paramomycin to the DNA:RNA hybrid inhibitsboth RNase H- and RNase A-mediated cleavage of the RNAstrand.

4.1.3. Minor groove bindingNaturally occurring polyamides, distamycin and netropsin,

have been investigated for their ability to bind DNA:RNA hy-brid Okazaki fragments [105,106]. Lexitropsins were designedto bind A/T stretches of DNA, displacing the spine of hydra-tion, deep in the minor groove [107]. Distamycin derivativeswere developed through various strategies [108], creatinga number of distamycin dimers. Discussion of these com-pounds can be found in Section 4.2.2.

4.1.4. Dual recognitionRecently we have attempted to exploit the unique binding

characteristics displayed by ethidium bromide and neomycinin the design of a ligand capable of binding DNA:RNA hy-brids with high affinity [107]. Neomycin was covalently linkedto 6-(4-carboxyphenyl)-3,8-diamino-5-methylphen-anthridi-nium chloride, a derivative of ethidium bromide, through theformation of an amide bond, to afford a neomycin-methidium

chloride conjugate, NM (see Table 2). Competition meltingexperiments and competition dialysis experiments revealeda preferential binding of NM to DNA:RNA hybrid structures,of which d(Pu):r(Py) type, poly(dA):poly(rU), was preferredabove d(Py):r(Pu) type, poly(rA):poly(dT). The thermal stabil-ity afforded by NM on the preferred hybrid, poly(dA):po-ly(rU), was 26 �C, much greater than the stabilizationafforded by neomycin (7.8 �C), ethidium bromide (1.1 �C) orboth neomycin and ethidium bromide 7.3 �C, at equivalentratios.

Association constants, KT(25 �C) values were determined uti-lizing UV, CD and calorimetry. Association constants demon-strate NM binds poly(dA):poly(rU) at higher affinities thanneomycin or ethidium bromide. While the binding constantfor neomycin closely resembles the binding of neomycin tothe RNA backbone HIV-1 chimera [26], 9.93 � 106 M�1,and the binding of ethidium bromide was in close agreementwith previously reported results [94] at 9.28 � 106 M�1, NMbinds poly(dA):poly(rU) at a much higher association constantof 4.77 � 1010 M�1. The binding constant of NM far exceedsneomycin or ethidium bromide clearly demonstrating the po-tential power of ligand conjugation. This conjugate providesan aminoglycoside based approach to the high affinity bindingof DNA:RNA hybrids and is the highest binding constant ob-served for non-oligonucleotide based targeting of theDNA:RNA hybrid.

4.2. Targeting biologically significant DNA:RNA hybrids

This section examines reports of ligand targetingDNA:RNA hybrids of biological significance. If one considersthe list of ligands capable of binding DNA:RNA hybrids asshort, the list of accounts focusing on ligands binding biolog-ically significant DNA:RNA hybrids is even shorter.

4.2.1. PPT of HIV-1Targeting the polypurine tract, as previously mentioned, is

an attractive method for inhibition of reverse transcription ofHIV-1. Formation of a triplex can be achieved through theuse of a single-stranded compliment designed to target theDNA:RNA hybrid present on the PPT of HIV-1. Triple helixformation can alternatively be formed using a duplex targetedat the single-stranded RNA strand of the PPT. Regardless, tri-plex forming oligonucleotides (TFOs) prevent RNase H cleav-age of the RNA strand of the PPT and congruently inhibitinitiation of plus-strand DNA synthesis in vitro [110]. It hasbeen determined that a 25 mer TFO directed against thePPT is more inhibitory for RNA dependent DNA synthesisthan antisense approaches directed at other regions of RNA[36]. Cell culture experiments indicate one TFO, forming anRNA:RNA:DNA hybrid triplex, is efficient in inhibiting retro-virus replication by preventing cleavage at the PPT site byRNase H, in turn inhibiting plus-strand DNA synthesis[110]. Furthermore, studies have suggested this uniqueDNA:RNA hybrid triplex can be induced and even stabilizedby the presence of ligands such as 40,6-diamidino-2-phenylin-dole (DAPI) [111] and the aminoglycoside, neomycin [100].


We first reported the ability of neomycin to stabilizeDNA:RNA hybrids and even induce hybrid triplex formation[100]!

This work was further expanded by Pilch and coworkers toinclude the aminoglycosides neomycin and ribostamycin, fo-cusing on DNA:RNA hybrid chimeras. These chimeras weredesigned as constructs of the polypurine tract (PPT) of HIV-1,found at two unique steps in the reverse transcription ofHIV-1 [26]. Containing the same DNA:RNA hybrid portion,these chimeras differ in the composition, RNA or DNA, of thecompliment strand. As reported earlier, RNase H cleavage ofthe DNA:RNA hybrid chimeras is inhibited by the presence ofthe aminoglycosides and a number of small molecule non-nucleoside reverse transcriptase inhibitors (NNRTIs) [112]. Infact, of the aminoglycosides surveyed, neomycin is far superiorat inhibiting RNase H cleavage in both chimeras. Furthermore,thermal stabilization of the chimeras, afforded by the presenceof aminoglycosides, reveals that neomycin increases the stabil-ity of both chimeras to a larger extent than paramomycin, whichare both much greater than ribostamycin. Isothermal titrationcalorimetry derived binding constants for the aminoglycosidesbinding the two chimeras are located in Table 2. In both chi-meras, the association constant for neomycin is greater thanthe other aminoglycosides studied. Furthermore, it is clearthat the addition of ring four to the 2-deoxy streptose moietyis paramount to high affinity binding of aminoglycosides toDNA:RNA hybrid structures.

4.2.2. Okazaki fragmentsA series of conformationally restrained bis-distamycin

compounds, dimerized through the ortho/para or meta posi-tions of benzene and pyridine (2,5- and 2,4- type, respec-tively), were synthesized [108]. Ortho/para bis-distamycinswere capable of binding both DNA duplexes as well as theOkazaki fragment at similar binding constants, w108 M�1

and w107 M�1 range, respectively. Surprisingly, mono-dista-mycins, linked to the benzene moiety, showed no binding tothe duplexes. The meta pyridyl bis-distamycins were also stud-ied, however, affinity for the Okazaki duplex is in thew106 M�1 range.

4.2.3. TelomeraseDNA:RNA hybrid targeting with ethidium bromide has

been applied to targeting telomerase through its DNA:RNAhybrid duplex. Friedman and coworkers have surveyed a num-ber of compounds in an attempt to inhibit telomerase activitythrough DNA:RNA hybrid binding [41,113]. Surveying a num-ber of intercalators, four ligands (ethidium bromide, rivanol,acridine orange and yellow) were found to inhibit telomeraseactivity at IC50 values in the low micromolar range. Further-more, telomerase inhibition through the binding of these li-gands to G-quadraplex structures, another substantiatedmethod for achieving telomerase inhibition, was not observed[41]. Affinity chromatography isolated the high affinityDNA:RNA binding species from a homogeneous mixture ofbinding ligands. It was found that ethidium bromide uniquelybinds a DNA:RNA hybrid duplex, derived from the hybrid

formed during the catalytic cycle of telomerase, with the high-est affinity, even in the presence of other binding ligands[113]. This work identified ethidium bromide as a lead com-pound for therapeutic approaches to DNA:RNA hybrid target-ing of telomereres.

5. Concluding remarks

Acting at the hub of biological information transfer,DNA:RNA hybrids provide a potential substrate for control-ling gene expression. Conformationally distinguishable frommore common nucleic acids, DNA and RNA, these hybrids ne-cessitate more attention when studying nucleic acids therapeu-tics. Even more enticing, due to their relatively low occurrencewithin biological systems, when compared to DNA and RNA,DNA:RNA hybrids provide a unique substrate for targetingwith small binding ligands. Despite their uniqueness,DNA:RNA hybrids have not received their share of attention.The attention DNA:RNA hybrids have received suggest thatDNA:RNA hybrids can be targeted by small ligands. However,more work must be done to show that selective targeting ofDNA:RNA hybrids can be attained in the presence ofa much higher concentration of duplex DNA and RNA struc-tures in the cell. Small molecule binding to DNA:RNA hybridshas shown the ability to disrupt the transfer of genetic infor-mation by disabling the proper function of enzymes. Morework still needs to be done to develop highly specific ligandscapable of binding the hybrids at association constants higherthan naturally occurring enzymes. We have begun to outlinethe principles required for high affinity and high specificitybinding of DNA:RNA hybrids by small ligands. Through theconjugation of an aminoglycoside to an DNA.RNA hybridspecific intercalator, DNA:RNA hybrids of distinctly differentsequence can be distinguished from one another as well asother nucleic acid structures. More work is being done to de-velop more selective molecules such that DNA:RNA hybridtargeting can become a noteworthy therapeutic endeavor.

Acknowledgements

PI is thankful to NSF (CHE 034972) for financial support.We also thank Prof. Meredith Newby, Clemson University, forhelp with proofreading this manuscript.

References

[1] J.D. Watson, F.H. Crick, Molecular structure of nucleic acids; a structure

for deoxyribose nucleic acid, Nature 171 (1953) 737e738.

[2] A. Rich, An analysis of the relation between DNA and RNA, Ann. N.Y.

Acad. Sci. 81 (1959) 709e722.

[3] A. Rich, Discovery of the hybrid helix and the first DNAeRNA hybrid-

ization, J. Biol. Chem. 281 (2006) 7693e7696.

[4] A. Rich, A hybrid helix containing both deoxyribose and ribose polynu-

cleotides and its relation to the transfer of information between the nu-

cleic acids, Proc. Natl. Acad. Sci. USA 46 (1960) 1044e1053.

[5] C.L. Schildkraut, J. Marmur, P. Doty, The formation of hybrid DNA

molecules and their use in studies of DNA homologies, J. Mol. Biol.

3 (1961) 595e617.


[6] B.D. Hall, S. Spiegelman, Sequence complementarity of T2-DNA and

T2-specific RNA, Proc. Natl. Acad. Sci. USA 47 (1961) 137e163.

[7] G. Milman, R. Langridge, M.J. Chamberlin, The structure of a DNAe

RNA hybrid, Proc. Natl. Acad. Sci. USA 57 (1967) 1804e1810.

[8] A.H. Wang, S. Fujii, J.H. van Boom, G.A. van der Marel, S.A. van Boeckel,

A. Rich, Molecular structure of r(GCG)d(TATACGC): a DNAeRNA hy-

brid helix joined to double helical DNA, Nature 299 (1982) 601e604.

[9] D.G. Reid, S.A. Salisbury, T. Brown, D.H. Williams, J.J. Vasseur,

B. Rayner, J.L. Imbach, Use of inter-proton nuclear Overhauser effects

to assign the nuclear magnetic resonance spectra of oligodeoxynucleo-

tide and hybrid duplexes in aqueous solution, Eur. J. Biochem 135

(1983) 307e314.

[10] G. Gupta, M.H. Sarma, R.H. Sarma, Secondary structure of the hybrid

poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and

stereochemical modeling within the constraints of NOE data, J. Mol.

Biol. 186 (1985) 463e469.

[11] J. Hurwitz, A. Bresler, R. Diringer, The enzymic incorporation of ribo-

nucleotides into polyribonucleotides and the effect of DNA, Biochem.

Biophys. Res. Commun 3 (1960) 15e18.

[12] H.M. Temin, S. Mizutani, RNA-dependent DNA polymerase in virions

of Rous sarcoma virus, Nature 226 (1970) 1211e1213.

[13] D. Baltimore, RNA-dependent DNA polymerase in virions of RNA tu-

mour viruses, Nature 226 (1970) 1209e1211.

[14] J.P. Bouche, K. Zechel, A. Kornberg, dnaG gene product, a rifampicin-

resistant RNA polymerase, initiates the conversion of a single-stranded

coliphage DNA to its duplex replicative form, J. Biol. Chem. 250 (1975)

5995e6001.

[15] N.A. Campbell, Biology, Benjamin Cummings, Menlo Park, CA, 1996.

[16] R.L.P. Adams, R.H. Burton, A.M. Campbell, D.P. Leader,

R.M.S. Smellie, The Biochemistry of the Nucleic Acids, Chapman

and Hall, New York, 1981.

[17] M.J. Chamberlin, RNA Polymerase, Cold Spring Harbor Laboratory,

Cold Spring Harbor, NY, 1976.

[18] B. Xu, D.A. Clayton, RNAeDNA hybrid formation at the human mito-

chondrial heavy-strand origin ceases at replication start sites: an impli-

cation for RNAeDNA hybrids serving as primers, EMBO J 15 (1996)

3135e3143.

[19] F.H. Martin, I. Tinoco Jr., DNA RNA hybrid duplexes containing oli-

go(dA:rU) sequences are exceptionally unstable and may facilitate ter-

mination of transcription, Nucleic Acids Res. 8 (1980) 2295e2299.

[20] K.R. Blake, A. Murakami, S.A. Spitz, S.A. Glave, M.P. Reddy,

P.O. Ts’o, P.S. Miller, Hybridization arrest of globin synthesis in rabbit

reticulocyte lysates and cells by oligodeoxyribonucleoside methyl-

phosphonates, Biochemistry 24 (1985) 6139e6145.

[21] K.R. Blake, A. Murakami, P.S. Miller, Inhibition of rabbit globin

mRNA translation by sequence-specific oligodeoxyribonucleotides,

Biochemistry 24 (1985) 6132e6138.

[22] H.M. Temin, Malignant transformation of cells by viruses, Perspect.

Biol. Med 14 (1970) 11e26.

[23] H.M. Temin, Origin of retroviruses from cellular moveable genetic el-

ements, Cell 21 (1980) 599e600.

[24] H.M. Temin, Reverse transcription in the eukaryotic genome: retrovi-

ruses, pararetroviruses, retrotransposons, and retrotranscripts, Mol.

Biol. Evol. 2 (1985) 455e468.

[25] W. Gilbert, Origin of life: the RNA world, Nature 319 (1986) 618.

[26] T.K. Li, C.M. Barbieri, H.C. Lin, A.B. Rabson, G. Yang, Y. Fan,

B.L. Gaffney, R.A. Jones, D.S. Pilch, Drug targeting of HIV-1

RNA.DNA hybrid structures: thermodynamics of recognition and im-

pact on reverse transcriptase-mediated ribonuclease H activity and viral

replication, Biochemistry 43 (2004) 9732e9742.

[27] B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molec-

ular Biology of the Cell, Garland Science, New York, 2002.

[28] D.A. Clayton, Replication of animal mitochondrial DNA, Cell 28

(1982) 693e705.

[29] R. Okazaki, T. Okazaki, K. Sakabe, K. Sugimoto, A. Sugino, Mecha-

nism of DNA chain growth. I. Possible discontinuity and unusual sec-

ondary structure of newly synthesized chains, Proc. Natl. Acad. Sci.

USA 59 (1968) 598e605.

[30] M.E. Schmitt, D.A. Clayton, Conserved features of yeast and mamma-

lian mitochondrial DNA replication, Curr. Opin. Genet. Dev. 3 (1993)

769e774.

[31] D.D. Chang, D.A. Clayton, Priming of human mitochondrial DNA rep-

lication occurs at the light-strand promoter, Proc. Natl. Acad. Sci. USA

82 (1985) 351e355.

[32] D.D. Chang, D.A. Clayton, Precise identification of individual pro-

moters for transcription of each strand of human mitochondrial DNA,

Cell 36 (1984) 635e643.

[33] T. Itoh, J. Tomizawa, Formation of an RNA primer for initiation of rep-

lication of ColE1 DNA by ribonuclease H, Proc. Natl. Acad. Sci. USA

77 (1980) 2450e2454.

[34] M.N. Prichard, S. Jairath, M.E. Penfold, S. St. Jeor, M.C. Bohlman,

G.S. Pari, Identification of persistent RNAeDNA hybrid structures

within the origin of replication of human cytomegalovirus, J. Virol.

72 (1998) 6997e7004.

[35] M. Tisdale, T. Schulze, B.A. Larder, K. Moelling, Mutations within

the RNase H domain of human immunodeficiency virus type 1 reverse

transcriptase abolish virus infectivity, J. Gen. Virol. 72 (Pt 1) (1991)

59e66.

[36] S. Volkmann, J. Dannull, K. Moelling, The polypurine tract, PPT, of

HIV as target for antisense and triple-helix-forming oligonucleotides,

Biochimie 75 (1993) 71e78.

[37] N.W. Kim, M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West,

P.L. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, J.W. Shay, Spe-

cific association of human telomerase activity with immortal cells and

cancer, Science 266 (1994) 2011e2015.

[38] S.P. Lichtsteiner, J.S. Lebkowski, A.P. Vasserot, Telomerase. A target

for anticancer therapy, Ann.. N.Y. Acad. Sci. 886 (1999) 1e11.

[39] C. Autexier, Telomerase as a possible target for anticancer therapy,

Chem. Biol. 6 (1999) R299eR303.

[40] A. Siddiqa, D.A. Cavazos, R.A. Marciniak, Targeting telomerase, Reju-

venation Res. 9 (2006) 378e390.

[41] R. Francis, C. West, S.H. Friedman, Targeting telomerase via its key

RNA/DNA heteroduplex, Bioorg. Chem. 29 (2001) 107e117.

[42] S.H. Hung, Q. Yu, D.M. Gray, R.L. Ratliff, Evidence from CD spectra

that d(purine).r(pyrimidine) and r(purine).d(pyrimidine) hybrids are in

different structural classes, Nucleic Acids Res. 22 (1994) 4326e4334.

[43] M.J. Chamberlin, D.L. Patterson, Physical and chemical characteriza-

tion of the ordered complexes formed between polyinosinic acid, poly-

cytidylic acid and their deoxyribo-analogues, J. Mol. Biol. 12 (1965)

410e428.

[44] M. Riley, B. Maling, Physical and chemical characterization of two- and

three-stranded adenine-thymine and adenine-uracil homopolymer com-

plexes, J. Mol. Biol. 20 (1966) 359e389.

[45] D.N. Dubins, A. Lee, R.B. Macgregor Jr., T.V. Chalikian, On the stabil-

ity of double stranded nucleic acids, J. Am. Chem. Soc. 123 (2001)

9254e9259.

[46] K.B. Hall, L.W. McLaughlin, Thermodynamic and structural properties

of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of iden-

tical sequence, Biochemistry 30 (1991) 10606e11613.

[47] E.A. Lesnik, S.M. Freier, Relative thermodynamic stability of DNA,

RNA, and DNA:RNA hybrid duplexes: relationship with base composi-

tion and structure, Biochemistry 34 (1995) 10807e10815.

[48] R.W. Roberts, D.M. Crothers, Stability and properties of double and tri-

ple helices: dramatic effects of RNA or DNA backbone composition,

Science 258 (1992) 1463e1466.

[49] E.J. O’Brien, A.W. MacEwan, Molecular and crystal structure of the

polynucleotide complex: polyinosinic acid plus polydeoxycytidylic

acid, J. Mol. Biol. 48 (1970) 243e261.

[50] M. Chamberlin, P. Berg, Mechanism of RNA polymerase action: forma-

tion of DNAeRNA hybrids with single-stranded templates, J. Mol.

Biol. 8 (1964) 297e313.

[51] M.J. Tunis, J.E. Hearst, Optical rotatory dispersion of DNA in concen-

trated salt solutions, Biopolymers 6 (1968) 1218e1223.

[52] D.M. Gray, R.L. Ratliff, Circular dichroism spectra of poly[d(AC):d(GT)],

poly[r(AC):r(GU)], and hybrids poly[d(AC):r(GU)] and poly[r(AC):

d(GT)] in the presence of ethanol, Biopolymers 14 (1975) 487e498.


[53] S.C. Erfurth, P.J. Bond, W.L. Peticolas, Characterization of the A in

equilibrium B transition of DNA in fibers and gels by laser Raman spec-

troscopy, Biopolymers 14 (1975) 1245e1257.

[54] H. Shindo, U. Matsumoto, Direct evidence for a bimorphic structure of

a DNAeRNA hybrid, poly(rA).poly(dT), at high relative humidity, J.

Biol. Chem. 259 (1984) 8682e8684.

[55] S.B. Zimmerman, B.H. Pheiffer, A RNA.DNA hybrid that can adopt

two conformations: an x-ray diffraction study of poly(rA).poly(dT) in

concentrated solution or in fibers, Proc. Natl. Acad. Sci. USA 78

(1981) 78e82.

[56] J.M. Benevides, G.J. Thomas Jr., A solution structure for poly(rA).

poly(dT) with different furanose pucker and backbone geometry in rA

and dT strands and intrastrand hydrogen bonding of adenine 8CH, Bio-

chemistry 27 (1988) 3868e3873.

[57] H.T. Steely Jr., D.M. Gray, R.L. Ratliff, CD of homopolymer DNAe

RNA hybrid duplexes and triplexes containing A-T or A-U base pairs,

Nucleic Acids Res. 14 (1986) 10071e10090.

[58] T. Fujiwara, H. Shindo, Phosphorus-31 nuclear magnetic resonance of

highly oriented DNA fibers. 2. Molecular motions in hydrated DNA,

Biochemistry 24 (1985) 896e902.

[59] S. Arnott, R. Chandrasekaran, R.P. Millane, H.S. Park, DNAeRNA hy-

brid secondary structures, J. Mol. Biol. 188 (1986) 631e640.

[60] A. Pardi, F.H. Martin, I. Tinoco Jr., Comparative study of ribonucleo-

tide, deoxyribonucleotide, and hybrid oligonucleotide helices by nu-

clear magnetic resonance, Biochemistry 20 (1981) 3986e3996.

[61] J.I. Gyi, A.N. Lane, G.L. Conn, T. Brown, Solution structures of

DNA.RNA hybrids with purine-rich and pyrimidine-rich strands: com-

parison with the homologous DNA and RNA duplexes, Biochemistry 37

(1998) 73e80.

[62] J.I. Gyi, G.L. Conn, A.N. Lane, T. Brown, Comparison of the thermo-

dynamic stabilities and solution conformations of DNA.RNA hybrids

containing purine-rich and pyrimidine-rich strands with DNA and

RNA duplexes, Biochemistry 35 (1996) 12538e12548.

[63] E. Hantz, V. Larue, P. Ladam, L. Le Moyec, C. Gouyette, T. Huynh

Dinh, Solution conformation of an RNAeDNA hybrid duplex contain-

ing a pyrimidine RNA strand and a purine DNA strand, Int. J. Biol.

Macromol 28 (2001) 273e284.

[64] G.L. Conn, T. Brown, G.A. Leonard, The crystal structure of the

RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) shows sig-

nificant differences to that found in solution, Nucleic Acids Res. 27

(1999) 555e561.

[65] Y. Xiong, M. Sundaralingam, Crystal structure of a DNA.RNA hybrid

duplex with a polypurine RNA r(gaagaagag) and a complementary pol-

ypyrimidine DNA d(CTCTTCTTC), Nucleic Acids Res. 28 (2000)

2171e2176.

[66] G.W. Han, Direct-methods determination of an RNA/DNA hybrid dec-

amer at 1.15 A resolution, Acta Crystallogr. D 57 (2001) 213e218.

[67] L. Ratmeyer, R. Vinayak, Y.Y. Zhong, G. Zon, W.D. Wilson, Sequence

specific thermodynamic and structural properties for DNA.RNA du-

plexes, Biochemistry 33 (1994) 5298e5304.

[68] O.Y. Fedoroff, Y. Ge, B.R. Reid, Solution structure of r(gaggacug):

d(CAGTCCTC) hybrid: implications for the initiation of HIV-1

(þ)-strand synthesis, J. Mol. Biol. 269 (1997) 225e239.

[69] X. Gao, P.W. Jeffs, Sequence-dependent conformational heterogeneity of

a hybrid DNA.RNA dodecamer duplex, J. Biomol. NMR 4 (1994) 367e384.

[70] S.H. Chou, P. Flynn, B. Reid, High-resolution NMR study of a synthetic

DNAeRNA hybrid dodecamer containing the consensus pribnow pro-

moter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG), Bio-

chemistry 28 (1989) 2435e2443.

[71] A.N. Lane, S. Ebel, T. Brown, NMR assignments and solution confor-

mation of the DNA.RNA hybrid duplex d(GTGAACTT).r(AAGUU-

CAC), Eur. J. Biochem. 215 (1993) 297e306.

[72] N.C. Horton, B.C. Finzel, The structure of an RNA/DNA hybrid: a sub-

strate of the ribonuclease activity of HIV-1 reverse transcriptase, J. Mol.

Biol. 264 (1996) 521e533.

[73] M. Salazar, O.Y. Fedoroff, J.M. Miller, N.S. Ribeiro, B.R. Reid, The

DNA strand in DNA.RNA hybrid duplexes is neither B-form nor A-

form in solution, Biochemistry 32 (1993) 4207e4215.

[74] O. Fedoroff, M. Salazar, B.R. Reid, Structure of a DNA:RNA hybrid

duplex. Why RNase H does not cleave pure RNA, J. Mol. Biol. 233

(1993) 509e523.

[75] E. Selsing, R.D. Wells, T.A. Early, D.R. Kearns, Two contiguous con-

formations in a nucleic acid duplex, Nature 275 (1978) 249e250.

[76] E. Selsing, R.D. Wells, Polynucleotide block polymers consisting of

a DNA.RNA hybrid joined to a DNA.DNA duplex. Synthesis and charac-

terization of dGn.rCidCk duplexes, J. Biol. Chem. 254 (1979) 5410e5416.

[77] M. Egli, N. Usman, S.G. Zhang, A. Rich, Crystal structure of an Oka-

zaki fragment at 2-A resolution, Proc. Natl. Acad. Sci. USA 89 (1992)

534e538.

[78] D.G. Sanford, K.J. Kotkow, B.D. Stollar, Immunochemical detection of

multiple conformations within a 36 base pair oligonucleotide, Nucleic

Acids Res. 16 (1988) 10643e10655.

[79] J.R. Mellema, C.A. Haasnoot, G.A. van der Marel, G. Wille, C.A. van

Boeckel, J.H. van Boom, C. Altona, Proton NMR studies on the cova-

lently linked RNAeDNA hybrid r(GCG)d(TATACGC). Assignment of

proton resonances by application of the nuclear Overhauser effect, Nu-

cleic Acids Res. 11 (1983) 5717e5738.

[80] T. Nishizaki, S. Iwai, T. Ohkubo, C. Kojima, H. Nakamura, Y. Kyogoku,

E. Ohtsuka, Solution structures of DNA duplexes containing

a DNA � RNA hybrid region, d(GG)r(AGAU)d(GAC) x

d(GTCATCTCC) and d(GGAGA)r(UGAC) � d(GTCATCTCC), Bio-

chemistry 35 (1996) 4016e4025.

[81] S.T. Hsu, M.T. Chou, J.W. Cheng, The solution structure of

[d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA du-

plexes, Nucleic Acids Res. 28 (2000) 1322e1331.

[82] L. Zhu, M. Salazar, B.R. Reid, DNA duplexes flanked by hybrid du-

plexes: the solution structure of chimeric junctions in [r(cgcg)d(TA-

TACGCG)]2, Biochemistry 34 (1995) 2372e2380.

[83] M. Salazar, J.J. Champoux, B.R. Reid, Sugar conformations at hybrid

duplex junctions in HIV-1 and Okazaki fragments, Biochemistry 32

(1993) 739e744.

[84] M. Salazar, O. Fedoroff, L. Zhu, B.R. Reid, The solution structure of the

r(gcg)d(TATACCC):d(GGGTATACGC) Okazaki fragment contains two

distinct duplex morphologies connected by a junction, J. Mol. Biol. 241

(1994) 440e455.

[85] O. Fedoroff, M. Salazar, B.R. Reid, Structural variation among retrovi-

ral primer-DNA junctions: solution structure of the HIV-1 (�)-strand

Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC), Biochemistry 35

(1996) 11070e11080.

[86] A. Rich, The double helix: a tale of two puckers, Nat. Struct. Biol. 10

(2003) 247e249.

[87] B.N. Conner, T. Takano, S. Tanaka, K. Itakura, R.E. Dickerson, The

molecular structure of d(ICpCpGpG), a fragment of right-handed dou-

ble helical A-DNA, Nature 295 (1982) 294e299.

[88] S. Arnott, D.W. Hukins, Refinement of the structure of B-DNA and im-

plications for the analysis of x-ray diffraction data from fibers of bio-

polymers, J. Mol. Biol. 81 (1973) 93e105.

[89] S. Arnott, D.W. Hukins, Optimised parameters for A-DNA and B-DNA,

Biochem. Biophys. Res. Commun. 47 (1972) 1504e1509.

[90] W. Muller, D.M. Crothers, Interactions of heteroaromatic compounds

with nucleic acids. 1. The influence of heteroatoms and polarizability

on the base specificity of intercalating ligands, Eur. J. Biochem. 54

(1975) 267e277.

[91] J. Ren, J.B. Chaires, Sequence and structural selectivity of nucleic acid

binding ligands, Biochemistry 38 (1999) 16067e16075.

[92] X. Qu, J.B. Chaires, Analysis of drug-DNA binding data, Methods En-

zymol. 321 (2000) 353e369.

[93] I. Haq, T.C. Jenkins, B.Z. Chowdhry, J. Ren, J.B. Chaires, Parsing free

energies of drugeDNA interactions, Methods Enzymol. 323 (2000)

373e405.

[94] J. Ren, X. Qu, N. Dattagupta, J.B. Chaires, Molecular recognition of

a RNA:DNA hybrid structure, J. Am. Chem. Soc. 123 (2001) 6742e6743.

[95] X. Shi, J.B. Chaires, Sequence- and structural-selective nucleic acid

binding revealed by the melting of mixtures, Nucleic Acids Res. 34

(2006) e14.


[96] J.P. McDonnell, A.C. Garapin, W.E. Levinson, N. Quintrell, L. Fanshier,

J.M. Bishop, DNA polymerases of Rous sarcoma virus: delineation of

two reactions with actinomycin, Nature 228 (1970) 433e435.

[97] W.E. Muller, R.K. Zahn, H.J. Seidel, Inhibitors acting on nucleic acid syn-

thesis in an oncogenic RNA virus, Nat. New Biol. 232 (1971) 143e145.

[98] W. Chu, K. Shigehiro, M. Shinomiya, R.G. Carlson, F. Takusagawa, To-

ward the design of an RNA:DNA hybrid binding agent, J. Am. Chem.

Soc. 116 (1994) 2243e2253.

[99] F. Takusagawa, K.T. Takusagawa, R.G. Carlson, R.F. Weaver, Selectiv-

ity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia

activity, Bioorg. Med. Chem. 5 (1997) 1197e1207.

[100] D.P. Arya, R.L. Coffee Jr., I. Charles, Neomycin-induced hybrid triplex

formation, J. Am. Chem. Soc. 123 (2001) 11093e11094.

[101] Q. Chen, R.H. Shafer, I.D. Kuntz, Structure-based discovery of ligands

targeted to the RNA double helix, Biochemistry 36 (1997) 11402e11407.

[102] D.P. Arya, L. Xue, B. Willis, Aminoglycoside (neomycin) preference is

for A-form nucleic acids, not just RNA: results from a competition di-

alysis study, J. Am. Chem. Soc. 125 (2003) 10148e10149.

[103] D.P. Arya, Aminoglycoside Antibiotics: From Chemical Biology to

Drug Discovery, Wiley Series in Drug Discovery and Development,

John Wiley & Sons, Inc., Hoboken, NJ, 2007.

[104] C.M. Barbieri, T.K. Li, S. Guo, G. Wang, A.J. Shallop, W. Pan, G. Yang,

B.L. Gaffney, R.A. Jones, D.S. Pilch, Aminoglycoside complexation

with a DNA.RNA hybrid duplex: the thermodynamics of recognition

and inhibition of RNA processing enzymes, J. Am. Chem. Soc. 125

(2003) 6469e6477.

[105] W.H. Gmeiner, W. Cui, S. Sharma, A.M. Soto, L.A. Marky, J.W. Lown,

Shape-selective binding of geometrically-constrained bis-distamycins to

a DNA duplex and a model Okazaki fragment of identical sequence,

Nucleosides Nucleotides Nucleic Acids 19 (2000) 1365e1379.

[106] W.H. Gmeiner, W. Cui, D.E. Konerding, P.A. Keifer, S.K. Sharma,

A.M. Soto, L.A. Marky, J.W. Lown, Shape-selective recognition of

a model Okazaki fragment by geometrically-constrained bis-distamy-

cins, J. Biomol. Struct. Dyn. 17 (1999) 507e518.

[107] C.L. Kielkopf, S. White, J.W. Szewczyk, J.M. Turner, E.E. Baird,

P.B. Dervan, D.C. Rees, A structural basis for recognition of A.T and

T.A base pairs in the minor groove of B-DNA, Science 282 (1998)

111e115.

[108] N. Neamati, A. Mazumder, S. Sunder, J.M. Owen, M. Tandon,

J.W. Lown, Y. Pommier, Highly potent synthetic polyamides, bisdista-

mycins, and lexitropsins as inhibitors of human immunodeficiency virus

type 1 integrase, Mol. Pharmacol. 54 (1998) 280e290.

[109] N. Shaw, H. Xi, D.P. Arya, Molecular recognition of a DNA:RNA hy-

brid: sub-nanomolar binding by a neomycin-methidium conjugate, Bio-

org. Med. Chem. Lett. (in press).

[110] S. Volkmann, J. Jendis, A. Frauendorf, K. Moelling, Inhibition of HIV-1

reverse transcription by triple-helix forming oligonucleotides with viral

RNA, Nucleic Acids Res. 23 (1995) 1204e1212.

[111] Z. Xu, D.S. Pilch, A.R. Srinivasan, W.K. Olson, N.E. Geacintov,

K.J. Breslauer, Modulation of nucleic acid structure by ligand binding:

induction of a DNA.RNA.DNA hybrid triplex by DAPI intercalation,

Bioorg. Med. Chem. 5 (1997) 1137e1147.

[112] J.Q. Hang, Y. Li, Y. Yang, N. Cammack, T. Mirzadegan, K. Klumpp,

Substrate-dependent inhibition or stimulation of HIV RNase H activity

by non-nucleoside reverse transcriptase inhibitors (NNRTIs), Biochem.

Biophys. Res. Commun. 352 (2007) 341e350.

[113] C. West, R. Francis, S.H. Friedman, Small molecule/nucleic acid affin-

ity chromatography: application for the identification of telomerase in-

hibitors which target its key RNA/DNA heteroduplex, Bioorg. Med.

Chem. Lett. 11 (2001) 2727e2730.

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