Quick viewing(Text Mode)

Recognition of the Unique Structure of DNA:RNA Hybrids

Recognition of the Unique Structure of DNA:RNA Hybrids

Available online at www.sciencedirect.com

Biochimie 90 (2008) 1026e1039 www.elsevier.com/locate/biochi Review Recognition of the unique of DNA:RNA hybrids

Nicholas N. Shaw, Dev P. Arya*

Laboratory of Medicinal , 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 using small routinely uses the end products in the conversion pathway of ‘‘DNA to RNA, RNA to ’’. However, the intermediate processes in this path have not always been targeted. The DNAeRNA interaction, specifically DNA:RNA hybrid formation, provides a unique target for controlling the transfer of genetic through binding by small molecules. Not only do DNA:RNA hybrids differ in conformation from widely targeted DNA and RNA, the low occurrence within biological systems further validates their 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 in ligand targeting, are discussed. Ó 2008 Published by Elsevier Masson SAS.

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

1. History as a product of hybridization between a single-stranded DNA template strand and a newly formed RNA strand, was also Six years after the report on the double-helical structure of hypothesized. Experimental data supported this hypothesis in DNA by Watson and Crick [1], the DNA:RNA hybrid was 1960 [11]. This was 10 years before Temin [12] and Baltimore proposed to address the interaction between DNA and RNA [13], independently, yet simultaneously, reported the use of [2]; a wonderful retrospective account can be found in the in the synthesis of a new DNA strand, following reference [3]. The first synthetic DNA:RNA hybrid via RNA template strand. Originally proposed by Rich [2], structure was formed in 1960 through the reaction of oligo- this became known as reverse , the second deoxythymidylic with polyriboadenylic acid [4].A example of DNA:RNA hybrid formation. In 1975, the first mere year later, the first DNA:RNA hybrid was formed example of DNA:RNA hybrid formation in the replication of through the novel annealing of a RNA strand, with a comple- DNA was discovered [14]. Preceded by their biological signif- mentary DNA strand [5,6]. X-ray fiber diffraction studies first icance, DNA:RNA hybrids as therapeutic targets logically appeared in 1967 [7], and confirmed CD spectroscopy obser- followed. vations that the conformation of the DNA:RNA hybrid was different from B-form DNA. The crystal structure of 2. Importance a DNA:RNA hybrid was solved in 1982 [8], while solution based conformational analysis, first used to study a DNA:RNA 2.1. Transcription [15,16] hexamer [9] was followed shortly by polymeric DNA:RNA hybrids in 1985 [10]. The DNA mediated synthesis of RNA is a finite process In the initial report where the presence of DNA:RNA which initiates at sites within the DNA template, hybrids was suggested [2], the mechanism of their formation, proceeds in the 50 to 30 direction and terminates at sites within the DNA template. RNA binds to the * Corresponding author. Tel.: þ1 864 6561106. initiation site, unwinds a helical turn of duplex DNA and be- E-mail address: [email protected] (D.P. Arya). gins to incorporate ribonulecotides sequentially to the 30 end

0300-9084/$ - see front Ó 2008 Published by Elsevier Masson SAS. doi:10.1016/j.biochi.2008.04.011 N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039 1027 of the growing RNA strand. Ribonuclotide selection involves in the replication of DNA can be found in as the interaction of the incoming base with the template DNA well [33]. strand and the ability of the base to fit the of the en- zyme [17]. Termination is less well understood; the termina- 2.5. DNA:RNA hybrids of therapeutic interest tion process involves (i) cessation of RNA chain growth, (ii) the release of the RNA strand from the DNA template and Despite reports of the existence of DNA:RNA hybrids of (iii) the release of RNA polymerase from the DNA [18].It potential therapeutic importance (reports of the persistent ex- has been suggested that termination concludes with the release istence of DNA:RNA hybrids in cytomegalovirus of the RNA strand from an unstable DNA:RNA hybrid [19]. have been made [34]); extensive studies reporting the targeting Furthermore, the lability of some hybrid sequences may aid of these DNA:RNA hybrids as therapeutic strategies have not the dissociation of the RNA strand from the DNA template yet been examined, with the following notable exceptions. [20,21]. The reverse transcription of HIV-1 has attracted substantial attention [27,35]. HIV-1 contains two identical polypurine 2.2. Reverse transcription tracts (PPTs). The first PPT only contains two ba- ses and extends to 25 bases from the 30 The RNA mediated synthesis of DNA was originally pro- end of the viral RNA. The second PPT lies further towards posed [22] and substantiated [23] as an integral part of the ret- the 50 end of the viral RNA and closer to the primer binding roviral cycle. Expanded to eukaryotic cells [25], as much site used by tRNA to initiate minus strand DNA synthesis. as 10% of the eukaryotic is a direct product of reverse Both of these PPTs are identical and 18 in length transcription [24]. Mechanistically similar to the transcription [36]. Reverse transcription (minus strand DNA synthesis) of process, reverse transcription relies on the dual activity of re- both PPTs occurs after DNA strand transfer by reverse tran- verse transcriptase (RT) [24,26]. DNA:RNA hybrid formation scriptase, providing a unique r(Pu):d(Py) type DNA:RNA hy- can be found following RT mediated DNA synthesis and prior brid [26]. This unique PPT DNA:RNA hybrid exists until to RT mediated RNase H cleavage of the template RNA cleavage of the RNA template strand (50 to the PPT) and cleav- strand. Since reverse transcription relies on the dual role of age of the tRNA and PPT. Targeting of DNA:RNA hybrids are RT, the intermediate DNA:RNA hybrid form provides an en- discussed in subsequent sections of the manuscript. ticing target for targeting by small molecules, prior to reattach- activity, seemingly synonymous with G-quad- ment and subsequent cleavage by RT. raplex formation, is a well understood process, of which DNA:RNA hybrid targeting is an equally viable approach 2.3. DNA replication [27] for telomerase inhibition. Telomerase activity is associated with cellular [37] and its potential as a universal The discussion of DNA replication may seem unnecessary target has been proposed [38,39]. exist as when addressing DNA:RNA hybrid duplexes, however a protein coupled DNA complex with long repeats of a simple DNA:RNA hybrids play an important role in DNA replication. 6 base sequence, d(TTAGGG)n which becomes shortened with DNA replication begins with the unwinding of duplex DNA each cellular division [40].The30 tail of the parent DNA telo- by DNA followed by priming of the DNA strands mere is longer than its compliment and forms a DNA:RNA hy- by and [18] and proceeds in: (1) a heavy brid upon binding to the active site of telomerase. Binding of 0 0 (H)-strand origin (OH), 5 to 3 , leading strand synthesis and small molecules to the DNA:RNA hybrid can potentially offer 0 0 (2) a light (L)-strand (OL), 3 to 5 , which begins replication a therapeutic method for controlling telomerase activity, pre- upon exposure to the single-stranded region of DNA [28], lag- venting extension, by distorting the substrate/ ging strand synthesis. Lagging strand synthesis utilizes Oka- interaction, or preventing dissociation of the enzyme from the zaki fragments [29]. This process is repeated, forming substrate [41]. DNA:RNA hybrids every 100e200 nucleotides on the DNA lagging strand. The DNA polymerase deletes the RNA primers 3. Stability and conformation which are then replaced by DNA using DNA . 3.1. Stability 2.4. Mitochondrial DNA DNA:RNA hybrid composition can be isolated to homopurine Human mitochondrial DNA (mtDNA) is present in the mi- and homopyrimide strands, which gives rise to deoxyribo(pyrimi- tochondria of the . Circular in conformation, mtDNA en- dine):ribo(), d(Py):r(Pu), and deoxyribo(purine):ribo(pyri- for the remaining machinery of protein synthesis not midine), d(Pu):r(Py) type hybrids. The designation of pyrimidine encoded for in nuclear DNA [27]. The mechanism of replica- (Py) and purine (Pu) bases is consistent through this manuscript, tion is similar to that of nuclear DNA, however, the priming of analogous to (Y) and (R), found in other accounts, respectively. mtDNA for DNA replication requires the sole use of RNA Homopurine:homopyrimidine and oligomeric primers [30]. DNA:RNA hybrids are prerequisite to the initia- decrease in stability in the order of r(Pu):r(Py) > r(Pu): tion of transcription and serve as primers for DNA replication d(Py) > d(Py):d(Pu) > r(Py):d(Pu), as suggested by Hung et al. of the leading strand [18,31,32]. Similar use of RNA primers [42,43]. However, the introduction of long stretches of 1028 N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039

(A)n:(Tor U)n reverses the trend, an observation made by Dubins et al. [44]. The polymeric duplexes containing stretches of (A)n:(Tor U)n and at least one homoribonucleic acid strand follow the general hierarchy for stability DNA:DNA > DNA:RNA type d(Py):r(Pu) > RNA:RNA > DNA:RNA type d(Pu):r(Py). Oligmeric DNA:RNA hybrids containing homopurine and homopyrimidine tracts, stabilized by a single GeC at 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 and McLaughlin [46] demonstrate that of mixed base sequences, not isolated to purine or pyrimidine strands, show exceptions to the previously described trends. In these excep- Fig. 1. A and B form nucleic acids. tions, homoduplexes, d(Pu):d(Py) and r(Pu):r(Py), are more stable than heteroduplexes, d(Pu):r(Py) and d(Py):r(Pu). 3.2.1. Polymeric DNA:RNA hybrids An extensive survey and review of DNA:RNA hybrid sta- The first DNA:RNA hybrid structure [7] resisted conforma- bility while varying (1) the percentage of (A)n:(T or U)n con- tional changes upon relative humidity changes, a technique tent, (2) oligomeric length and (3) the percentage of d(Py) commonly used to drive the A- to B- transition in duplex content in each of the strands, offers the most complete anal- DNA. The observation, by Milman et al. [7], that DNA:RNA ysis of hybrid stability [47]. Lesnik and Freier demonstrate hybrids resembled A-form DNA while differing from the that in general: (i) an inverse relationship is displayed between RNA duplex, coupled with solution studies of DNA:RNA hy- the percentages of (A)n:(T or U)n content in the hybrid duplex brids [50,51], spurned a 20 year debate on the conformation and the thermal stability. (ii) Hybrid duplexes of equal d(Py) and structure of DNA:RNA hybrids. X-ray fiber diffraction and d(Pu) content are thermally similar when comparing the studies of poly(rA):poly(dT) suggest a structure similar to d(Pu):r(Py) and d(Py):r(Pu) forms. (iii) Decreasing oligomeric that observed for B-form DNA duplexes when the hybrid length decreases the thermal stability. (iv) Isolation of (A)n: was in a highly solvated state [7] suggest a structure similar (T or U)n content to continuous tracts results in a lower ther- to that observed for B-form DNA duplexes. Parallel circular mally stable duplex than when the same percentage of (A)n: dichorism (CD) [52] and Raman [53] experiments refute (Tor U)n content is dispersed throughout the sequence. (v) Con- a complete B-form model for the DNA:RNA hybrid. Further 31 tinuous tract (A)n:(T or U)n content results in d(Py):r(Pu) analysis by P solid state NMR suggests poly(rA):poly(dT) thermal stability lower than the corresponding d(Pu):r(Py) is capable of existing in an A-like conformation at relatively form, at physiological conditions, as expected. low humidity [54] while increasing the from 87% It has also been suggested that DNA:RNA hybrid stability to 92% yields a B-like conformation [55]. Equilibrium Raman is related to the ability of the hybrid duplex to associate spectroscopy further clarifies the conformational differences with a third single strand [43,48]. The d(Py):r(Pu) hybrid are a result of differences in furanose conformation, does not associate with a third strand of DNA, while unique to each respective and strand d(Pu):r(Py) hybrids easily accommodate a third strand to [56], of which the global B-like conformation is dictated by form d(Pu):r(Py):d(Pu) [43]. Furthermore, with the exception the burial of the methyl [48] in the d(Py) strand of long stretches of (A)n:(T or U)n, the stability of duplexes [57]. Steely et al. suggest [10] that ‘the d(Py) strand contrib- can also be attributed to the relative stability of the ribose utes to the overall B-like conformation and concurrently stabi- chain. Unfavorable van der Waals contacts [49] have been ob- lizes the duplex’. Currently, it is generally accepted that the served between the sugar and pyrimidine nucleotides, result- conformation of poly(rA):poly(dT) is polymorphic, capable ing in the expectation that within a double helix, of undergoing an A- to B- type transition upon relative humid- a pyrimidine is less stable that a purine nucleotide. ity changes [57,58]. Polymorphic structures, type d(Pu):r(Py), poly(dA): 3.2. Conformation poly(rU) and poly(dI):poly(rC) further broaden the conforma- tional spectrum in which DNA:RNA hybrid structures exist. For the sake of this discussion, we examine DNA:RNA hy- The model for poly(dA):poly(rU) suggests a ‘heteromerous’ brid conformation by individually focusing on DNA:RNA hy- structure [59]. This term was used to describe the overall con- brids of polymeric length as well as DNA:RNA hybrids of formation of the duplex in which the one strand, the d(Pu) oligomeric length, further defined by homo ribose:deoxyribose strand, displays B-form characteristics while the other strand, oligomers and chimeric oligomers. For comparison purposes, the r(Py) strand, displays A-form characteristics. The global the conformation of various DNA:RNA hybrids will be com- conformation of poly(dA):poly(rU) is strongly driven by the pared to conformationally distinct A and B form helices r(Py) strand and the global conformation of the hybrid is an (Fig. 1). A-type duplex closer in conformation to the A-form of RNA N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039 1029

[48]. Congruently, the analogous DNA:RNA hybrid, poly(dI): DNA:RNA hybrid of 33% purine content in the DNA strand poly(rC) displayed similar ‘heteromerous’ conformation [69,70]. Increasing DNA purine content increases the debate [48,49]. However, the r(Py) has a diminutive effect on the in the conformational contributions from the respective overall conformation of the poly(rI):poly(dC) duplex, when DNA:RNA strands. One report suggests the DNA strand con- compared to poly(dA):poly(rU) [59]. tributes B-form characteristics [71], while other reports sug- Mixed base polymeric DNA:RNA hybrids poly(dAC):poly gest the DNA strand contributes A-form and B-form (rGU) and poly(dGU):poly(rAC) have also been extensively characteristics, a result of homologous sugar pucker studied by Gray [52]. Poly(dAC):poly(rGU) and poly(dGU): [70,72,73] or distribution of independent sugar pucker in the poly(rAC) are more similar to the analogous A-form RNA DNA strand [74]. duplex. Furthermore, conformational differences between the individual heteroduplexes mirror differences observed in the 3.2.3. Chimeric DNA:RNA hybrids polymeric analogs poly(dA):poly(rU) and poly(rA):poly(dT). Chimeric DNA:RNA hybrids are formed when DNA:RNA The authors suggest poly(dGU):poly(rAC) is more A-like hybrid helix is joined to double-helical DNA. Studies on than poly(dAC):poly(rGU). These duplexes are also suscepti- dGi:rCkdCn confirmed the presence of a bend joining two dis- ble to dehydration driving the conformation to complete A- tinctly unique conformations within the polymer [75,76]. form conformation. Crystallized in 1982, the [r(GCG)d(TATACGC)]2 [8] suggests the global conformation adopted by the duplex 3.2.2. Oligomeric DNA:RNA hybrids is close to the A-form RNA double helix, although discrep- Pardi et al. used seven-mer oligomeric DNA:RNA hybrids ancies exist [8,77]. Subsequent conformational treatments re- containing an A5:(T or U)5 stretch, sealed by terminal G-C vealed that chimeric hybrids are polymorphic with base pairs as the first instance of oligomeric studies of conformationally distinct contributions from each portion DNA:RNA hybrids by NMR [60]. The authors suggest [78]. Furthermore, the RNA influence from the ribonucleic DNA:RNA hybrid conformation belonging to the A-form, is strand in DNA:RNA hybrids is greatly diminished in chimeric driven by the A-form conformation of the RNA strand, while duplexes [79]. Solution studies on chimeras reveal that regard- the DNA strand is predominantly B-form. Unfortunately, par- less of flanking DNA:RNA hybrids with DNA duplexes or the tial triplex formation of d(CA5G):r(GU5C) precludes the as- contrary flanking of a central core DNA by DNA:RNA hy- signment of this hybrid to A-form or B-form. brids, the portion of the hybrid retains A-form characteristics Oligomeric DNA:RNA hybrids, restricted in base composi- that lie closer to B-form, while the DNA portion of the duplex tion to homopurine and homopyrimidine strands, have been exists as predominant B-form [80e82]. Even single junction studied by both solution [61e63] and crystal [64e66] tech- hybrids composed of a DNA duplex and DNA:RNA hybrid niques. Solution studies suggest a ‘heteromerous’ duplex con- duplex displayed the dual conformation characterized by taining A-form conformation in the RNA strand while the a DNA:RNA hybrid:DNA bend [83e85]. DNA strand conformation was closer to B-form [63].How- ever, crystal structure observations [64e66] seemingly dispel 3.3. Sugar conformation the notion that the conformation is ‘heteromerous’ by suggest- ing the conformation is homogeneous and closer to A-form. The ribose sugar pucker is a major contributor to the helical The most complete analysis of the conformational relation- parameters of nucleic acids. Ribose sugar pucker of ribonu- ships between homopyrimidine and homopurine DNA:RNA cleic acids are generally found in the C30-endo (‘north’ or hybrids and their corresponding DNA and RNA duplexes equivalent C20-exo) conformation, an accommodation made 0 0 0 0 was made comparing duplexes 5 -(GAAG)3-3 and 5 - by the sugar to facilitate the 2 -hydroxyl [55]. The C30-endo ri- 0 (CTTC)3-3 [67]; thymidine bases were replaced by in bose drives the conformation of homoribonucleic strands to- ribose strands. The DNA:RNA hybrid, d(Pu):r(Py) is closer wards A-form, affecting most helical parameters, giving rise in global conformation to B-form than d(Py):r(Pu); in fact to the ‘heteromerous’ of DNA:RNA hybrids. Deoxyri- the CD spectra closely resembles the CD spectra of bose conformation is much more accommodating to a number d(Py):d(Pu). The opposite was true for the d(Py):r(Pu) hybrid; of different conformations and generally assumes a C20-endo the CD closely resembles the duplex RNA analog r(Py):r(Pu), (‘south’ or equivalent C30-exo) conformation. This conforma- which further perpetuates the idea that d(Py):r(Pu) oligomeric tion is characteristic of B-form. Another sugar pucker, an in- hybrids are closer to A-form than analogous d(Py):r(Pu) du- termediate between the north and south conformations, is plexes. In fact, the authors suggest predictions of the global C40-endo and arises at various points in the subsequent discus- conformation of duplexes can be made based on whether or sion on the effect of sugar pucker on DNA:RNA hybrids heli- not the hybrid duplex contains a DNA or RNA purine strand. cal parameters (see Figs. 2 and 3). A survey of mixed base DNA:RNA hybrids suggests the conformation varies depending upon the percentage of purine 3.3.1. Polymeric DNA:RNA hybrids content in the DNA strand. Fedoroff et al. utilized a mixed Polymeric DNA:RNA hybrids generally follow two trends. base DNA:RNA hybrid and described its conformation as an Refinement of the secondary structure of poly(rA):poly(dT) intermediate between A-form and B-form [68]. The ‘hetero- suggests the sugar puckers, regardless of strand, are a slight merous’ nature of DNA:RNA hybrids reprises in the variant of the C20-endo conformation [10,54]. However, Gupta 1030 N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039

Fig. 2. Sugar conformations found in DNA:RNA hybrids. Adapted from ‘Principles of Structure’, Wolfram Saenger, p. 18. and coworkers make the case that ‘in order to facilitate the 20- 3.3.2. Oligomeric DNA:RNA hybrids hydroxyl, a displacement of the RNA bases from the helical Oligomeric DNA:RNA hybrids generally reflect the trends axis is observed. This displacement results in a concomitant observed in polymeric hybrids. However, discrepancies arise change in the tilt and twist of these bases as well as a change in the conformation of the sugar pucker. C30-endo sugar pucker in a backbone torsion angle [10].’ However, there exists is generally observed in the RNA strand, while variation in the a ‘high energy barrier’ through which the ribose sugar of interpretation of the deoxyribose conformation as an equilib- RNA must pass in order to convert to C20-endo, a barrier sig- rium between C30-endo/C20-endo or alternatively interpreted nificantly lower for DNA ribose [86]. Dehydration converts as C40-endo [73,74]. Further assessment of sugar pucker sug- the global ribose sugar pucker to C30-endo and results in pa- gests that the flexible DNA strand exists with a fraction of de- rameters closer in agreement to A-form [55]. Unlike the oxyribose in the C30-endo form, increasing in the order case of d(Py):r(Pu), in which global conformational changes of DNA:DNA duplexes, d(Pu):r(Py) and d(Py):r(Pu), which can be accommodated in both strands of the duplex, may be attributed to oligomeric d(Py):r(Pu) type hybrids bear- d(Pu):r(Py) type polymeric duplexes are not as forgiving. ing more A-form characteristics [61,62]. Mixed purine/pyrim- Driven strongly by the conformational stubbornness of the idine bases tend to assume the same general trends as observed r(Py) strand to exist in an A-form state with C30-endo sugar in homopurine/homopyrimidine hybrids. Fedoroff et al. pro- pucker, helical parameters tend to be more A-form in charac- pose ‘DNA:RNA structure parameters (roll and tip) can be at- teristic, with deviations arising from the d(Pu) strand tributed to DNA strand propensity to roll about the long base [59,62,63]. pair axis while RNA susceptibility to rotate along the short base pair axis affects parameters such as tilt and inclination [74].’ Also, interpretation of DNA:RNA hybrid sugar pucker as global C30-endo has been reported [64,65,87].

3.3.3. Chimeric DNA:RNA hybrids Chimeric DNA:RNA hybrids are structurally unique, as al- ready discussed. The extent of sugar pucker on the overall structure 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-endo conformation exclusively [81]. DNA duplexes flanked by DNA:RNA hybrids have been suggested to have a global sugar pucker of C 0-endo with a conformation characteristic of Fig. 3. Definition of helical parameters pitch (P), axial rise per residue (h), 3 x-displacement (x-disp.), inclination (inc.) and residues per turn (shown with A-form [88,89]. However, more recent studies have demon- n ¼ 6 residues per turn), found in Table 1, adapted from ‘Principles of Nucleic strated that DNA residues adopt a conformation close in agree- Acid Structure’, Wolfram Saenger, p. 25. ment to C40-endo [83], as alluded to by Mellema et al. [79]. N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039 1031

4. Targeting DNA:RNA hybrids ligand [91]. An ensuing report assayed 85 ligands against var- ious nucleic acids and focused on binding to poly(rA):po- Although polymorphic in structure, as demonstrated in pre- ly(dT). This confirmed the presence of five ligands: vious sections, DNA:RNA hybrids offer a unique structure to , ethidium bromide, coralyne, propidium and target and potentially inhibit biological functions. However, TAS103, which recognized the poly(rA):poly(dT) structure. a survey of the literature reveals the existence of less than Of these ligands, ethidium bromide showed the highest prefer- ten ligands which specifically bind DNA:RNA hybrids. In ence for poly(rA):poly(dT). Complete thermodynamic profiles the course of this section, the binding of these ligands to for ellipticine, propidium and ethidium bromide binding to DNA:RNA hybrids is examined. Attention is paid to ligand poly(rA):poly(dT) yield binding constants for the three ligands binding class as well as targeting biologically significant (see Table 2). Inasmuch, an inverse relationship between the DNA:RNA hybrids. Structures, targets, binding constants IC50 (inhibitory concentration giving 50% inhibition of poly and buffer conditions can be found in Table 2. (rA):poly(dT) degradation by RNase H) and the observed ligand binding constant [92e94] is observed. This assay was 4.1. Ligand classes expanded to simultaneously provide the thermal stabilization afforded by the ligand on the preferentially targeted nucleic An examination of various ligand binding modes to acid structure, while in competition with other nucleic acid DNA:RNA hybrids is conducted in the subsequent section. In- structures [95]. Ethidium bromide competition dialysis find- terestingly enough, DNA:RNA binding by small molecules ings were corroborated in this mixed experiment. has been achieved via all available modes: intercalation, Actinomycin D (AMD) has demonstrated the ability to in- groove binding as well as dual recognition. It should be noted hibit reverse transcriptase activity [96,97] through the inhibi- that although the binding of antisense to tion of double-stranded DNA synthesis [96]. Comprised of mRNA forms DNA:RNA hybrids, this therapeutic approach an intercalating phenoxazome ring, substitution for the targets single-stranded RNA templates and is excluded from at the 8-position of the planar ring system by in- the following discussion, however, the use of antisense oligo- creases the selectivity of the compound for DNA:RNA hybrids nucleotides is addressed in the section covering the PPT of [98]. Unfortunately, affinity of N8AMD (Table 2) for the HIV-1. DNA:RNA hybrid is slightly lower than the . Of note, the authors suggest that AMD actually binds 4.1.1. Intercalation DNA:RNA hybrids, structures previously believed to be un- Using a technique developed by Muller and Crothers [90], able to bind AMD. However, the preference of AMD for Chaires et al. identified the intercalator, ethidium bromide, as DNA duplexes dominates. Similarly, substitution at the sp2 a DNA:RNA hybrid, poly(rA):poly(dT), preferential binding carbon at position 8, by fluorine increases the preference of

Table 1 Helical parameters displayed by a wide array of DNA:RNA hybrid type duplexes Helix Pitch Axial Residues Inclination ()Dx(A˚ )a Ref. type height (A˚ ) rise/residue (A˚ ) per turn B-DNA B 33.7 3.37 10 5.9 e [59,87,88] Polymeric DNA:RNA hybrids poly(rA):poly(dT) Hydrated B 33.7 3.46 9.7 ee[55] 79% rel. humidity A 36.0 3.03 11.9 ee[55] poly(rI):poly(dC) A 35.6 2.97 12 þ5.68 e [49] poly(dI):poly(rC) A 31.3 e 10 þ15 e [59] poly(dA):poly(rU) A 33.7 e 11 þ13 e [59] Oligomeric DNA:RNA hybrids d(CTCTTCTTC):r(GAAGAAGAG) A 34.2 2.9 11.8 þ7.9 4.0 [65] d(GTGAACTT):r(AAGUUCAC) A e 3.2 11 þ17 4.5 [71] d(CAGTCCTC):r(GAGGACUG)b A e 3.2 10.9 þ6.8 3.7 [68] d(CGCGCCCCAA):r(UUCGGGCGCC) A e 2.9 10.9 þ13.9 3.3 [72] Chimeric DNA:RNA hybrids

[d(CGC)r(AAA)-d(TTTGCG)]2 A/B e 3.1 11 1.3 1.8 [81] r(GCG)d(TATACGC) A/B 33 2.6 10.9 þ20 2.0 [8] A-DNA A 28.2 2.56 11.0 þ20.0 e [88] A-RNA A 30.0 2.73 11.0 þ17.0 e [89] Adapted and expanded from ‘Principles of ’, Wolfram Saenger, p. 235. a x-displacement. b Average values. Table 2 1032 Structures, targets, binding constants and buffer conditions for ligands and their respective DNA:RNA hybrid targets

Structure Target Ka Ref.

- + 5 Ethidium bromide Br N poly(rA):poly(dT): 7 mM Na2HPO4, 2 mM NaH2PO4, 1.1 10 ; [91] 180 mM NaCl, 1 mM EDTA, pH 7.0e7.1 fluorescence poly(dA):poly(rU): 10 mM sodium calcodylate, 9.3 106; [109] H N NH 2 2 0.5 mM EDTA, 100 mM NaCl, pH 5.5 calorimetric

N+ ..Sa,DP ra/Bohme9 20)1026 (2008) 90 Biochimie / Arya D.P. Shaw, N.N. 5 Propidium iodide N+ . poly(rA):poly(dT): 7 mM Na HPO ,2mM 3.3 10 ; [91] 2 I- 2 4 NaH2PO4, 180 mM NaCl, 1 mM EDTA, pH 7.0e7.1 fluorescence

H2N NH2

CH3

N Ellipticine poly(rA):poly(dT): 10 mM sodium calcodylate, 4.4 105; [91] 0.5 mM EDTA, 100 mM NaCl fluorescence N

CH3

N N R R1 1 e 1039 X N NH2 R2

O O d(GGAGCAGGAC):r(CUCCUGCUCC): 20 mM 3.4 104; [98] , 300 mM NaCl, pH 7.0 UV-spectroscopic CH3 CH3

R = -Thr- Val-Pro-Sar-MeVal 1 D O

R1 = C; X = H (AMD)

0 0 a 5 R1 ¼ C; X ¼ H (AMD) 5 -AUAUAUGCAUAUTTTTTATATGCATATAT-3 : 2.6 10 ; [99] 50 mM TriseHCl, 10 mM MgCl2, 10 mM KCl, pH 7.5 UV-spectroscopic 4 R1 ¼ N (N8AMD) d(GGAGCAGGAC):r(CUCCUGCUCC); 20 mM 1.6 10 ; [98] phosphate, 300 mM NaCl, pH 7.0 UV-spectroscopic 0 0 a 5 R1 ¼ C; X ¼ F (F8AMD) 5 -AUAUAUGCAUAUTTTTTATATGCATATAT-3 : 1.4 10 ; [99] 50 mM TriseHCl, 10 mM MgCl2, 10 mM KCl, pH 7.5 UV-spectroscopic

O R H N R = H X N a 7 N H d(CAAAGATTCCTC):(GTTTCTAAGGAG): 200 mM 5.4 10 ; [105] O N N H TriseHCl, 50 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, pH 7.6 electrophoretic R O N N N O O X = CH (MT-9)

X ¼ N (MT-15) d(CAAAGATTCCTC):(GTTTCTAAGGAG):a 200 mM w106; [105] TriseHCl, 50 mM MgCl , 1 mM DTT, 0.1 mM EDTA, pH 7.6 electrophoretic

2 1026 (2008) 90 Biochimie / Arya D.P. Shaw, N.N.

OH

O HO HO H2N H2N O NH2 HO O O OH Paramomycin d(GCCACTGC):r(GCAGUGGC): 10 mM sodium calcodylate, [104] 0.1 mM EDTA, total Naþ 60 mM (pH 6.0) 4.3 106 NH 6 2 (pH 7.0) 1.2 10 ; O OH calorimetric 0 0 0 5 5 -CGGGCGCCACTGCTAGAG-3 ;3- 4.2 10 ; [26] e O 1039 GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS, 0.1 mM calorimetric H N OH 2 EDTA, total Naþ 100 mM, pH 7.5 OH 50-CGGGCGCCACTGCTAGAG-30;30- 1.8 105; [26] GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS, 0.1 mM calorimetric EDTA, total Naþ 100 mM, pH 7.5

NH2

O HO HO H2N H2N O 10 Ribostamycin NH2 50-CGGGCGCCACTGCTAGAG-30;30- 1.1 10 ; [26] HO O 0 a O OH GCCCGCGGUGACGAUCUC-5 : 10 mM EPPS, 0.1 mM calorimetric EDTA, total Naþ 100 mM, pH 7.5 50-CGGGCGCCACTGCTAGAG-30;30- 1.5 104; [26] GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS, 0.1 mM calorimetric þ OH OH EDTA, total Na 100 mM, pH 7.5 (continued on next page) 1033 Table 2 (continued) 1034

Structure Target Ka Ref.

NH2

O HO HO H2N H N 2 O NH2 HO O O OH Neomycin 50-CGGGCGCCACTGCTAGAG-30;30- 1.1 106; [26] GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS, calorimetric 0.1 mM EDTA, total Naþ 100 mM, pH 7.5 5 NH2 50-CGGGCGCCACTGCTAGAG-30;30- 1.4 10 ; [26] O OH GCCCGCGGUGACGAUCUC-50:a 10 mM EPPS, calorimetric 0.1 mM EDTA, total Naþ 100 mM, pH 7.5 O 6 b poly(rA):poly(dT): 10 mM sodium calcodylate, 1.7 10 ; 1026 (2008) 90 Biochimie / Arya D.P. Shaw, N.N. H N OH 2 0.5 mM EDTA, 100 mM NaCl, pH 6.8 calorimetric/UV OH poly(dA):poly(rU): 10 mM sodium calcodylate, 9.9 106; [109] 0.5 mM EDTA, 100 mM NaCl, pH 5.5 calorimetric/UV

NH2 HO O O H HO H2N H N N 2 O NH NM S O 2 poly(rA):poly(dT): 10 mM sodium calcodylate, 1.9 108; b O OH 0.5 mM EDTA, 100 mM NaCl, pH 6.8 calorimetric/UV poly(dA):poly(rU): 10 mM sodium calcodylate, 4.8 1010; [109] NH N+ O OH 2 0.5 mM EDTA, 100 mM NaCl, pH 5.5 calorimetric/UV

O OH H N NH H2N 2 2 OH a In all cases, bold denotes RNA nucleotides. Buffer conditions and method of binding constant determination are noted throughout.

b e

Unpublished results. 1039 N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039 1035 the ligand for DNA:RNA hybrids, although not notably higher chloride conjugate, NM (see Table 2). Competition melting than N8AMD. The binding constant of F8AMD towards a - experiments and competition dialysis experiments revealed pin DNA:RNA hybrid, 50-aua uau gca uau-TTT TTA TAT a preferential binding of NM to DNA:RNA hybrid structures, GCA TAT AT-30, containing a five loop (RNA bases of which d(Pu):r(Py) type, poly(dA):poly(rU), was preferred denoted in lowercase), is nominally lower than AMD (see above d(Py):r(Pu) type, poly(rA):poly(dT). The thermal stabil- Table 2). However, both conjugates of AMD display a unique ity afforded by NM on the preferred hybrid, poly(dA):po- selectivity for cells, inhibiting cellular growth by ly(rU), was 26 C, much greater than the stabilization 50% at less than 1.0 nM! Although the binding mode ulti- afforded by neomycin (7.8 C), ethidium bromide (1.1 C) or mately leading to retardation of leukemia is not both neomycin and ethidium bromide 7.3 C, at equivalent known, the authors hypothesize these conjugates bind ratios. DNA:RNA hybrids and validate further development [99]. Association constants, KT(25 C) values were determined uti- lizing UV, CD and calorimetry. Association constants demon- 4.1.2. Major groove binding strate NM binds poly(dA):poly(rU) at higher affinities than Aminoglycosides have been recognized as A-form nucleic neomycin or ethidium bromide. While the binding constant acid major groove binders [100e103] and shown to signifi- for neomycin closely resembles the binding of neomycin to cantly stabilize DNA:RNA hybrids [103]. Among all amino- the RNA backbone HIV-1 chimera [26], 9.93 106 M1, glycosides studied, neomycin showed the maximum and the binding of ethidium bromide was in close agreement stabilization for DNA:RNA hybrid duplexes and even induced with previously reported results [94] at 9.28 106 M1,NM the formation of hybrid triplexes [100]. Later, paramomycin, binds poly(dA):poly(rU) at a much higher association constant an aminoglycoside, and its complexation with DNA:RNA hy- of 4.77 1010 M1. The binding constant of NM far exceeds brids was studied by Barbieri et al. [104]. Thermal stabiliza- neomycin or ethidium bromide clearly demonstrating the po- tion of a mixed base RNA 8-mer duplex, afforded upon tential power of ligand conjugation. This conjugate provides paramomycin complexation, is 6.3 C, not surprising since an aminoglycoside based approach to the high affinity binding aminoglycosides traditionally bind RNA. However, thermal of DNA:RNA hybrids and is the highest binding constant ob- stabilization of the DNA:RNA hybrid analog was on par served for non- based targeting of the with the RNA duplex at 6.2 C. Furthermore, the binding of DNA:RNA hybrid. paramomycin to the hybrid duplex induces a shift in the hybrid duplex to a more A-form like conformation. Additionally, it 4.2. Targeting biologically significant DNA:RNA hybrids was found that paramomycin binds the RNA duplex at higher binding affinities than the corresponding DNA:RNA hybrid This section examines reports of ligand targeting (Table 2). Discrimination between paramomycin binding the DNA:RNA hybrids of biological significance. If one considers RNA duplex and hybrid duplex is enthalpy driven. However, the list of ligands capable of binding DNA:RNA hybrids as the difference between binding constants between the RNA short, the list of accounts focusing on ligands binding biolog- duplex and DNA:RNA hybrid is diminutive in comparison to ically significant DNA:RNA hybrids is even shorter. the analogous DNA duplex. Finally, the authors suggest the binding of paramomycin to the DNA:RNA hybrid inhibits 4.2.1. PPT of HIV-1 both RNase H- and RNase A-mediated cleavage of the RNA Targeting the polypurine tract, as previously mentioned, is strand. an attractive method for inhibition of reverse transcription of HIV-1. Formation of a triplex can be achieved through the 4.1.3. Minor groove binding use of a single-stranded compliment designed to target the Naturally occurring polyamides, distamycin and netropsin, DNA:RNA hybrid present on the PPT of HIV-1. have been investigated for their ability to bind DNA:RNA hy- formation can alternatively be formed using a duplex targeted brid [105,106]. Lexitropsins were designed at the single-stranded RNA strand of the PPT. Regardless, tri- to bind A/T stretches of DNA, displacing the spine of hydra- plex forming oligonucleotides (TFOs) prevent RNase H cleav- tion, deep in the minor groove [107]. Distamycin derivatives age of the RNA strand of the PPT and congruently inhibit were developed through various strategies [108], creating initiation of plus-strand DNA synthesis [110]. It has a number of distamycin dimers. Discussion of these com- been determined that a 25 mer TFO directed against the pounds can be found in Section 4.2.2. PPT is more inhibitory for RNA dependent DNA synthesis than antisense approaches directed at other regions of RNA 4.1.4. Dual recognition [36]. experiments indicate one TFO, forming an Recently we have attempted to exploit the unique binding RNA:RNA:DNA hybrid triplex, is efficient in inhibiting retro- characteristics displayed by ethidium bromide and neomycin replication by preventing cleavage at the PPT site by in the design of a ligand capable of binding DNA:RNA hy- RNase H, in turn inhibiting plus-strand DNA synthesis brids with high affinity [107]. Neomycin was covalently linked [110]. Furthermore, studies have suggested this unique to 6-(4-carboxyphenyl)-3,8-diamino-5-methylphen-anthridi- DNA:RNA hybrid triplex can be induced and even stabilized nium chloride, a derivative of ethidium bromide, through the by the presence of ligands such as 40,6-diamidino-2-phenylin- formation of an amide bond, to afford a neomycin-methidium dole (DAPI) [111] and the aminoglycoside, neomycin [100]. 1036 N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039

We first reported the ability of neomycin to stabilize formed during the catalytic cycle of telomerase, with the high- DNA:RNA hybrids and even induce hybrid triplex formation est affinity, even in the presence of other binding ligands [100]! [113]. This work identified ethidium bromide as a lead com- This work was further expanded by Pilch and coworkers to pound for therapeutic approaches to DNA:RNA hybrid target- include the aminoglycosides neomycin and ribostamycin, fo- ing of telomereres. cusing on DNA:RNA hybrid chimeras. These chimeras were designed as constructs of the polypurine tract (PPT) of HIV-1, 5. Concluding remarks found at two unique steps in the reverse transcription of HIV-1 [26]. Containing the same DNA:RNA hybrid portion, these chimeras differ in the composition, RNA or DNA, of the Acting at the hub of biological information transfer, compliment strand. As reported earlier, RNase H cleavage of DNA:RNA hybrids provide a potential substrate for control- the DNA:RNA hybrid chimeras is inhibited by the presence of ling expression. Conformationally distinguishable from the aminoglycosides and a number of small non- more common nucleic acids, DNA and RNA, these hybrids ne- reverse transcriptase inhibitors (NNRTIs) [112].In cessitate more attention when studying nucleic acids therapeu- fact, of the aminoglycosides surveyed, neomycin is far superior tics. Even more enticing, due to their relatively low occurrence at inhibiting RNase H cleavage in both chimeras. Furthermore, within biological systems, when compared to DNA and RNA, thermal stabilization of the chimeras, afforded by the presence DNA:RNA hybrids provide a unique substrate for targeting of aminoglycosides, reveals that neomycin increases the stabil- with small binding ligands. Despite their uniqueness, ity of both chimeras to a larger extent than paramomycin, which DNA:RNA hybrids have not received their share of attention. are both much greater than ribostamycin. Isothermal titration The attention DNA:RNA hybrids have received suggest that calorimetry derived binding constants for the aminoglycosides DNA:RNA hybrids can be targeted by small ligands. However, binding the two chimeras are located in Table 2. In both chi- more work must be done to show that selective targeting of meras, the association constant for neomycin is greater than DNA:RNA hybrids can be attained in the presence of the other aminoglycosides studied. Furthermore, it is clear a much higher concentration of duplex DNA and RNA struc- that the addition of ring four to the 2-deoxy streptose moiety tures in the cell. binding to DNA:RNA hybrids is paramount to high affinity binding of aminoglycosides to has shown the ability to disrupt the transfer of genetic infor- DNA:RNA hybrid structures. mation by disabling the proper function of enzymes. More work still needs to be done to develop highly specific ligands 4.2.2. Okazaki fragments capable of binding the hybrids at association constants higher A series of conformationally restrained bis-distamycin than naturally occurring enzymes. We have begun to outline compounds, dimerized through the ortho/para or meta posi- the principles required for high affinity and high specificity tions of and pyridine (2,5- and 2,4- type, respec- binding of DNA:RNA hybrids by small ligands. Through the tively), were synthesized [108]. Ortho/para bis-distamycins conjugation of an aminoglycoside to an DNA.RNA hybrid were capable of binding both DNA duplexes as well as the specific intercalator, DNA:RNA hybrids of distinctly different Okazaki fragment at similar binding constants, w108 M1 sequence can be distinguished from one another as well as and w107 M1 range, respectively. Surprisingly, mono-dista- other nucleic acid structures. More work is being done to de- mycins, linked to the benzene moiety, showed no binding to velop more selective molecules such that DNA:RNA hybrid the duplexes. The meta pyridyl bis-distamycins were also stud- targeting can become a noteworthy therapeutic endeavor. ied, however, affinity for the Okazaki duplex is in the w106 M1 range. Acknowledgements

4.2.3. Telomerase PI is thankful to NSF (CHE 034972) for financial support. DNA:RNA hybrid targeting with ethidium bromide has We also thank Prof. Meredith Newby, Clemson University, for been applied to targeting telomerase through its DNA:RNA help with this manuscript. hybrid duplex. Friedman and coworkers have surveyed a num- ber of compounds in an attempt to inhibit telomerase activity References through DNA:RNA hybrid binding [41,113]. Surveying a num- ber of intercalators, four ligands (ethidium bromide, rivanol, [1] J.D. Watson, F.H. Crick, Molecular structure of nucleic acids; a structure e orange and yellow) were found to inhibit telomerase for deoxyribose nucleic acid, Nature 171 (1953) 737 738. [2] A. Rich, An analysis of the relation between DNA and RNA, Ann. N.Y. activity at IC50 values in the low micromolar range. Further- Acad. Sci. 81 (1959) 709e722. more, telomerase inhibition through the binding of these li- [3] A. Rich, Discovery of the hybrid helix and the first DNAeRNA hybrid- gands to G-quadraplex structures, another substantiated ization, J. Biol. Chem. 281 (2006) 7693e7696. method for achieving telomerase inhibition, was not observed [4] A. Rich, A hybrid helix containing both deoxyribose and ribose polynu- [41]. Affinity chromatography isolated the high affinity cleotides and its relation to the transfer of information between the nu- cleic acids, Proc. Natl. Acad. Sci. USA 46 (1960) 1044e1053. DNA:RNA binding from a homogeneous mixture of [5] C.L. Schildkraut, J. Marmur, P. Doty, The formation of hybrid DNA binding ligands. It was found that ethidium bromide uniquely molecules and their use in studies of DNA homologies, J. Mol. Biol. binds a DNA:RNA hybrid duplex, derived from the hybrid 3 (1961) 595e617. N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039 1037

[6] B.D. Hall, S. Spiegelman, Sequence complementarity of T2-DNA and [30] M.E. Schmitt, D.A. Clayton, Conserved features of yeast and mamma- T2-specific RNA, Proc. Natl. Acad. Sci. USA 47 (1961) 137e163. lian mitochondrial DNA replication, Curr. Opin. Genet. Dev. 3 (1993) [7] G. Milman, R. Langridge, M.J. Chamberlin, The structure of a DNAe 769e774. RNA hybrid, Proc. Natl. Acad. Sci. USA 57 (1967) 1804e1810. [31] D.D. Chang, D.A. Clayton, Priming of human mitochondrial DNA rep- [8] A.H. Wang, S. Fujii, J.H. van Boom, G.A. van der Marel, S.A. van Boeckel, lication occurs at the light-strand promoter, Proc. Natl. Acad. Sci. USA A. Rich, Molecular structure of r(GCG)d(TATACGC): a DNAeRNA hy- 82 (1985) 351e355. brid helix joined to double helical DNA, Nature 299 (1982) 601e604. [32] D.D. Chang, D.A. Clayton, Precise identification of individual pro- [9] D.G. Reid, S.A. Salisbury, T. Brown, D.H. Williams, J.J. Vasseur, moters for transcription of each strand of human mitochondrial DNA, B. Rayner, J.L. Imbach, Use of inter-proton nuclear Overhauser effects Cell 36 (1984) 635e643. to assign the nuclear magnetic resonance spectra of oligodeoxynucleo- [33] T. Itoh, J. Tomizawa, Formation of an RNA primer for initiation of rep- tide and hybrid duplexes in aqueous solution, Eur. J. Biochem 135 lication of ColE1 DNA by H, Proc. Natl. Acad. Sci. USA (1983) 307e314. 77 (1980) 2450e2454. [10] G. Gupta, M.H. Sarma, R.H. Sarma, Secondary structure of the hybrid [34] M.N. Prichard, S. Jairath, M.E. Penfold, S. St. Jeor, M.C. Bohlman, poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and G.S. Pari, Identification of persistent RNAeDNA hybrid structures stereochemical modeling within the constraints of NOE data, J. Mol. within the of human cytomegalovirus, J. Virol. Biol. 186 (1985) 463e469. 72 (1998) 6997e7004. [11] J. Hurwitz, A. Bresler, R. Diringer, The enzymic incorporation of ribo- [35] M. Tisdale, T. Schulze, B.A. Larder, K. Moelling, within nucleotides into polyribonucleotides and the effect of DNA, Biochem. the RNase H of human immunodeficiency virus type 1 reverse Biophys. Res. Commun 3 (1960) 15e18. transcriptase abolish virus infectivity, J. Gen. Virol. 72 (Pt 1) (1991) [12] H.M. Temin, S. Mizutani, RNA-dependent DNA polymerase in virions 59e66. of , Nature 226 (1970) 1211e1213. [36] S. Volkmann, J. Dannull, K. Moelling, The polypurine tract, PPT, of [13] D. Baltimore, RNA-dependent DNA polymerase in virions of RNA tu- HIV as target for antisense and triple-helix-forming oligonucleotides, mour , Nature 226 (1970) 1209e1211. Biochimie 75 (1993) 71e78. [14] J.P. Bouche, K. Zechel, A. Kornberg, dnaG , a - [37] N.W. Kim, M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, resistant RNA polymerase, initiates the conversion of a single-stranded P.L. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, J.W. Shay, Spe- coliphage DNA to its duplex replicative form, J. Biol. Chem. 250 (1975) cific association of human telomerase activity with immortal cells and 5995e6001. cancer, 266 (1994) 2011e2015. [15] N.A. Campbell, Biology, Benjamin Cummings, Menlo Park, CA, 1996. [38] S.P. Lichtsteiner, J.S. Lebkowski, A.P. Vasserot, Telomerase. A target [16] R.L.P. Adams, R.H. Burton, A.M. Campbell, D.P. Leader, for anticancer therapy, Ann.. N.Y. Acad. Sci. 886 (1999) 1e11. R.M.S. Smellie, The of the Nucleic Acids, Chapman [39] C. Autexier, Telomerase as a possible target for anticancer therapy, and Hall, New York, 1981. Chem. Biol. 6 (1999) R299eR303. [17] M.J. Chamberlin, RNA Polymerase, Cold Spring Harbor Laboratory, [40] A. Siddiqa, D.A. Cavazos, R.A. Marciniak, Targeting telomerase, Reju- Cold Spring Harbor, NY, 1976. venation Res. 9 (2006) 378e390. [18] B. Xu, D.A. Clayton, RNAeDNA hybrid formation at the human mito- [41] R. Francis, C. West, S.H. Friedman, Targeting telomerase via its key chondrial heavy-strand origin ceases at replication start sites: an impli- RNA/DNA heteroduplex, Bioorg. Chem. 29 (2001) 107e117. cation for RNAeDNA hybrids serving as primers, EMBO J 15 (1996) [42] S.H. Hung, Q. Yu, D.M. Gray, R.L. Ratliff, Evidence from CD spectra 3135e3143. that d(purine).r(pyrimidine) and r(purine).d(pyrimidine) hybrids are in [19] F.H. Martin, I. Tinoco Jr., DNA RNA hybrid duplexes containing oli- different structural classes, Nucleic Acids Res. 22 (1994) 4326e4334. go(dA:rU) sequences are exceptionally unstable and may facilitate ter- [43] M.J. Chamberlin, D.L. Patterson, Physical and chemical characteriza- mination of transcription, Nucleic Acids Res. 8 (1980) 2295e2299. tion of the ordered complexes formed between polyinosinic acid, poly- [20] K.R. Blake, A. Murakami, S.A. Spitz, S.A. Glave, M.P. Reddy, cytidylic acid and their deoxyribo-analogues, J. Mol. Biol. 12 (1965) P.O. Ts’o, P.S. Miller, Hybridization arrest of globin synthesis in rabbit 410e428. lysates and cells by oligodeoxyribonucleoside methyl- [44] M. Riley, B. Maling, Physical and chemical characterization of two- and phosphonates, Biochemistry 24 (1985) 6139e6145. three-stranded -thymine and adenine-uracil homopolymer com- [21] K.R. Blake, A. Murakami, P.S. Miller, Inhibition of rabbit globin plexes, J. Mol. Biol. 20 (1966) 359e389. mRNA by sequence-specific oligodeoxyribonucleotides, [45] D.N. Dubins, A. Lee, R.B. Macgregor Jr., T.V. Chalikian, On the stabil- Biochemistry 24 (1985) 6132e6138. ity of double stranded nucleic acids, J. Am. Chem. Soc. 123 (2001) [22] H.M. Temin, of cells by viruses, Perspect. 9254e9259. Biol. Med 14 (1970) 11e26. [46] K.B. Hall, L.W. McLaughlin, Thermodynamic and structural properties [23] H.M. Temin, Origin of from cellular moveable genetic el- of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of iden- ements, Cell 21 (1980) 599e600. tical sequence, Biochemistry 30 (1991) 10606e11613. [24] H.M. Temin, Reverse transcription in the eukaryotic genome: retrovi- [47] E.A. Lesnik, S.M. Freier, Relative thermodynamic stability of DNA, ruses, pararetroviruses, , and retrotranscripts, Mol. RNA, and DNA:RNA hybrid duplexes: relationship with base composi- Biol. Evol. 2 (1985) 455e468. tion and structure, Biochemistry 34 (1995) 10807e10815. [25] W. Gilbert, Origin of life: the RNA world, Nature 319 (1986) 618. [48] R.W. Roberts, D.M. Crothers, Stability and properties of double and tri- [26] T.K. Li, C.M. Barbieri, H.C. Lin, A.B. Rabson, G. Yang, Y. Fan, ple helices: dramatic effects of RNA or DNA backbone composition, B.L. Gaffney, R.A. Jones, D.S. Pilch, Drug targeting of HIV-1 Science 258 (1992) 1463e1466. RNA.DNA hybrid structures: thermodynamics of recognition and im- [49] E.J. O’Brien, A.W. MacEwan, Molecular and crystal structure of the pact on reverse transcriptase-mediated activity and viral complex: polyinosinic acid plus polydeoxycytidylic replication, Biochemistry 43 (2004) 9732e9742. acid, J. Mol. Biol. 48 (1970) 243e261. [27] B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molec- [50] M. Chamberlin, P. Berg, Mechanism of RNA polymerase action: forma- ular Biology of the Cell, Garland Science, New York, 2002. tion of DNAeRNA hybrids with single-stranded templates, J. Mol. [28] D.A. Clayton, Replication of mitochondrial DNA, Cell 28 Biol. 8 (1964) 297e313. (1982) 693e705. [51] M.J. Tunis, J.E. Hearst, Optical rotatory dispersion of DNA in concen- [29] R. Okazaki, T. Okazaki, K. Sakabe, K. Sugimoto, A. Sugino, Mecha- trated salt solutions, 6 (1968) 1218e1223. nism of DNA chain growth. I. Possible discontinuity and unusual sec- [52] D.M. Gray, R.L. Ratliff, Circular dichroism spectra of poly[d(AC):d(GT)], ondary structure of newly synthesized chains, Proc. Natl. Acad. Sci. poly[r(AC):r(GU)], and hybrids poly[d(AC):r(GU)] and poly[r(AC): USA 59 (1968) 598e605. d(GT)] in the presence of , Biopolymers 14 (1975) 487e498. 1038 N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039

[53] S.C. Erfurth, P.J. Bond, W.L. Peticolas, Characterization of the A in [74] O. Fedoroff, M. Salazar, B.R. Reid, Structure of a DNA:RNA hybrid equilibrium B transition of DNA in fibers and gels by laser Raman spec- duplex. Why RNase H does not cleave pure RNA, J. Mol. Biol. 233 troscopy, Biopolymers 14 (1975) 1245e1257. (1993) 509e523. [54] H. Shindo, U. Matsumoto, Direct evidence for a bimorphic structure of [75] E. Selsing, R.D. Wells, T.A. Early, D.R. Kearns, Two contiguous con- aDNAeRNA hybrid, poly(rA).poly(dT), at high relative humidity, J. formations in a nucleic acid duplex, Nature 275 (1978) 249e250. Biol. Chem. 259 (1984) 8682e8684. [76] E. Selsing, R.D. Wells, Polynucleotide block consisting of [55] S.B. Zimmerman, B.H. Pheiffer, A RNA.DNA hybrid that can adopt a DNA.RNA hybrid joined to a DNA.DNA duplex. Synthesis and charac- two conformations: an x-ray diffraction study of poly(rA).poly(dT) in terization of dGn.rCidCk duplexes, J. Biol. Chem. 254 (1979) 5410e5416. concentrated solution or in fibers, Proc. Natl. Acad. Sci. USA 78 [77] M. Egli, N. Usman, S.G. Zhang, A. Rich, Crystal structure of an Oka- (1981) 78e82. zaki fragment at 2-A resolution, Proc. Natl. Acad. Sci. USA 89 (1992) [56] J.M. Benevides, G.J. Thomas Jr., A solution structure for poly(rA). 534e538. poly(dT) with different furanose pucker and backbone geometry in rA [78] D.G. Sanford, K.J. Kotkow, B.D. Stollar, Immunochemical detection of and dT strands and intrastrand hydrogen bonding of adenine 8CH, Bio- multiple conformations within a 36 base pair oligonucleotide, Nucleic chemistry 27 (1988) 3868e3873. Acids Res. 16 (1988) 10643e10655. [57] H.T. Steely Jr., D.M. Gray, R.L. Ratliff, CD of homopolymer DNAe [79] J.R. Mellema, C.A. Haasnoot, G.A. van der Marel, G. Wille, C.A. van RNA hybrid duplexes and triplexes containing A-T or A-U base pairs, Boeckel, J.H. van Boom, C. Altona, Proton NMR studies on the cova- Nucleic Acids Res. 14 (1986) 10071e10090. lently linked RNAeDNA hybrid r(GCG)d(TATACGC). Assignment of [58] T. Fujiwara, H. Shindo, -31 nuclear magnetic resonance of proton resonances by application of the nuclear Overhauser effect, Nu- highly oriented DNA fibers. 2. Molecular motions in hydrated DNA, cleic Acids Res. 11 (1983) 5717e5738. Biochemistry 24 (1985) 896e902. [80] T. Nishizaki, S. Iwai, T. Ohkubo, C. Kojima, H. Nakamura, Y. Kyogoku, [59] S. Arnott, R. Chandrasekaran, R.P. Millane, H.S. Park, DNAeRNA hy- E. Ohtsuka, Solution structures of DNA duplexes containing brid secondary structures, J. Mol. Biol. 188 (1986) 631e640. aDNA RNA hybrid region, d(GG)r(AGAU)d(GAC) x [60] A. Pardi, F.H. Martin, I. Tinoco Jr., Comparative study of ribonucleo- d(GTCATCTCC) and d(GGAGA)r(UGAC) d(GTCATCTCC), Bio- tide, , and hybrid oligonucleotide helices by nu- chemistry 35 (1996) 4016e4025. clear magnetic resonance, Biochemistry 20 (1981) 3986e3996. [81] S.T. Hsu, M.T. Chou, J.W. Cheng, The solution structure of [61] J.I. Gyi, A.N. Lane, G.L. Conn, T. Brown, Solution structures of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA du- DNA.RNA hybrids with purine-rich and pyrimidine-rich strands: com- plexes, Nucleic Acids Res. 28 (2000) 1322e1331. parison with the homologous DNA and RNA duplexes, Biochemistry 37 [82] L. Zhu, M. Salazar, B.R. Reid, DNA duplexes flanked by hybrid du- (1998) 73e80. plexes: the solution structure of chimeric junctions in [r(cgcg)d(TA- [62] J.I. Gyi, G.L. Conn, A.N. Lane, T. Brown, Comparison of the thermo- TACGCG)]2, Biochemistry 34 (1995) 2372e2380. dynamic stabilities and solution conformations of DNA.RNA hybrids [83] M. Salazar, J.J. Champoux, B.R. Reid, Sugar conformations at hybrid containing purine-rich and pyrimidine-rich strands with DNA and duplex junctions in HIV-1 and Okazaki fragments, Biochemistry 32 RNA duplexes, Biochemistry 35 (1996) 12538e12548. (1993) 739e744. [63] E. Hantz, V. Larue, P. Ladam, L. Le Moyec, C. Gouyette, T. Huynh [84] M. Salazar, O. Fedoroff, L. Zhu, B.R. Reid, The solution structure of the Dinh, Solution conformation of an RNAeDNA hybrid duplex contain- r(gcg)d(TATACCC):d(GGGTATACGC) Okazaki fragment contains two ing a pyrimidine RNA strand and a purine DNA strand, Int. J. Biol. distinct duplex morphologies connected by a junction, J. Mol. Biol. 241 Macromol 28 (2001) 273e284. (1994) 440e455. [64] G.L. Conn, T. Brown, G.A. Leonard, The crystal structure of the [85] O. Fedoroff, M. Salazar, B.R. Reid, Structural variation among retrovi- RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) shows sig- ral primer-DNA junctions: solution structure of the HIV-1 ()-strand nificant differences to that found in solution, Nucleic Acids Res. 27 Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC), Biochemistry 35 (1999) 555e561. (1996) 11070e11080. [65] Y. Xiong, M. Sundaralingam, Crystal structure of a DNA.RNA hybrid [86] A. Rich, : a tale of two puckers, Nat. Struct. Biol. 10 duplex with a polypurine RNA r(gaagaagag) and a complementary - (2003) 247e249. ypyrimidine DNA d(CTCTTCTTC), Nucleic Acids Res. 28 (2000) [87] B.N. Conner, T. Takano, S. Tanaka, K. Itakura, R.E. Dickerson, The 2171e2176. molecular structure of d(ICpCpGpG), a fragment of right-handed dou- [66] G.W. Han, Direct-methods determination of an RNA/DNA hybrid dec- ble helical A-DNA, Nature 295 (1982) 294e299. amer at 1.15 A resolution, Acta Crystallogr. D 57 (2001) 213e218. [88] S. Arnott, D.W. Hukins, Refinement of the structure of B-DNA and im- [67] L. Ratmeyer, R. Vinayak, Y.Y. Zhong, G. Zon, W.D. Wilson, Sequence plications for the analysis of x-ray diffraction data from fibers of bio- specific thermodynamic and structural properties for DNA.RNA du- polymers, J. Mol. Biol. 81 (1973) 93e105. plexes, Biochemistry 33 (1994) 5298e5304. [89] S. Arnott, D.W. Hukins, Optimised parameters for A-DNA and B-DNA, [68] O.Y. Fedoroff, Y. Ge, B.R. Reid, Solution structure of r(gaggacug): Biochem. Biophys. Res. Commun. 47 (1972) 1504e1509. d(CAGTCCTC) hybrid: implications for the initiation of HIV-1 [90] W. Muller, D.M. Crothers, Interactions of heteroaromatic compounds (þ)-strand synthesis, J. Mol. Biol. 269 (1997) 225e239. with nucleic acids. 1. The influence of heteroatoms and polarizability [69] X. Gao, P.W. Jeffs, Sequence-dependent conformational heterogeneity of on the base specificity of intercalating ligands, Eur. J. Biochem. 54 a hybrid DNA.RNA dodecamer duplex, J. Biomol. NMR 4 (1994) 367e384. (1975) 267e277. [70] S.H. Chou, P. Flynn, B. Reid, High-resolution NMR study of a synthetic [91] J. Ren, J.B. Chaires, Sequence and structural selectivity of nucleic acid DNAeRNA hybrid dodecamer containing the consensus pribnow pro- binding ligands, Biochemistry 38 (1999) 16067e16075. moter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG), Bio- [92] X. Qu, J.B. Chaires, Analysis of drug-DNA binding data, Methods En- chemistry 28 (1989) 2435e2443. zymol. 321 (2000) 353e369. [71] A.N. Lane, S. Ebel, T. Brown, NMR assignments and solution confor- [93] I. Haq, T.C. Jenkins, B.Z. Chowdhry, J. Ren, J.B. Chaires, Parsing free mation of the DNA.RNA hybrid duplex d(GTGAACTT).r(AAGUU- energies of drugeDNA interactions, Methods Enzymol. 323 (2000) CAC), Eur. J. Biochem. 215 (1993) 297e306. 373e405. [72] N.C. Horton, B.C. Finzel, The structure of an RNA/DNA hybrid: a sub- [94] J. Ren, X. Qu, N. Dattagupta, J.B. Chaires, of strate of the ribonuclease activity of HIV-1 reverse transcriptase, J. Mol. a RNA:DNA hybrid structure, J. Am. Chem. Soc. 123 (2001) 6742e Biol. 264 (1996) 521e533. 6743. [73] M. Salazar, O.Y. Fedoroff, J.M. Miller, N.S. Ribeiro, B.R. Reid, The [95] X. Shi, J.B. Chaires, Sequence- and structural-selective nucleic acid DNA strand in DNA.RNA hybrid duplexes is neither B-form nor A- binding revealed by the melting of mixtures, Nucleic Acids Res. 34 form in solution, Biochemistry 32 (1993) 4207e4215. (2006) e14. N.N. Shaw, D.P. Arya / Biochimie 90 (2008) 1026e1039 1039

[96] J.P. McDonnell, A.C. Garapin, W.E. Levinson, N. Quintrell, L. Fanshier, [106] W.H. Gmeiner, W. Cui, D.E. Konerding, P.A. Keifer, S.K. Sharma, J.M. Bishop, DNA of Rous sarcoma virus: delineation of A.M. Soto, L.A. Marky, J.W. Lown, Shape-selective recognition of two reactions with actinomycin, Nature 228 (1970) 433e435. a model Okazaki fragment by geometrically-constrained bis-distamy- [97] W.E. Muller, R.K. Zahn, H.J. Seidel, Inhibitors acting on nucleic acid syn- cins, J. Biomol. Struct. Dyn. 17 (1999) 507e518. thesis in an oncogenic RNA virus, Nat. New Biol. 232 (1971) 143e145. [107] C.L. Kielkopf, S. White, J.W. Szewczyk, J.M. Turner, E.E. Baird, [98] W. Chu, K. Shigehiro, M. Shinomiya, R.G. Carlson, F. Takusagawa, To- P.B. Dervan, D.C. Rees, A structural basis for recognition of A.T and ward the design of an RNA:DNA hybrid binding agent, J. Am. Chem. T.A base pairs in the minor groove of B-DNA, Science 282 (1998) Soc. 116 (1994) 2243e2253. 111e115. [99] F. Takusagawa, K.T. Takusagawa, R.G. Carlson, R.F. Weaver, Selectiv- [108] N. Neamati, A. Mazumder, S. Sunder, J.M. Owen, M. Tandon, ity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia J.W. Lown, Y. Pommier, Highly potent synthetic polyamides, bisdista- activity, Bioorg. Med. Chem. 5 (1997) 1197e1207. mycins, and lexitropsins as inhibitors of human immunodeficiency virus [100] D.P. Arya, R.L. Coffee Jr., I. Charles, Neomycin-induced hybrid triplex type 1 integrase, Mol. Pharmacol. 54 (1998) 280e290. formation, J. Am. Chem. Soc. 123 (2001) 11093e11094. [109] N. Shaw, H. Xi, D.P. Arya, Molecular recognition of a DNA:RNA hy- [101] Q. Chen, R.H. Shafer, I.D. Kuntz, Structure-based discovery of ligands brid: sub-nanomolar binding by a neomycin-methidium conjugate, Bio- targeted to the RNA double helix, Biochemistry 36 (1997) 11402e11407. org. Med. Chem. Lett. (in press). [102] D.P. Arya, L. Xue, B. Willis, Aminoglycoside (neomycin) preference is [110] S. Volkmann, J. Jendis, A. Frauendorf, K. Moelling, Inhibition of HIV-1 for A-form nucleic acids, not just RNA: results from a competition di- reverse transcription by triple-helix forming oligonucleotides with viral alysis study, J. Am. Chem. Soc. 125 (2003) 10148e10149. RNA, Nucleic Acids Res. 23 (1995) 1204e1212. [103] D.P. Arya, Aminoglycoside : From to [111] Z. Xu, D.S. Pilch, A.R. Srinivasan, W.K. Olson, N.E. Geacintov, Drug Discovery, Wiley Series in Drug Discovery and Development, K.J. Breslauer, Modulation of nucleic acid structure by ligand binding: John Wiley & Sons, Inc., Hoboken, NJ, 2007. induction of a DNA.RNA.DNA hybrid triplex by DAPI intercalation, [104] C.M. Barbieri, T.K. Li, S. Guo, G. Wang, A.J. Shallop, W. , G. Yang, Bioorg. Med. Chem. 5 (1997) 1137e1147. B.L. Gaffney, R.A. Jones, D.S. Pilch, Aminoglycoside complexation [112] J.Q. Hang, Y. Li, Y. Yang, N. Cammack, T. Mirzadegan, K. Klumpp, with a DNA.RNA hybrid duplex: the thermodynamics of recognition Substrate-dependent inhibition or stimulation of HIV RNase H activity and inhibition of RNA processing enzymes, J. Am. Chem. Soc. 125 by non-nucleoside reverse transcriptase inhibitors (NNRTIs), Biochem. (2003) 6469e6477. Biophys. Res. Commun. 352 (2007) 341e350. [105] W.H. Gmeiner, W. Cui, S. Sharma, A.M. Soto, L.A. Marky, J.W. Lown, [113] C. West, R. Francis, S.H. Friedman, Small molecule/nucleic acid affin- Shape-selective binding of geometrically-constrained bis-distamycins to ity chromatography: application for the identification of telomerase in- a DNA duplex and a model Okazaki fragment of identical sequence, hibitors which target its key RNA/DNA heteroduplex, Bioorg. Med. Nucleotides Nucleic Acids 19 (2000) 1365e1379. Chem. Lett. 11 (2001) 2727e2730.

Top View