Matthew D. Disney Jessica L. Childs Douglas H. Turner Departments of Chemistry and Pediatrics, and the Center for Human Genetics and Molecular Pediatric Disease, University of Rochester, Rochester, NY 14627-0216

New Approaches to Targeting RNA with Oligonucleotides: Inhibition of Group I Intron SelfSplicing

Received 29 August 2003; accepted 29 August 2003

Abstract: RNA is one class of relatively unexplored drug targets. Since RNAs play a myriad of essential roles, it is likely that new drugs can be developed that target RNA. There are several factors that make targeting RNA particularly attractive. First, the amount of information about the roles of RNA in essential biological processes is currently being expanded. Second, sequence information about targetable RNA is pouring out of genome sequencing efforts at unprecedented levels. Third, designing and screening potential oligonucleotide therapeutics to target RNA is relatively simple. The use of oligonucleotides in cell culture, however, presents several challenges such as oligonucleotide uptake and stability, and selective targeting of genes of interest. Here, we review investigations aimed at targeting RNA with oligonucleotides that can circumvent several of these potential problems. The hallmark of the strategies discussed is the use of short oligonucleotides, which may have the advantage of higher cellular uptake and improved binding selectivity compared to longer oligonucleotides. These strategies have been applied to Group I introns from the mammalian pathogens Pneumocystis carinii and Candida albicans. Both are examples of fungal infections that are increasing in number and prevalence. © 2003 Wiley Periodicals, Inc. Biopolymers 73: 151–161, 2004 Keywords: RNA; oligonucleotides; genome sequencing; Group I introns; Pneumocystis carinii; Candida albicans

INTRODUCTION Genome sequencing projects of many organisms are providing a basis for understanding the mechanisms of therapeutic intervention and for aiding development of new therapeutics. These efforts have resulted

in a huge increase in the amount of targetable nucleic acid sequence that is in public databases (Figure 1). In addition, new information is being discovered about the roles of RNA in biological systems. These include the discovery of catalytic RNAs,1–3 interfering RNAs,4,5 and micro RNAs,6,7 and the fact that un-

Correspondence to: Douglas H. Turner, Department of Chemistry, University of Rochester, Rochester, NY 14627-0216; email: [email protected] Biopolymers, Vol. 73, 151–161 (2004) © 2003 Wiley Periodicals, Inc.

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FIGURE 1 Number of base pairs deposited in public databases as a function of calendar year (www.ncbi.nlm.nih. gov/GenBank/genbankstats.html).

translated regions in mRNAs can regulate translation of the message without a protein mediator.8 –10 Since RNAs play many essential roles, they can be exploited as targets for therapeutic design. Several antibiotics that are used clinically bind to bacterial ribosomes and inhibit protein synthesis.11,12 Recently, the structures of many of these molecules bound to bacterial ribosomes and mimics of the rRNA binding sites have been reported.13,14 These molecules are typically discovered by screening combinatorial libraries or natural products, and are rarely designed due to a lack of understanding of small-molecule targeting of RNA. The most direct way to design therapeutics from the wealth of sequence information is to design oligonucleotides that form approximately 20-base pairs to an RNA of interest. Once the oligonucleotide binds to a mRNA in cells, it should affect expression of the encoded protein. This design strategy, called antisense, was developed in the late 1970s by Stephenson and Zamecnik15,16 as a means to inhibit Rous Sarcoma virus replication in fibroblast tissue culture, and by Weintraub to inhibit thymidine kinase.17 Typically, this strategy inactivates the target RNA by RNase H degradation of the newly formed hybrid duplex.18,19 Other types of nucleic acids have also been used to target RNA. These include the hammerhead20 and hairpin21 ribozymes and engineered Group I introns.22–24 These strategies differ from antisense in that these nucleic acids catalyze reactions with the target RNA without having to recruit protein. There are several advantages to using these strategies in addition to the simplicity of design. For example, it has been shown that uptake, metabolism, and side effects of oligonucleotides in mammals are relatively independent of oligonucleotide se-

quence.25–28 Thus, a pharmacokinetics profile for each newly designed antisense agent may not be required, which should facilitate approval of new drug candidates by the Food and Drug Administration (FDA). The use of antisense oligonucleotides as therapeutics, however, has several problems, which are centered around specificity, cellular uptake, and metabolic stability. Specificity is potentially a problem because antisense oligonucleotides are typically 20 nucleotides long. Oligonucleotides of this length may bind to both their intended target and bystander sites with similar binding constants.29 –31 Targeting unintended sites has the potential for inhibiting the function of an RNA needed for survival of healthy cells. Uptake of oligonucleotides of the length typically used in antisense experiments is also not very efficient in mammalian cells. These problems are alleviated by the use of transfection agents that facilitate uptake.32 Also, once oligonucleotides enter cells they are often concentrated in endosomes and degraded.33 Degradation problems are often addressed by using oligonucleotides with modified backbones that are not recognized by nucleases and are stable inside cells.34 The use of both transfection agents and oligonucleotides with modified backbones can also induce nonspecific effects in cells. For example, some transfection agents lose efficacy in the presence of serum and they often do not work for all cell types.32 In addition, oligonucleotides with CG steps can induce a problematic immune response.35 These problems, however, can be overcome. The antisense drug, Vitravene, which inhibits cytomegalovirus,36 has been approved by the FDA. Many more antisense therapeutics are in the pipeline, including an inhibitor of protein kinase c-␣ (PKC-␣), which is near approval for treatment of solid tumors that are resistant to standard treatments.37 These landmarks show that oligonucleotide therapeutics have the potential to treat a wide variety of diseases. In this article, we review new strategies for oligonucleotide targeting of RNA. The model systems for testing these strategies have been Group I introns from the mammalian pathogens Pneumocystis carinii38,39 and Candida albicans.40,41 These RNAs were chosen because their function is necessary for formation of active ribosomes42; Group I introns are not present in mammals, potentially decreasing the likelihood of side effects; there is a wealth of information about this RNA that can facilitate inhibitor design1; and activity assays are easy to perform.41,43,44 Both P. carinii and C. albicans are pathogenic fungi that are becoming increasingly drug resistant, thus kindling interest in discovery of new therapeutics for treatment.

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FIGURE 2 Calculation of BETI. The contribution from base pairing is determined from optical melting experiments of substrates bound to mimics of the Group I intron internal guide sequence, e.g., 5⬘GACUCU binding to 3⬘CGGAGG (left side). The total Kd of binding oligonucleotides to the Group I intron is determined from gel shift binding assays (right side) with the assumption that all bound oligomers participate in tertiary interactions.48

The basic premise for design of these potential therapeutics is to use short oligonucleotides (i.e., ⱕ 12-mers) to affect self-splicing in novel ways. The use of short oligonucleotides should alleviate many of the problems with current antisense technologies. For example, the cost of oligonucleotide synthesis decreases with length, shorter oligonucleotides may be taken into cells in higher concentrations than their larger counterparts, and the specificity for oligonucleotide inhibition can be enhanced when the mechanism of action depends on more than Watson–Crick base pairing.30,41,45

BINDING ENHANCEMENT BY TERTIARY INTERACTIONS (BETI) Many RNAs require a specific three-dimensional structure for function. BETI exploits tertiary interactions with these RNAs to increase the binding affinity and specificity of small oligonucleotides.30,41 That is, adding tertiary interactions to a small free energy contribution from base pairing allows a short oligonucleotide to bind tightly and specifically to an RNA. For the Group I intron, information about the tertiary

interactions necessary for recognition of the 3⬘-end of the 5⬘-exon is used to design inhibitors of self-splicing that can access tertiary interactions.41,46 – 49 There are several advantages to the BETI approach, including greater target specificity because recognition of the RNA target is driven by formation of tertiary contacts that depend on the target’s three-dimensional (3D) shape. Moreover, oligonucleotides that are four to six nucleotides in length are used, which potentially will increase uptake by cells, reduce the cost of synthesis, and cause fewer side effects. A quantitative measure of BETI can be defined to represent the increased specificity of an oligonucleotide for a 3D structure that allows tertiary interactions rather than only Watson–Crick base pairing. It is approximated (Figure 2) with the following equation:

BETI ⫽

Kd,B.P. Kd,Total

where Kd,B.P. is the dissociation constant for base pairing and Kd,Total is the dissociation constant for binding with tertiary interactions to the target RNA. The Kd,Total includes contributions from base pairing

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FIGURE 3 Molecular modeling of tertiary contacts in the P1 helix in the C. albicans48,49 and P. carinii39,46,102 Group I introns. Space-filling atoms are used for functional groups thought to form tertiary interactions. Question marks indicate only indirect evidence for tertiary interactions.

and tertiary interactions. The energetics of base pairings are determined by optical melting of a duplex with substrate bound to an oligonucleotide with the sequence of the binding site on the target RNA. Total binding free energy is determined by gel retardation.39,41,50 Note that the experi-

mental measurement is only a good measure of tertiary interactions when BETIⰇ1 for the oligonucleotide binding since the gel shift assay probably does not discriminate between complexes with and without tertiary interactions. For the P. carinii Group I intron, the formation of a G 䡠 u base pair (Figure 3) between a 5⬘-exon mimic (lowercase) and the Group I intron’s internal guide sequence (IGS) (uppercase) gives rise to BETI. Oligonucleotides accommodating this tertiary interaction can be more specific for the Group I intron by up to 376,000fold compared to simple base pairing to a singlestranded hexamer.30,46 For the C. albicans Group I intron (Figure 3), there are five functional groups that contribute to tertiary binding of a 5⬘-exon mimic. These include the exocyclic amines of G’s in the P1 helix (49) from an imino G 䡠 a pair (⫺1.4 kcal/mol) at position ⫺5 and G 䡠 u pair (⫺2.9 kcal/mol) at the splice site, as well as the 2⬘-OH’s groups48 of u-3 (⫺1.5 kcal/mol), c-2 (⫺1.0 kcal/mol), and u-1 (⫺0.8 kcal/mol). Design of 5⬘-exon mimics that accommodate formation of the imino G 䡠 a pair and the G 䡠 u pair tertiary interactions increase BETI by 11-fold and 135-fold, respectively. The 2⬘OHs of u-3, c-2, and u-1 increase BETI by 13-, 5-, and 4-fold, respectively.48 Table I summarizes the contributions of functional groups to tertiary binding and the BETIs of selected 5⬘-exon mimics binding to a ribozyme derived from the C. albicans Group I intron. Figure 4 shows the secondary structure of the C. albicans Group I intron48 and tertiary contacts by analogy to the Tetrahymena thermophila Group I intron. The variety of functional groups participating in these tertiary contacts expands the possibility of

Table I Contributions of Functional Groups to Binding of r(GACUCU) to the C. albicans Group I Ribozyme and the BETIs of Selected 5ⴕ-Oligonucleotide Mimics with 2ⴕO-methyl (m) and ribophosphoramidate (rn) backbones41,48,49 Tertiary Contact G 䡠 a NH2 G 䡠 u NH2 u-3 2⬘-OH c-2 2⬘-OH u-1 2⬘-OH

Oligonucleotide r(GACUCU) GmAmCm(rU)Cm(rU) GmCmCm(rU)Cm(rU) rn(GACUC)r(U) rn(GCCUC)r(U)

⫺⌬G°37 (kcal/ mol)

BETI

1.4 2.9 1.5 1.0 0.8

11 135 13 5 4

Kd of Binding to Ribozyme (Total) (nM)

Kd of Binding to r(GGAGGC) (Base Pairing) (mM)

BETI

6.9 ⫾ 0.9 15 ⫾ 2 0.7 ⫾ 0.2 6.4 ⫾ 0.3 1.0 ⫾ 0.3

(1.1) 0.25 6.2 ⫻ 10⫺4 0.22 2.1 ⫻ 10⫺5

159000 17000 890 34000 21

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FIGURE 4 Tertiary interactions in the C. albicans Group I intron by analogy to the T. thermophila Group I intron. The intron and truncated exons are depicted in uppercase and lowercase letters, respectively.

BETI.39,46 For example, numerous base triples are formed, which require use of purine N7s.51,52 Other tertiary contacts, for example to backbone phosphates, could also be exploited to bind short oligonucleotides tightly to a 3D architecture. Exploitation of tertiary interactions for increased affinity to a 3D target is not limited to Group I introns. BETI could be applied to other RNAs with specific 3D shapes, including Group II introns,53–55 untranslated regions in mRNAs,56 –58 rRNAs,14,59 – 61 and RNase P RNAs.62– 66

its substrate.41,44 For example, Figure 5 is a schematic of the Group I intron self-splicing reaction (cis-splicing) and suicide inhibition of this reaction (transsplicing). Group I intron splice site recognition occurs in two steps: base pairing of the substrate to the

SUICIDE INHIBITION Suicide inhibition uses the catalytic potential of an RNA to trick the RNA into reacting with a mimic of

FIGURE 5 Schematics of suicide inhibition (trans-splicing) of Group I introns (top) and Group I intron self-splicing (cis-splicing) (bottom).

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FIGURE 6 Suicide inhibition of the C. albicans Group I intron as a function of Mg2⫹ concentration.41 The trans-splicing outcompetes cis-splicing at 1–2 mM Mg2⫹. The oligonucleotides have ribophosphoramidate (rn), deoxyphosphoramidate (dn), and ribose (r) nucleotides. When [Mg2⫹] ⱖ 5 mM, products from hydrolysis may overlap with products from trans-splicing.

internal guide sequence to form the P1 helix and docking of the P1 helix into the catalytic core with formation of tertiary interactions.67,68 In trans-splicing, which mimics the second step of self-splicing, a 5⬘-exon mimic competes with the endogenous substrate for binding to the Group I intron active site. The intron then ligates the 5⬘-exon mimic to the endogenous 3⬘-exon, resulting in dead end products. Since suicide inhibitors (5⬘- exon mimics) bypass the first step of self-splicing, they may have an advantage over the endogenous substrate for reaction with the 3⬘exon. Using information from BETI experiments, oligonucleotides were designed that suicide inhibit Group I intron self-splicing. Oligonucleotides were screened for inhibition of self-splicing as a function of Mg2⫹ concentration (Figure 6) since Mg2⫹ is required for formation of tertiary interactions and for catalysis. As shown in Figure 6, oligonucleotide mimics of the 5⬘-exon are able to compete with C. albicans Group I intron self-splicing at 1–2 mM Mg2⫹,41 which is about the estimated intracellular Mg2⫹ concentration.69 The intron may be folded more compactly at higher Mg2⫹, thus rendering the active site inaccessible for binding exogenous substrates. Alternatively, higher concentrations of Mg2⫹ ions may be required to fill catalytic sites for the first step of self-splicing70

than for the second step.71–75 At low Mg2⫹ concentrations, this would provide an advantage for suicide inhibitors that mimic the second step of self-splicing. The most efficient inhibitor of C. albicans Group I intron self-splicing at 1 mM Mg2⫹ is rn(GCCUC)rU where rn is a ribophosphoramidate linkage and r is a RNA linkage (Figure 7). Trans-splicing competes with cis-splicing even at 50 nM oligonucleotide concentration.41 For the P. carinii Group I intron, the 5⬘-exon mimic dn(ATGAC)rU, where dn is a deoxyphosphoramidate linkage and r is RNA (Figure 7), inhibits 50% of self-splicing at 200 nM oligonucleotide concentration in 4 mM Mg2⫹.44 The dn(ATGAC)rU and rn(GCCUC)rU 5⬘-exon mimics are 1100- and 21-fold more specific for the P. carinii and C. albicans Group I introns, respectively, than for a single stranded hexamer target. BETI and suicide inhibition results suggest that inhibition of Group I intron self-splicing in vivo may be possible. At intracellular Mg2⫹ concentrations, oligonucleotide mimics of the 5⬘-exon are able to compete with the endogenous substrate. In vivo inhibition has not yet been observed, however.76 Suicide inhibition is not limited to Group I introns.77,78 It has the potential to be applied to other catalytic RNAs such as Group II introns,53–55

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FIGURE 7 Chemical structures of DNA, RNA, and modified residues.

rRNAs,60,61,79,80 and RNase P RNAs,62– 64 all of which are found in human pathogens.

OLIGONUCLEOTIDE DIRECTED MISFOLDING OF RNA (ODMIR) Many RNAs require proper secondary or tertiary folds to function. Examples include Group I77,78 and Group II introns,53–55 mRNAs with regulatory untranslated regions,56 –58 rRNAs,60,61,79,80 and RNase P RNAs.62– 66 ODMiR exploits the proclivity of RNA to fold into multiple structures with similar free energies. Some of these structures are inactive, and can be trapped kinetically.81– 85 Proof of principle for the ODMiR strategy has been demonstrated by adding short oligonucleotides (8- to 12-mers) to in vitro transcription mixtures to direct the folding of an RNA into an inactive structure.86 Inactive folds due to kinetic traps have been observed in studies of the T. thermophila Group I intron,81,83,87,88 the hammerhead ribozyme,89 the hepatitis ␦ ribozyme,85,90 and RNase P RNAs.91–93 Although these kinetic traps are disadvantageous for folding studies,

they can be exploited to design or screen potential therapeutics to inhibit RNA function. Secondary structure prediction94 can give insights into potential inactive folds that can lead to kinetic traps,83,85,95 and therefore suggest oligonucleotides that can direct RNA folding into an inactive structure.86 Figure 8 shows the phylogenetic structure of the C. albicans Group I intron and a potential misfolded motif from secondary structure prediction. The misfolded structure replaces part of P3 with an asymmetric internal loop, and is only 2.2 kcal/mol less favorable than the predicted lowest free energy structure. The formation of the P3/P7 pseudoknot is the slow step in the folding of the full length T. thermophila Group I intron,96 and is therefore a region likely to be susceptible to misfolding.83,95 Therefore, an oligonucleotide designed to target the longer side of the asymmetric loop has the potential of competing with pseudoknot formation, trapping the intron in an inactive structure. Indeed, 150 nM of a locked nucleic acid (LNA, L) analog (Figure 7) of appropriate sequence, L (TACCTTTC), inhibits 50% of self-splicing in a transcription mixture.86

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FIGURE 8 The phylogenetic structure of the C. albicans Group I intron and a predicted possible misfolded P3/P7 region. Boxed nucleotides are folded differently in the two structures. An LNA oligonucleotide, L(TACCTTTC), favors the misfolded structure and inhibits self-splicing. The intron and truncated exons are depicted in uppercase and lowercase letters, respectively. Arrows point to the splice sites.

As an alternative to rational design, DNA oligonucleotides complementary to contiguous 12 nucleotide sections of the intron were screened for inhibition. The most efficient inhibitor of self-splicing binds to the sequence separating P4/P6 and P3/P7, which contains important tertiary interactions as well as the G-binding site.52,97,98 A DNA:LNA chimera with this sequence, TLCTLACLGALCGLGCLC, where LX represents an LNA residue, inhibits 50% of self-splicing at about 30 nM.86

Both L(TACCTTTC) and TLCTLACLGALCGLGCLC induce structural changes of important tertiary interactions, suggesting the overall fold of the intron has been altered. Interestingly, both of these oligonucleotides affect long-range interactions, particularly pseudoknot formation. Pseudoknots may therefore be potential targets for therapeutic intervention.86 Though the ODMiR strategy was demonstrated on a Group I intron, it should be applicable to any RNA that requires a specific structure to function.

Targeting RNA with Oligonucleotides

In addition to those listed previously, regulatory elements8 –10 and RNAs that interact with other biomolecules are potential targets for the ODMiR strategy.

SUMMARY AND FUTURE PROSPECTIVES RNA is emerging as an important therapeutic target.99,100 Discovery of the roles of RNA in biology and subsequent computational screening of genome sequences will reveal potential therapeutic targets. Improvements in prediction of secondary structure and in selection of optimal targets should eventually allow computational screening of genome sequences for designing oligonucleotides that will affect only a particular RNA. For example, search tools of the type reported for Group I introns101 could be expanded and applied to genomes of pathogens to find other RNAs that can be targeted with BETI, suicide inhibition, or ODMiR strategies. Sequences could then be compared to the human genome to design inhibitors that are unlikely to induce side effects by binding to human RNA. The BETI, suicide inhibition, and ODMiR strategies use relatively short oligonucleotides (4- to 12-mers), and they are less likely to affect bystander RNAs, because their effect depends on specific structures and functions of the RNA targets. Shorter oligonucleotides are also likely to be more easily taken up into cells and are less expensive to synthesize.

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