Kinetics and Mechanism of RNA Binding by Triplex Tethered Oligonucleotide Probes

We have described a series of tethered oligonucleotide probes (triplex TOPs) that recognize one singlestranded and one double-stranded region of an RNA simultaneously through the formation of Watson-Crick and Hoogsteen base pairs, respectively. Here we describe studies on the kinetics and mechanism of triplex TOP‚RREAU association and dissociation. Because triplex TOP‚RREAU complexes cannot be observed by direct electrophoretic methods, kinetics was monitored by use of a competitive electrophoretic mobility shift assay that quantified the effect of a triplex TOP on the association and dissociation rates of an electrophoretically stable TOP‚RREAU complex. Association and dissociation rate constants of triplex TOP‚RREAU complexes were extracted from the experimental data by numerical integration. Triplex TOP‚RREAU association reactions at 25 °C were characterized by rate constants between (7.8 ( 2.0) × 103 and (16 ( 3) × 103 M-1 s-1, while dissociation reactions were characterized by rate constants between (3.3 ( 1.0) × 10-4 and (5.4 ( 2.0) × 10-2 s-1. Rate constants for association of triplex TOP‚RREAU complexes were insensitive to the length and sequence of the 3′-oligonucleotide that mediates triple helix formation. Rate constants for dissociation of triplex TOP‚RREAU complexes were sensitive to changes in tether length as well as the length and composition of the 3′-oligonucleotide. Taken together, these data suggest that triplex TOPs follow a kinetic pathway for binding RREAU in which duplex formation is rate-limiting and precedes triple helix formation. The implication of our data with regard to the kinetics of triple helix association within the context of a highly structured RNA is discussed. Large, structured RNA molecules play critical roles in cellular and viral life cycles.1-3 A considerable body of biochemical structure-mapping data,4-6 along with several high-resolution structures,7-15 indicate that many large RNAs are composed of irregular arrangements of short singleand double-stranded regions joined by structures such as loops, bulges, and pseudoknots.16-19 These irregular architectures present complex functional group arrays that are recognized in nature by proteins,20-23 other nucleic acids,2,24 and small organic molecules.25 There is considerable current interest in the design of molecules that mimic the properties of these natural ligands, not only to inhibit the translation of messenger RNAs but also to interrupt the functions of catalytic and regulatory RNAs.26-32 Several strategies have been presented for the recognition of large, structured RNA molecules. 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For example, various rhodium complexes recognize expanded RNA major grooves48,49 or G‚U mismatches,50 and the organometallic complexes methidiumpropyl-EDTA and bis(phenanthroline)copper(II) show some preference for junctions between duplexes and singlestranded loops or bulges.51,52 The aminoglycoside antibiotic neomycin B recognizes a distinct stem-loop structure within the group I ribozyme,53 the hammerhead enzyme-substrate complex,31 the Rev response element RNA,54 and, under certain conditions, 16S rRNA.55,56 Although combinatorial methods may prove extremely useful for identifying small molecule ligands for specific RNAs,57 few general strategies exist for the design of molecules capable of binding large, globular RNAs in a sequenceand structure-dependent fashion. Several years ago our laboratory presented a general strategy through which the structure and sequence of an RNA might be recognized (Figure 1).47,58-62 Our strategy is based on a class of molecules called tethered oligonucleotide probes (TOPs). A TOP consists of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. Each of the oligonucleotides within a TOP recognizes a single, accessible sequence within the target RNA, and the tether traverses the distance between the two sequences. In contrast to traditional oligonucleotides which recognize a single, contiguous RNA sequence, TOPs recognize two short, noncontiguous sequences that are proximal in the folded RNA. Because TOPs bind simultaneously to two accessible sequences, rather than one long sequence that may not be fully accessible, TOPs exhibit very high affinities for structured RNA targets and bind these targets more rapidly than molecules that must disrupt structure in order to bind.47,60 Initially, we evaluated TOPs that recognized two noncontiguous single-stranded sequences within a target RNA through the formation of standard Watson-Crick base pairs.47,58-60 More recently, we evaluated TOPs that recognized one single-stranded and one double-stranded region simultaneously through the formation of Watson-Crick and Hoogsteen base pairs, respectively.61,62 These molecules were termed triplex TOPs. The RNA target for our triplex TOPs was a modified version of the HIV-1 Rev response element (RRE) (Figure 2A) in which twelve contiguous A-U base pairs replaced a portion of stemloop IIB (RREAU, Figure 2B). The 5′-oligonucleotide within each triplex TOP recognized the accessible, single-stranded region between positions 69 and 76 of RREAU (site 1) to form a duplex and the 3′-oligonucleotide recognized the AU-rich duplex (site 4) to form a triple helix. Triplex TOPs designed to recognize RREAU sites 1 and 4 formed complexes with nanomolar dissociation constants61 and were effective inhibitors of Rev‚RREAU complexation at equilibrium in Vitro.62 Here we characterize the kinetics and mechanism of triplex TOP‚RREAU association and dissociation. Because triplex TOP‚RREAU complexes cannot be observed by direct electrophoretic methods,61 kinetics was monitored by use of a competitive electrophoretic mobility shift assay63-65 that quanti(41) Uhlenbeck, O. C. J. Mol. Biol. 1972, 65, 25. (42) Freier, S. M.; Tinoco, I. J. Biochemistry 1975, 14, 3310. (43) Freier, S. M.; Lima, W. F.; Sanghvi, Y. S.; Vickers, T.; Zounes, M.; Cook, P. D.; Ecker, D. J. RaVen Press Ser. Mol. Cell. Biol. 1992, 1, 95. 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Soc., Vol. 119, No. 48, 1997 Moses and Schepartz