RNA molecules are believed to possess highly complex structures containing singleand double-stranded regions as well as loops, bulges, and pseudoknots.1 This structural complexity severely complicates efforts to design structureand/or sequenceselective RNA-binding molecules.2,3 Recently we described a family of molecules for the sequenceand structure-specific recognition of RNA.3 These molecules, called tethered oligonucleotide probes (TOPs), are comprised of two short oligonucleotides separated by a flexible, synthetic tether (Figure 1). These oligonucleotides hybridize in a Watson-Crick4 sense to two noncontiguous, single-stranded regions of a target RNA; the tether traverses the distance between the two regions. TOPs designed to recognize the L. collosoma spliced leader RNA and the HIV-1 Rev Response Element RNA (RRE) bind their targets with nanomolar affinity and high specificity.3d Here we describe TOPs that hybridize to singleand double-stranded regions of a target RNA in a Watson-Crick4 and Hoogsteen5 sense, respectively.6 These triplex TOPs form high affinity RNA complexes whose stabilities are considerably less sensitive than model triple helices to changes in temperature and monovalent cation concentration. Because the wtRRE (Figure 2A) lacks an extended polypurine-polypyrimidine site suitable for triple helix formation, we designed a variant RRE containing a 12 base-pair third-strand recognition site (RREAU, Figure 2B). The natural RRE ligand Rev binds to discrete residues in stem IIB of the wtRRE.7 We reasoned that lengthening stem IIB while maintaining these residues would retain RRE fold and function. RREAU and wtRRE bind Rev with similar affinities, with half maximal binding at 20 nM Rev. TOPs designed to recognize RREAU consist of a 5′ oligodeoxyribonucleotide complementary in a Watson-Crick sense to the bases of RREAU site 1 and a 3′ oligoribonucleotide complementary in a Hoogsteen sense to the extended region of stem IIB (site 4).3d To determine whether RREAU could be recognized with high affinity through the formation of Watson-Crick and Hoogsteen base pairs, we measured the equilibrium dissociation constants of its complexes with TOPs 1-4 through the use of a competition electrophoretic mobility shift assay (Figure 3).3c,8 TOP‚RREAU stabilities at 4 °C ranged from -8.7 kcal‚mol-1 (Kd ) 113 nM) for TOP 1 to -6.9 kcal‚mol-1 (Kd ) 3.6 μM) for TOP 4. All TOPs tested bound RREAU with higher affinities than oligonucleotides that recognized RREAU through WatsonCrick or Hoogsteen base pairs alone: S1‚RREAU exhibited a binding free energy of -6.1 kcal‚mol-1 (Kd ) 14 μM) and S4‚RREAU exhibited a binding free energy that could only be estimated with this assay (∆Gobs > -5 kcal‚mol-1, Kd > 100 μM). TOPs 5 or 6, containing three or 12 mutations in the 3′ oligonucleotide, bound RREAU with approximately 2.2 and 2.9 kcal‚mol-1 lower affinity, respectively, than TOP 1. TOP 1 bound poorly (∆Gobs ) -6.5 kcal‚mol-1, Kd ) 7.8 μM) to an † Phone: 203-432-5094. Fax: 203-432-6144. email: alanna@milan.chem. yale.edu. (1) Tinoco, I. J.; Davis, P. W.; Hardin, C. C.; Puglisi, J. D.; Walker, G. T.; Wyatt, J. Cold Spring Harbor Symp. Quant. Biol. 1987, 52, 135. (2) (a) Bass, B. L.; Cech, T. R. Nature (London) 1984, 308, 820. (b) Bass, B. L.; Cech, T. R. Biochemistry 1986, 25, 4473. (c) Moazed, D.; Noller, H. F. Nature (London) 1987, 327, 389. 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