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Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports. We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro. The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters. Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (approximately 7,500 nt). During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length. Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans. In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans. This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

We describe the synthesis, structures, and DNA incorporation of deoxyribonucleosides carrying polycyclic aromatic hydrocarbons as the DNA "base" analogue. The new polycyclic compounds are 1-naphthyl, 2-naphthyl, 9-phenanthrenyl, and 1-pyrenyl deoxynucleosides. The compounds are synthesized using a recently developed C-glycosidic bond formation method involving organocadmium derivatives of the aromatic compounds coupling with a 1α-chlorodeoxyribose precursor. The principal products of this coupling are the α-anomers of the deoxyribosides. An efficient method has also been developed for epimerization of the α-anomers to β-anomers by acid-catalyzed equilibration; this isomerization is successfully carried out on the four polycyclic nucleosides as well as two substituted phenyl nucleosides. The geometry of the anomeric substitution is derived from (1)H NOE experiments and is also correlated with a single-crystal X-ray structure of one α-isomer. Three of the polycyclic C-nucleoside derivatives are incorporated into DNA oligonucleotides via their phosphoramidite derivatives; the pyrenyl and phenanthrenyl derivatives are shown to be fluorescent in a DNA sequence. The results (1) broaden the scope of our C-glycoside coupling reaction, (2) demonstrate that (using a new acid-catalyzed epimerization) both α- and β-anomers are easily synthesized, and (3) constitute a new class of deoxynucleoside derivatives. Such nucleoside analogues may be useful as biophysical probes for the study of noncovalent interactions such as aromatic π-stacking in DNA. In addition, the fluorescence of the phenanthrene and pyrene nucleosides may make them especially useful as structural probes.

We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis. Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand. Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide. This results in a considerably more homogeneous oligonucleotide being produced. Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P). Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis. Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis. It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template. Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.

We report that small, single-stranded circular DNA oligonucleotides 26 to 74 nucleotides (nt) in size can behave as catalytic templates for DNA synthesis by several DNA polymerase enzymes. The DNA products are repeating end-to-end multimeric copies of the synthetic circular DNAs, and range from 1 000 to > 12 000 nucleotides in length. Several aspects of this reaction are unusual: first, the synthesis proceeds efficiently despite the curvature and small size of the circles, some of which have diameters significantly smaller than that of the enzyme itself. Second, the synthesis can proceed hundreds of times around the circle, while rolling replication of larger circular plasmid DNAs requires other proteins for processive synthesis. Finally, the synthesis scheme produces multiple copies of the template without the requirement for either heating or cooling cycles and requires less than stoichiometric amounts of primer, unlike other DNA synthesis methods. We report on the scope of this reaction, and demonstrate that the multimeric products can be cleaved enzymatically to short, sequence-defined oligodeoxynucleotides. This new approach to DNA synthesis may be a practical way to produce useful repeating DNAs, and combined with DNA cleavage strategies, it may represent a useful enzymatic approach to short, sequence-defined oligodeoxynucleotides.

Recent progress in the synthesis and properties of circular oligonucleotides as ligands for DNA and RNA and as templates for polymerase enzymes is described. Small synthetic circular DNAs, RNAs, and chimeric analogues ranging from 28 to 74 nucleotides in size have been synthesized with the use of nonenzymatic ligation strategy. Some of these were designed to undergo triplex formation with single-stranded DNA and RNA targets, and many bind with affinities and sequence selectivities considerably greater than those seen for linear oligonucleotides. Design strategies and modes of binding are discussed in the light of possible use of such molecules as hybridization probes, molecular diagnostics, and sequence-specific inhibitors of gene expression. Small circular oligonucleotides have also been shown to act as unusually efficient templates for DNA and RNA polymerases, which produce long, repeating copies of the circular sequence by a rolling circle process.

Circular nucleic acid molecules can have chemical and biological properties very different from those of the corresponding linear nucleic acid polymers. Described here are methods used recently for construction of such circular molecules, and some of the properties that can arise from making this topological change. Among the unusual properties found for circular nucleic acids are: strong resistance to degradation in biological media; high affinity of binding to other nucleic acids; high sequence selectivity in nucleic acid binding; topological linkage to biomolecules; and the ability to template the synthesis of specific repeating nucleic acid and protein polymers. These properties may be useful in biochemical, medical diagnostic and therapeutic applications.

We report the synthesis and DNA incorporation of a novel C-5 thiopropyne-substituted thymidine derivative which can be used to bring about covalent crosslinks between two noncomplementary DNA strands. This modified thymine pairs normally with adenine in duplex DNA and is shown not to be destabilizing to DNA double helices. Placement of the thiol-nucleotide near the center of opposing pyrimidine strands in pyr·pur·pyr triple helices results in crosslinking of the pyrimidine strands under aerobic conditions. Thermal melting studies at neutral pH show that such crosslinked ligands bind complementary purine strands with higher affinity than is possible with simple Watson-Crick recognition alone. In addition, we describe the construction of a triplex-forming circular oligonucleotide which contains a similar disulfide link across the center. This macrobicyclic ligand binds with extremely high affinity and sequence selectivity to a complementary purine DNA strand. The formation of crosslinks across two noncomplementary strands represents a new strategy for increasing affinity and selectivity of DNA recognition.

We report a novel convergent approach to the construction of circular DNA oligonucleotides from two smaller linear precursors. Circular DNAs 34-74 nucleotides (nt) in size are constructed non-enzymatically in a single step from two half-length oligomers. A DNA template is used to assemble the constituent parts into a triple helical complex which brings the four reactive ends together for chemical ligation with BrCN/imidazole/Ni2+. A homodimerization reaction strategy is successfully used on a small scale to construct circles 42, 58 and 74 nt in size. In addition, a heterodimerization strategy is successfully used in two cases to construct circular 34mers from different 16mer and 18mer precursors. Measurement of preparative yields for one biologically active 34mer circle shows that the dimerization strategy gives a yield higher than that from conventional cyclization and nearly as high as that for a normally synthesized linear DNA, establishing that there is not necessarily a yield penalty for circle construction. Six additional preparative circle constructions, giving conversions of approximately 33-85% from precursors to circular product, are also described. Convergent strategies allow the construction of medium and large size DNA molecules in higher yields than can be achieved by standard linear synthesis alone.

Recent reports describe a new strategy for the binding of single-stranded pyrimidine sequences by triple helix formation. In this approach, a double-length purine-rich oligonucleotide binds a target strand, folding back to form an antiparallel pur.pur.pyr triple helix. We now describe a series of studies in which sequence and structural variations are made in such purine-rich ligands, in an effort to optimize binding properties. Comparison is made between the use of two separate strands and the use of single two-domain ligands; the latter are found to bind more tightly and to aggregate less in media containing Na+ or K+. Placement of mismatched bases in the target shows that sequence selectivity of binding is as high as that for Watson-Crick duplex formation. Variation of the lengths and sequences of loops bridging the binding domains demonstrates that dinucleotide loops composed of pyrimidines give the highest stability. Oligoethylene glycol-derived loop replacements are shown to give good binding affinity as well. The binding of an RNA target is shown to occur with the same affinity as the binding of DNA. In general, it is found that circular variants bind more tightly than do either separate strands or singly-linked ligands and unlike linear oligomers, the circular compounds do not aggregate to a large extent even in buffers containing 100 mM K+. Such structurally optimized ligands are useful in expanding the number of possible naturally-occurring sequences which can be targeted by triplex formation.

Stopped-flow UV kinetics and thermal denaturation experiments are used to examine the origins of high sequence selectivity and binding affinity of circular triplex-forming oligonucleotides with single-stranded DNA/RNA targets. These 34-nt probes are hybridized to a series of 12-nt target sequences which are fully complementary or which contain a single mismatch. Also studied for comparison are standard 12-nt Watson-Crick DNA or RNA complements. Several novel findings are described: (1) Circular triplex-forming oligomers bind targets with very high thermodynamic selectivity (up to 8-10 kcal/mol against a single-nucleotide mismatch), while linear strands show only 2-3 kcal/mol selectivity. (2) Rates for triplex formation by circular ligands are much greater than other reported triplex formation modes and are nearly the same as for Watson-Crick duplex formation. (3) DNA-DNA and RNA-RNA hybridization rates are similar for both duplex and triplex formation. (4) For both modes of binding, hybridization rates do not vary when a mismatch is introduced into the target, and, therefore, binding selectivity is reflected in large variations in dissociation, rather than association rates. Finally, (5) binding selectivity of circular ligands becomes significantly greater as pH is lowered; results indicate that the high sequence selectivity of the circular DNA ligand is due in large part to the special stability of the protonated C+G-C triad relative to unprotonated mismatched triads. The results are useful in the understanding of properties of nucleic acid complexes in general and give insight into optimum design for synthetic DNA-binding ligands.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.

Recent studies have shown that there can be large differences in the stability of double and triple helical nucleic acid complexes, depending on whether RNA or DNA strands are involved. These differences have been attributed to structural differences in the sugar-phosphate backbone of these two polymers. However, since there are in fact two structural features which distinguish DNA from RNA (the 2'-hydroxyl and C-5 methyl groups), the stability differences may arise from either or both of these factors. We have separated effects of the 2'-hydroxyl and C-5 methyl groups by synthesizing nucleic acid strands which contain all possible combinations with and without these groups. Studies of the stabilities of double and triple helices involving these strands show that in fact the C-5 methyl group of thymine and the 2'-OH group of ribose have equally large effects on stability. The two effects vary with secondary structure and can be reinforcing or even opposing in their influence on stability. Three types of complexes are specifically examined: bimolecular pyrimidine.purine duplexes, termolecular pyrimidine.purine.pyrimidine triplexes, and bimolecular triplexes formed from circular pyrimidine oligonucleotides with purine target strands. It is found in general that the two types of substitutional effects are independent of one another and that C-5 methyl groups are in all cases stabilizing, while 2'-OH groups can be stabilizing or destabilizing, depending on the type of complex. In addition, studies with partially methylated duplexes lend evidence that the largest contribution to stabilization by the methyl group arises from increased base stacking ability rather than from a favorable hydrophobic methyl-methyl contact.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the properties of hydrophobic isosteres of pyrimidines and purines in synthetic DNA duplexes. Phenyl nucleosides 1 and 2 are nonpolar isosteres of the natural thymidine nucleoside, and indole nucleoside 3 is an analog of the complementary purine 2-aminodeoxyadenosine. The nucleosides were incorporated into synthetic oligodeoxynucleotides and were paired against each other and against the natural bases. Thermal denaturation experiments were used to measure the stabilities of the duplexes at neutral pH. It is found that the hydrophobic base analogs are nonselective in pairing with the four natural bases but selective for pairing with each other rather than with the natural bases. For example, compound 2 selectively pairs with itself rather than with A, T, G, or C; the magnitude of this selectivity is found to be 6.5-9.3 °C in Tm or 1.5-1.8 kcal/mol in free energy (25 °C). All possible hydrophobic pairing combinations of 1, 2, and 3 were examined. Results show that the pairing affinity depends on the nature of the pairs and on position in the duplex. The highest affinity pairs are found to be the 1-1 and 2-2 self-pairs and the 1-2 heteropair. The best stabilization occurs when the pairs are placed at the ends of duplexes rather than internally; the internal pairs may be destabilized by imperfect steric mimicry which leads to non-ideal duplex structure. In some cases the hydrophobic pairs are significantly stabilizing to the DNA duplex; for example, when situated at the end of a duplex, the 1-1 pair is more stabilizing than a T-A pair. When situated internally, the affinity of the 1-1 pair is the same as, or slightly better than, the analogous T-T mismatch pair, which is known to have two hydrogen bonds. The studies raise the possibility that hydrogen bonds may not always be required for the formation of stable duplex DNA-like structure. In addition, the results point out the importance of solvation and desolvation in natural base pairing, and lend new support to the idea that hydrogen bonds in DNA may be more important for specificity of pairing than for affinity. Finally, the study raises the possibility of using these or related base pairs to expand the genetic code beyond the natural A-T and G-C pairs.

Described are studies systematically exploring structural effects in he use of ethylene glycol (EG) oligomers as non-nucleotide replacements for nucleotide loops in duplex and triplex DNAs. The new structurally optimized loop replacements are more stabilizing in duplexes and triplexes than previously described EG-based linkers. A series of compounds ranging in length from tris(ethylene glycol) to octakis(ethylene glycol) are derivatized as monodimethoxytrityl ethers on one end and phosphoramidites on the other, to enable their incorporation into DNA strands by automated methods. These linker molecules span lengths ranging from 13 to 31 Å in extended conformation. They are incorporated into a series of duplex-forming and triplex-forming sequences, and the stabilities of the corresponding helixes are measured by thermal denaturation. In the duplex series, results show that the optimum linker is the one derived from heptakis(ethylene glycol), which is longer than most previous loop replacements studied. This affords a helix with greater thermal stability than one with a natural T(4) loop. In the triplex series, the loop replacements were examined in four separate situations, in which the loop lies in the 5' or 3' orientation and the central purine target strand is short or extends beyond the loop. Results show that in all cases the loop derived from octakis(ethylene glycol) (EG(8)) gives the greatest stability. In the cases where the target strand is short, the EG(8)-linked probe strands bind with affinities in some cases greater than those with a natural pentanucleotide (T(5)) loop. For the cases where the target strand extends beyond the linker, the EG(8)-linked strands are much lower in the 5' loop orientation than in the 3' loop orientation. It is found that extension by one additional nucleotide in one of the bonding domains in the EG-linked series can result in considerably greater stabilities with long target strands. Overall, the data show that optimum loop replacements are longer than would be expected from simple distance analysis. The results are discussed in relation to expected lengths and geometries for double and triple helixes. The findings will be usefull in the design of synthetically modified nucleic acids for use as diagnostic probes, as biochemical tools, and as potential therapeutic agents.

Described are the design, synthesis, and structures of three nonpolar nucleoside isosteres to be used as probes of noncovalent bonding in DNA and as isosteric replacements for the natural nucleosides in designed nucleic acid structures. Reaction of substituted aryl Grignards with 3',5'-bis-O-toluoyl-α-deoxyibofuranosyl chloride and subsequent deprotection with sodium methoxide in methanol afforded the two β-C-nucleoside pyrimidine analogs 1 and 2. The dimethylindolyl nucleoside 3, a purine isostere, was obtained by a nucleophilic displacement on α-chlorodeoxyribofuranose by the sodium salt of 4,6-dimethylindole, followed by deprotection. Regio- and stereochemistry of the products were established with NOE difference spectra and (1)H NMR splitting patterns. Analogs 1 and 2 are nonpolar isosteres of thymidine, and nucleoside 3 is an isostere of 2-aminodeoxyadenosine, the triply-bonded Watson-Crick partner of thymidine. Semiempirical AM1 calculations were carried out to provide bond length information to assess structural similarities between the isosteres and their natural counterparts.

We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD >> RRR > RDR >> DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones. Triplex-forming circular RNAs represent a novel and potentially useful strategy for high-affinity binding of RNA.

The effects of changing solution conditions on the strength of triple helical complexes formed between pyrimidine-rich circular DNA oligonucleotides and their homopurine complements are examined. The complexes display properties different from those seen for other types of DNA triplexes.

Recent studies of DNA hairpin loops have shown considerable dependence of the stability on the sequence of the loop [Senior, M., Jones, R. A., & Breslauer, K. J. (1988a) Proc. Natl. Acad. Sci. U.S.A. 85, 6242-6246; Xodo, L. E., Manzini, G., Quadrifoglio, F., van der Marel, G., & van Boom, J. H. (1989) Biochimie 71, 793-803; Hirao, I., Nishimura, Y., Tagawa, Y., Watanabe, K., & Miura, K. (1992) Nucleic Acids Res. 20, 3891-3896]. Analogous studies have not, until now, been carried out with loops in triple helices. We report the results from experiments in which we examine the relative stabilities of pentanucleotide loops that bridge between the pyrimidine strands in DNA pyr.pur.pyr triple helices. There are two types of loops that are defined by the relative orientation of the purine strand: a 5'-loop and a 3'-loop. The sequences examined in this study are the bimolecular triplexes formed between 5'-dTTCTTTTCL1TTTL5CTTTTCTT (loop nucleotides are underlined, and L1 and L5 represent varied nucleotides) and the two purine strands, 5'-dAAGAAAAG-3' and 5'-dGAAAAGAA-3'. The first and last nucleotides in the loop are varied, since stacking interactions may be strongest at these positions [Senior et al., 1988a; Senior, M., Jones, R. A., & Breslauer, K. J. (1988b) Biochemistry 27, 3879-3885], and we examine 14 sequence combinations for each loop type. Thermal denaturation studies carried out at pH 7.0 indicate considerable variation in the stabilities of these loops.(ABSTRACT TRUNCATED AT 250 WORDS)

Bimolecular triple-helical DNA complexes recently have found use in a new strategy for the recognition of single-stranded nucleic acids, in which circular (Kool, 1991; Prakash & Kool, 1992) or hairpin-shaped (Giovannangeli et al., 1991; D'Souza & Kool, 1992) oligonucleotides bind these single strands by triplex formation. Bimolecular triplexes may also be formed in vivo as H-DNA, where this structure may potentially play a role in gene expression and recombination (Belotserkovskii et al., 1990; Hanvey et al., 1989; Shimizu et al., 1989). In all of these complexes, the central strand of the triplex must pass beyond the loop that bridges the outer two strands, and models and preliminary experiments have indicated that there may be important interactions between this central strand and the loop (Prakash & Kool, 1992). We now report thermal denaturation studies carried out specifically to investigate these interactions in detail, using as a model the 5'-loop and 3'-loop complexes formed between 14 pyrimidine oligodeoxynucleotides having the sequence 5'-dTTCTTTTCL1TTTL5CTTTTCTT, where L1 and L5 represent varied nucleotides in the loop (which is underlined), and eight target strands having the sequence 5'-dCCCCFAAGAAAAG-3' or 5'-dGAAAAGAAFCCCCC-3', where F is a varied nucleotide flanking the triplex in the central strand. Results correlated from 64 different sequence combinations show that there is wide variation in the stabilities of the complexes, indicating specific and substantial interactions between the nucleotides at the L1, F, and L5 positions. Melting temperatures at pH 7.0 range from 17.0 degrees C to 34.6 degrees C, and free energies (37 degrees C) range from -3.2 to -7.8 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA8 were studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr.pur.pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 degrees C higher than Watson-Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.

It was recently reported that certain pyrimidine-rich circular DNA oligomers can bind strongly and specifically to purine-rich DNA or RNA strands by forming bimolecular triple helical complexes. 1-3 In this study are investigated the effects of structural variations on the strength of binding for this new class of nucleotide-binding ligand. The number of loop nucleotides (nt) which is optimum for bridging the two binding domains of a circle is examined. Comparing loop sizes of 3, 4, 5, 6, and 10 nt, the optimum number of nucleotides in a loop is found to be five for the sequences studied. In order to test the method of construction and the ability of these compounds to bind sites of varied length, we attempted to synthesize circles of varied size. Circles over the size range 24-46 nt were successfully constructed. Varying the target site length shows that oligomers of four, eight, twelve, and eighteen nucleotides can be complexed strongly by circles, with melting temperatures ( T m ) 17° to >33 °C higher at pH 7.0 than the corresponding Watson-Crick duplexes of the same length. Also studied is the effect of the covalently closed circular structure in comparison to linear oligomers having the same sequence; it is shown that a covalently closed circle has considerably higher binding affinity than do three different "nicked" circles (linear oligomers) which contain the same bases. The high binding affinities of these circles are thus attributed to the entropic benefit of preorganization. Finally, the ability of such circles to bind to complementary sites within longer oligomers, the ends of which must pass beyond the loops of a circle, is confirmed by melting studies with synthetic target strands 36 bases in length.

Pyrimidine-rich circular DNA oligonucleotides 1 and 2 display very high binding affinities for complementary DNA and RNA oligomers by forming bimolecular triple helical complexes.