The high specificity of incorporation of nucleotides into DNA by polymerase enzymes is crucial for maintaining fidelity of information transfer in cellular replication. The initial insertion event is the first point at which mutations of the genome are avoided. 1,2 Mismatched pairing at this step occurs on the level of only ∼1 in 10 3 -10 5 insertions, indicating a selectivity of at least 4 kcal/mol. 1 It is clear that polymerases enhance the selectivity of nucleotide choice at the active site relative to the much lower pairing differences observed at the duplex terminus in the absence of enzyme. 2 While many of the kinetic details of replication have been studied in recent years, 3 the precise physical origins of this selectivity enhancement are poorly understood. Mechanisms involving both kinetic and binding selectivity between correct and incorrect nucleotides have been proposed. 3 Base-base hydrogen bonding, base stacking, base pair geometry, and interactions between the enzyme, DNA, and nucleotides have all been invoked as potentially important interactions; however, the relative importance of these different effects remains unclear. Many DNA nucleotide analogs with altered or reduced H-bonding potential have been examined as substrates for polymerases; 4 most of those analogs are quite poor substrates, and result in less discriminate incorporation fidelity than do the natural nucleotides. This general finding has been used as evidence that the number and strength of hydrogen bonds in a given pair determine efficiency and fidelity of DNA synthesis. Indeed, most if not all current models for replication fidelity hold that the specificity of hydrogen bonds formed in the new base pair is a central contributor to the observed selectivity.Here we present evidence, however, that a DNA polymerase can exert high fidelity even when a base pair completely lacks conventional hydrogen bonds. The difluorotoluene nucleoside 1 has recently been constructed as a nonpolar shape mimic for natural thymidine (2). 5,6 Its "base" moiety cannot measurably form paired complexes with adenine derivatives even in chloroform, a solvent in which H-bonded complexes are much more stable than they are in water. 7 When placed within a DNA strand paired opposite adenine, moreover, it actually destabilizes the helix by ∼4-5 kcal relative to thymine at the same position. In addition, 1 shows no inherent pairing selectivity among the four natural bases, also consistent with its nonpolar, nonhydrogen-bonding nature (ref 7 and work in progress). We felt therefore that 1 would serve as a good test for the importance of thymidine's hydrogen bonding groups on fidelity, because 1 lacks the strongly localized charges but retains nearly the exact steric shape of the natural molecule. If, as current models suggest, such polar interactions are important for achieving high fidelity, then 1 would be expected to be very inefficient and highly nonselective as a template for replication.© 1997 American Chemical Society * Author to whom correspondence should...

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 acidcatalyzed 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.

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.

The sequence-specific binding of RNA and DNA by synthetic oligonucleotides and analogues is important as a diagnostic tool in the study of naturally ocurring polynucleotides and is the subject of intense research as a potential therapeutic strategy as well. [1][2][3] Two limitations of natural DNA oligomers in these applications are their low binding affinity [2] and also their limited stability, due to cleavage by exo-and endonucleases which occur in natural systems. [4,5] We report here the construction of circular hybrid molecules which contain two oligonucleotide domains bridged by two oligoethylene glycol chains. These molecules bind with high affinity to complementary strands of RNA and DNA and display exceptional resistance to degradation by nucleases.Oligoethylene glycol chains have been successfully used as simple linking groups which replace nucleotide units in linear oligonucleotide chains [6] and in hairpin-shaped oligonucleotides in duplexes and triplexes as well. [7] We wished to determine whether such spacers could be incorporated into circular structures and what the effect on their recognition properties and stability would be. We have previously shown that circular oligonucleotides can display very strong binding affinity [8] and high sequence selectivity [9] for singlestranded DNA and RNA by forming bimolecular triple helices. These macrocycles bind by forming bonds on two sides of a linear target strand, and their unusual recognition characteristics arise from the preorganized circular structure. [10] Examination of models [11] and published data on DNA triple helices [12] revealed that a group 19-21 Å long would be ideal for bridging the outer pyrimidine strands, and that fivebase nucleotide loops might be replaced with penta-or hexaethylene glycol chains. In addition to confirming our model for the binding, such a replacement might lead to favorable properties, such as lower cost and higher synthesis yield (by decreasing the number of nucleotide units), longer lifetimes in biological media, and possibly, improved membrane permeability (by decreasing total negative charge).The linear precursors to the compounds investigated in this study 1-3 were constructed by automated DNA synthesis methods [13] (Experimental Procedure) with the introduction of the synthetic dimethoxytrityl hexa-or pentaethylene glycol (HEG or PEG) phosphoramidites [7] at two positions. The oligomers d(A) 12 (for 1) and

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.