Structural Optimization of Non-Nucleotide Loop Replacements for Duplex and Triplex DNAs

Abstract

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.

Figure 1

Structures and lengths of the six oligo(ethylene glycol)-based loop replacements in this study. The compounds are shown as the dimethoxytrityl (DMT) phosphoramidite derivatives which are used for incorporation into the DNA.

Figure 2

Haasnoot-type plots of the bridging distance between 5′-oxygens as a function of the base steps involved for B-duplex DNA, A-duplex RNA, and an A’-regular triplex, For duplexes, the distances are from one strand to the other; for pyr·pur·pyr triplex, the distance shown is from one pyrimidine strand to the other. The measurement starts from the * position in one strand and bridges to various 5′-O positions in the opposite strand. As an example, in a triplex, the distance from * to the −2 position (shown as a dashed line) is 15 Å.

Figure 3

Sequences and structures of complexes in this study (curved lines indicate ethylene glycol-derived loop replacements of varied length): (A) DNA duplexes with natural (left) and nonnatural (right) loops; (B) DNA triplexes formed between hairpin-type pyrimidine ligands and minimal eight-base purine target strands; (C) similar DNA triplexes formed with longer targets which extend beyond the loop.

Figure 4

Representative thermal denaturation curves of hyperchromicity (percent increase in absorbance at 260 nm) vs temperature for two types of complexes in this study: (A) DNA duplexes with EG_n linkers where n = 3–8 (see Figure 3A); (B) DNA triplexes formed between the probe strands 5′-dTTCTTTC-EG_n-CTTTTCTT (where n = 4–8) and the 8-mer target 5′-dGAAAAGAA.

Figure 5

Thermal melting temperature (T_m) versus the number of ethylene glycol units, n, for DNA hairpins formed from the sequence 5′-dCGAACG-EG_n-CGTTCG. The horizontal line (at 67.8 °C) indicated T_m fo the same sequence but bridged with a natural T₄ loop. See Table 1 for conditions.

Figure 6

T_m versus number of ethylene glycol (EG) units for triplexes formed between the probe strands 5′-dTTCTTTTC-EG_n-CTTTTCTT (where n = 4–8) and 8-mer target sequences at pH 7.0: (A) short target sequences, 5′-dAAGAAAAG (□) and dGAAAAGAA (○); (B) extended target sequences, 5′-dAAGAAAAGACCCCC (□) and 5′-dCCCCCAGAAAAGAA (○).

Figure 7

Effect of linker length (EG_n) on the binding of purine-rich targets by loop-modified circular DNAs at pH 7.0: (A) sequences of two complexes formed with short (top) and extended (bottom) target strands; (B) T_m versus number of EG units (n) for the two complexes (short targets, □; long targets, ○). See Table 4 for conditions.

Figure 8

Melting temperature versus number of ethylene glycol (EG) units for complexes formed between triplex-forming probe strands and short or extended target sequences at pH 7.0: (A) the binding of 5′-dGAAAAGAA by probe strands 5′-dTTCTTTTC-EG_n-CTTTTCTT (□), 5′-dTTCTTTTC-EG_n-T-CTTTTCTT (○), and 5′-dTTCTTTTC-T-EG_n-CTTTTCTT (×); (B) the binding of 5′-dAAGAAAAG by the same three probe strands; (C) the binding of 5′-dCCCCCAGAAAAGAA by probe strands 5′-dTTCTTTTC-EG_n-CTTTTCTT (□), 5′-dTTCTTTTTC-EG_n-T-CTTTTCTT (○), and 5′-dTTCTTTTC-T-EG_n-CTTTTCTT (×); (D) the binding of 5′-dAAGAAAAGAACCCCC by the same three probe strands.