This report analyzes the theoretical limits of DNA sequence discrimination by linked …

• Abstract
• Introduction
• Methods
• Results and Discussion
• References
• Figures

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Figures

- The theoretical limits of DNA sequence discrimination by linked polyamides

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Fig. 1.   Linked polyamides comprised of imidazole and pyrrole rings have been synthesized by using two methods: with a hairpin linkage (a) or stapled through a central linkage (b). When bound in the DNA minor groove, the amide groups form hydrogen bonds with the bases, positioning the heterocyclic rings directly adjacent to the edges of the bases. Hydrogen bonding and steric contacts between the rings and the bases give each ring its particular base specificity. The molecules shown here contain two imidazole rings (stippled) and four pyrrole rings (unstippled). The hairpin-linked polyamide binds to sequences of the form C-A/T-G, where A/T refers to degenerate binding to either adenine or thymine. The stapled polyamide with the same rings will bind with a shifted phasing of the rings to sequences of the form C-A/T-A/T-G. In both cases, the actual target sequence will include an additional A/T bp at each end (in parentheses), which is recognized by atoms in the tails and linkers of the polyamides.

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Fig. 2.   Binding fraction for polyamides composed of two different types of rings. (a) For polyamides composed of a ring that recognizes adenine and a ring that recognizes thymine, target sequences composed of only AT bp may be recognized with a binding fraction close to one for rings with strong base specificity. A given level of binding may be obtained with an adenine-specific ring and a thymine-specific ring of identical specificity (point marked "A") or with a placeholder and an adenine-specific ring of twice that strength ("B"). (b) For polyamides composed of a ring that recognizes adenine and a ring that recognizes guanine, binding to a sequence with 4-AT and 1-GC bp, the maximal binding fraction is 0.5. To achieve this fraction, the polyamide must be designed from a strong adenine-specific ring and a weaker guanine-specific ring ("A"). If the guanine-specific ring is replaced by a placeholder, the maximal binding fraction drops to 0.25 ("B"). If the guanine-specific ring is stronger than the adenine-specific ring, the binding fraction drops to close to zero ("C").

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Fig. 3.   Binding fraction for polyamides composed of imidazole- and pyrrole-type rings. (a) Binding to a sequence with 4-AT and 1-GC bp. (b) Binding to a sequence with 1-AT and 4-GC bp. The value for the optimal polyamide composed of pyrrole and imidazole rings, with imidazole placed next to each guanine and pyrrole next to adenine, cytosine, or thymine in the target sequence, is shown with an "X."

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Fig. 4.   Performance of polyamides composed of an adenine-specific ring, a guanine-specific ring, and a placeholder ring. Contours of 0.5 for the binding fraction are shown for sequences with different AT and GC content. The best choice for a multifunctional set of rings is at the point where the curves cross, with adenine- and guanine-specific rings of equal strength.

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Fig. 5.   Effect of polyamide length on binding fraction. A polyamide with three pairs of rings is compared with a polyamide with four pairs of rings. (a) Values of ₃ and ₄ for different values of the base specificity . (b) Values of ₃/₄ for two comparisons: with equal concentrations of the two polyamides, [P₃] = [P₄], with values chosen such that the saturation of the target site ₃ is 0.97 (at this concentration, ₄ = 0.999); and at equal saturation of target sites, ₃ = ₄ = 0.97. Values of ₃/₄ greater than four indicate that the shorter molecule is the optimal choice in the comparison. The complex nature of the equal concentration ₃/₄ graph at low  values, where ₃ and ₄ are close to zero, is due to a set of stepwise increases of ₃ and ₄ with very small magnitude. These points are not relevant to polyamide design, as the polyamides would have very low specificity.

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