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. 2001 Jul 18;123(28):6961-3.
doi: 10.1021/ja015873n.

The generality of DNA-templated synthesis as a basis for evolving non-natural small molecules

Z J Gartner  1 , D R Liu
Affiliations

The generality of DNA-templated synthesis as a basis for evolving non-natural small molecules

Z J Gartner et al. J Am Chem Soc. .
No abstract available

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Figures

Figure 1
Figure 1
Synthesis directed by hairpin (H) and end-of-helix (E) DNA templates. Reactions were analyzed by denaturing PAGE after the indicated reaction times. Lanes 3 and 4 contained templates quenched with excess β-mercaptoethanol prior to reaction.
Figure 2
Figure 2
Matched (M) or mismatched (X) reagents linked to thiols (S) or primary amines (N) were mixed with 1 equiv of template functionalized with the variety of electrophiles shown. Reactions with thiol reagents were conducted at pH 7.5 under the following conditions: SIAB and SBAP: 37 °C, 16 h; SIA: 25 °C, 16 h; SMCC, GMBS, BMPS, SVSB: 25 °C, 10 min. Reactions with amine reagents were conducted at 25 °C, pH 8.5 for 75 min.
Figure 3
Figure 3
(a) H templates linked to α-iodoacetamide group were reacted with thiol reagents containing 0, 1, or 3 mismatches at 25 °C. (b) Reactions in (a) were repeated at the indicated temperature for 16 h. Calculated reagent Tm: 38 °C (matched), 28 °C (single mismatch).
Figure 4
Figure 4
(a) The depicted reaction was performed using a 41-base E template and a 10-base reagent designed to anneal 1–30 bases from the 5′ end of the template. The kinetic profiles in the graph show the average of two trials (deviations < 10%). The “n = 1, mis” reagent contains three mismatches. (b) The n = 10 reaction in (a) was repeated using templates in which the nine bases following the 5′-NH2-dT were replaced with the backbone analogues shown. Five equivalents of a DNA oligonucleotide complementary to the intervening bases was added to the “DNA + clamp” reaction. Reagents were matched (0) or contained three mismatches (3). The gel shows reactions at 25 °C after 25 min.
Figure 5
Figure 5
The n = 1, n = 10, and n = 1 mismatched (mis) reactions described in Figure 4a were repeated with template and reagent concentrations of 12.5, 25, 62.5, or 125 nM.
Figure 6
Figure 6
A model translation, selection, and amplification of synthetic molecules that bind streptavidin from a DNA-encoded library.
Figure 7
Figure 7
(a) Lanes 1 and 5: PCR: amplified library before stretptavidin binding selection. Lanes 2 and 6: PCR amplified library after selection. Lanes 3 and 7: PCR amplified authentic biotin-encoding template. Lane 4: 20 bp ladder. Lanes 5–7 were digested with Tsp45I. DNA sequencing traces of the amplified templates before and after seletion are also shown, together with the sequences of the non-biotin-encoding and biotin-encoding templates. (b) General scheme for the creation and evolution of libraries of non-natural molecules using DNA-templated synthesis, where –R1 represents the library of product functionality transferred from reagent library 1, and –R1B represents a selected product.
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