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Synthetic Genetic Polymers Capable of Heredity and Evolution

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Science  20 Apr 2012:
Vol. 336, Issue 6079, pp. 341-344
DOI: 10.1126/science.1217622

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  1. Fig. 1

    Engineering XNA polymerases. (A) Sequence alignments showing mutations from Tgo consensus in polymerases Pol6G12 (red), PolC7 (green), and PolD4K (blue). (B) Mutations are mapped on the structure of Pfu (Protein Data Bank identification code: 4AIL). Yellow, template; dark blue, primer; orange, mutations present in the parent polymerase TgoT.

  2. Fig. 2

    HNA synthesis, MS analysis, and reverse transcription. (A) Structure of 1,5-anhydrohexitol (HNA) nucleic acids (B, nucleobase). (B) Pol6G12 extends the primer (p) incorporating 72 hNTPs against template T1 (table S3) to generate a full-length hybrid molecule with a 37,215-dalton expected molecular mass (27). MW, ILS 600 molecular weight marker. P, primer-only reactions. (C) Matrix-assisted laser desorption/ionization–time-of-flight spectrum of a full-length HNA molecule showing a measured HNA mass of 37,190 ± 15 daltons (n = 3 measurements). a.u., arbitrary units; m/z, mass-to-charge ratio. (D) HNA reverse transcription (DNA synthesis from an HNA template). Polymerase-synthesized HNA (from template YtHNA4) (table S3) is used as template by RT521 for HNA-RT (-* denotes a no HNA synthesis control to rule out template contamination).

  3. Fig. 3

    XNA genetic polymers. Structures, polyacrylamide gel electrophoresis (PAGE) of synthesis (+72 xnt), and reverse transcription (+93 nt) of (A) CeNA, (B) ANA, (C) FANA, and (D) TNA. (E) PAGE of LNA synthesis [primer (41 nt) + 72 lnt] and LNA-RT (red) resolved by alkali agarose gel electrophoresis (AAGE). LNA synthesis (green) migrates at its expected size (113 nt) and comigrates with reverse transcribed DNA (red) synthesized from primer PRT2 (20 nt) (fig. S8 and table S3). (F) AAGE of XNA and DNA polymers of identical sequence. MW, ILS 600 molecular weight markers. Equivalent PAGE is shown in fig. S5. (G) XNA RT–polymerase chain reaction (MW, New England Biolabs low molecular weight marker; NT, no template control). Amplification products of expected size (133 base pairs) are obtained only with both XNA forward synthesis and RT (RT521 or RT521K) (fig. S12).

  4. Fig. 4

    Characterization of HNA aptamers. Anti-TAR aptamer T5-S8-7 (HNA: 6’-AGGTAGTGCTGTTCGTTCATCTCAAATCTAGTTCGCTATCCAGTTGGC-4’) and anti-HEL aptamer LYS-S8-19 (HNA: 6’-AGGTAGTGCTGTTCGTTTAAATGTGTGTCGTCGTTCGCTATCCAGTTGGC-4’) were characterized by ELONA (27). (A and B) Aptamer binding specificity against TAR variants (red, sequence randomized but with base-pairing patterns maintained) and different protein antigens (human lysozyme, HuL; cytochrome C, CytC; streptavidin, sAV; biotinylated-HEL bound to streptavidin, sAV-bHEL). OD, optical density. (C) Affinity measurements of aptamer binding by SPR. RU, response units. (D) FACS analysis of fluorescein isothiocyanate (FITC)–labeled aptamers binding to plasmacytoma line J558L with and without expression of membrane-bound HEL (mHEL) (27). wt, wild type.