An in vitro translation, selection and amplification system for peptide nucleic acids

Abstract

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template, (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures, (iii) selection of synthetic polymer libraries for desired binding or catalytic properties and (iv) amplification of template sequences that survive selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of 12 PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection and amplification on a library of 4.3 x 10(8) PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.

Figure 1. Strategies to evolve biological and synthetic polymers

(a) Evolution of a biopolymer in nature or in the laboratory. Biological polymers are translated from and spatially associated with an information carrier (typically DNA). The biopolymers undergo selection based on their functional properties. The information encoding surviving biopolymers replicates and mutates inside or outside of cells, resulting in a second generation of biopolymer variants related to those that survived selection. (b) Synthetic polymers can in principle undergo a similar evolutionary process. Translation could be effected by enzymatic or non-enzymatic templated synthesis in a manner that associates each synthetic polymer with its information carrier. Following selection, the information carriers encoding surviving synthetic polymers are amplified and mutated to generate templates for a subsequent round of translation. (c) Representative polymerization of a PNA aldehyde building block (H₂N-acact-CHO) on a complementary DNA template. Fig. 1a and Fig. 1b are adapted from Ref. .

Figure 2. A PNA genetic code that exhibits uniform DNA-templated polymerization

Ten consecutive DNA-templated reductive amination couplings result in PNA 50-mers containing secondary amine linkages between every fifth nucleotide. The efficiency of this process is shown at 25 °C, 45 °C and 60 °C for the 12 PNA pentamer aldehydes shown. Reaction conditions: 16 μM PNA aldehyde and 0.4 μM DNA templates were heated to the specified temperature. NaBH₃CN was added to 80 mM. After 20 min, the reactions were analyzed by denaturing PAGE and visualized by staining with ethidium bromide. Except for the cases noted below in which repetitive template sequences adopted strong secondary structures, the coding region of the DNA templates contained ten consecutive repeats of the codon complementary to the building block shown. *DNA template: (AACGTAACAC)₅; **DNA template: (AGTACAACCA)₅; ^†DNA template: (ATGCAAACCA)₅; ^‡DNA template: (ATGCAAACCA)₅. In these four cases, 8 μM of the assayed building block was supplemented with 8 μM of the H₂N-tggtt-CHO building block.

Figure 3. Sequence-specific DNA-templated PNA polymerization using all 12 building blocks in a variety of contexts

The four templates shown each contain six different codons and collectively use each of the 12 codons in two different contexts. Each gel lane shows a polymerization reaction containing 11 free amine PNA aldehyde building blocks and one capped (N-acetylated) PNA building block. Because the capped building block uniquely terminates polymerization, the length of the resulting PNA oligomer reveals the sequence specificity of the building block’s coupling. Reaction conditions: 7 μM of each PNA aldehyde and 0.4 μM DNA templates were heated to 95 °C and cooled to 37 °C. NaBH₃CN was added to 80 mM. After 60 min at 37 °C the reactions were quenched and analyzed by denaturing PAGE and visualized by staining with ethidium bromide.

Figure 4. Translation of a DNA template into PNA and displacement of the resulting PNA strand

Following PNA polymerization (lane 5) under conditions described in Figure 2, the PNA strand is displaced by primer extension of the 3′ hairpin using Herculase DNA polymerase II and dNTPs (lane 6). DNA polymerization to the end of the template strand (through the 5′ stem-loop) generates an 84-base pair DNA duplex that can form as a mutually exclusive alternative to the 40-base pair PNA-DNA heteroduplex and 24-base pair DNA-DNA stem. Successful strand displacement creates a double-stranded BccI restriction endonuclease cleavage site. The BccI-mediated cleavage of the primer extension product (lanes 4 and 8) is therefore consistent with displacement of the PNA strand. The red dot represents a Cy3 backbone spacer that blocks subsequentual DNA polymerization. The green dot represents a fluorescein group. The denaturing PAGE gel shown is visualized by imaging fluorescein fluorescence. Reactions shown in lanes labeled “translation–” lacked NaCNBH₃; those in lanes labeled “displacement–” lacked DNA polymerase; those in lanes labeled “restriction digestion–” lacked BccI.

Figure 5. A full cycle of translation, displacement, simulated selection, and PCR amplification for a single DNA template encoding a PNA 40-mer

Double-stranded DNA template (lane 1) was immobilized on streptavidin-linked beads and the non-biotinylated coding strand (lane 2) was eluted. Two hairpins were ligated onto the coding strand (lane 5) to generate a template ready for translation into PNA (lane 6) and displacement of the resulting PNA strand (lane 7). A small amount (1/15,000th) of the translated and displaced material was subjected to PCR, regenerating the double-stranded template (lane 8). Lanes 3 and 4 show hairpin ligation reactions with only one hairpin. The denaturing PAGE gel shown was visualized by ethidium bromide staining.

Figure 6. Model selection of a DNA-templated PNA 40-mer library

To a library of DNA templates encoding 4.3 × 10⁸ PNA 40-mers containing eight consecutive building blocks was added 10⁻², 10⁻⁴, or 10⁻⁶ equivalents of a DNA template uniquely containing an AATCC codon (red) that translates into a biotinylated H₂N-ggatt-CHO PNA building block. The positive control DNA template, but not the other templates, contains an MspI cleavage site. The resulting library of DNA templates was subjected to multiple iterated rounds of translation, PNA strand displacement, in vitro selection for binding to immobilized streptavidin, and amplification by PCR (Fig. 5). MspI digestion of the PCR-amplified DNA before and after selection reveals that the selection resulted in > 1,000,000-fold enrichment for the positive control sequence encoding the biotinylated PNA. A similar model selection using 10⁻² equivalents of the positive control template but omitting the biotinylated building block during translation (-biotin lanes) results in no enrichment of the positive control sequence. Y′ = mixture of AC, GT, and TG; X′ = mixture of AC, GT, TG, and CA. The images shown are from 2.5% agarose gels stained with ethidium bromide. The complete set of gel images analyzing all model selections are provided in Supplementary Fig. 3.

Figure 7