An in vitro selection system for TNA

Abstract

(3'-2')-alpha-l-Threose nucleic acid (TNA) is an unnatural polymer that possesses the rare ability to base-pair with RNA, DNA, and itself. This feature, coupled with its chemical simplicity, makes TNA of interest as a possible progenitor of RNA during the early history of life. To evaluate the functional potential of TNA, we have developed a system for the in vitro selection of TNA. We identified the Therminator DNA polymerase as a remarkably efficient DNA-dependent TNA polymerase capable of polymerizing more than 50 tNTPs. We have also developed a method of covalently linking a DNA template to the TNA strand that it encodes, thus obviating the need for a TNA-dependent DNA polymerase during cycles of selection.

Figure 1

DNA-dependent TNA polymerization with the Therminator DNA polymerase. (A) Structures of TNA and RNA (B) Template sequence. Red = primer binding site; black = ssDNA template. (C) Time course of TNA polymerization. Reaction was performed with 5 nM primer/template, 60 μM tDTP, 31 μM tTTP, 18 μM tCTP, 2 μM tGTP, 1 unit ofTth pyrophosphatase, and 0.5 units of Therminator polymerase in 10 μL of 1× thermopol buffer (NEB) at 75 °C. Samples were boiled in 7 M urea and 400 mM NaOH and analyzed by 20% denaturing PAGE.

Figure 2

DNA-displayed TNA molecules. (A) In vitro selection scheme for TNA. (B) Synthetic DNA construct. Red represents loop region/strand displacement primer binding site. (C) TNA transcription of hairpin construct. 50 nM hairpin was transcribed with tNTPs for 12 h (∼13% fully extended) as in Figure 1C. Control reaction (+dNTPs) was performed for 10 min. Reactions were analyzed by 12% native PAGE. (D) Strand displacement (s.d.) of TNA strand. Full-length TNA-transcribed hairpins were gel-purified by native PAGE. End-labeled DNA primer (0.1 nM) was annealed to 1 nM transcribed hairpin by heating to 55 °C for 1 min in 1.1× thermopol buffer and 250 μM of each dNTP. One unit of Therminator was added, and the reactions were incubated for 30 min at 55 °C followed by 90 min at 75 °C. Restriction digests:  strand displacement reactions were diluted 4-fold in 1× thermopol buffer, and 10 units of restriction enzyme were added for 2 h at 37 °C. Reactions were analyzed by 20% denaturing PAGE. “No hairpin” lane is a primer extension without the hairpin template. “+ control” lane is a primer extension on a single-stranded version of the hairpin constuct.