Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2'-5' Linkages

Abstract

Information-bearing nucleic acids display universal 3'-5' linkages, but regioisomeric 2'-5' linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2'-5' linkages by an engineered polymerase using 3'-deoxy- or 3'-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3'-5' linked DNA with good fidelity. This enables a fast and simple method for "structural mutagenesis" by the position-selective incorporation of 2'-5' linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23 RNA endonuclease DNAzyme.

Keywords: DNAzyme; nucleic acids; nucleotides; polymerase; regioselectivity.

Figure 1

Structure of partially substituted a) 2′‐5′ DNA and b) 2′‐5′ RNA, with 3′O‐methyl groups as synthesized by polymerase TGLLK.

Figure 2

Enzymatic synthesis of partially substituted a) 2′‐5′ DNA b) 3′‐5′ RNA by TGLLK on a 57 nt template (TempN, see Supporting Information) encoding all possible dinucleotide combinations. Reactions in (a) involve dNTPs apart from 3′dA/G as indicated. Reactions in (b) involve NTPs apart from 3′OMeA/G as indicated. c),d) Error spectra of c) 3′dG/dHTP synthesis (aggregate misincorporation rate 5.08×10⁻⁴) and d) 3′OMe‐A/BTP synthesis (aggregate misincorporation rate 7.18×10⁻⁴). The columns show the misincorporation frequency for each incorrect nucleotide.

Figure 3

HPLC analysis of a fluorescent oligonucleotide that was synthesized by TGLLK either using 2′dATP or 3′dATP. Additional traces are corresponding chemically synthesized 2′‐5′ (3′dA)/3′‐5′ (2′dA) standards and an enzymatically synthesized 2′dA control. Standards and polymerase‐synthesized oligonucleotides were mixed in equimolar ratio and separated by HPLC under denaturing conditions.

Figure 4

Structural mutagenesis of enzymatically synthesized 10–23 DNAzyme variants. a) 10–23 DNAzyme/substrate pair used in this study. b) Cleavage of the fluorescent RNA (200 nm) after 1 h at 37 °C by different DNAzyme variants (40 nm) containing 2′‐5′ linkages downstream of G6 or G14 shown in panel (a). Blue boxes indicate 2′‐5′ linkages after 3′dG. c) Cleavage reaction progress of DNAzymes with increasing amounts of 3′dG substitutions. d) Cleavage after 1 h by different DNAzyme variants containing single 2′‐5′ linkages produced by position‐selective enzymatic synthesis.