Enzymatic synthesis of biphenyl-DNA oligonucleotides

Highlights

• Synthesis of biphenyl-modified nucleoside triphosphate.

• Stereoelectronic environment of distal ring in template influences incorporation.

• Two-step protocol for enzymatic synthesis of oligonucleotides containing C-nucleotides.

• TdT-mediated tailing reactions.

Abstract

The incorporation of nucleotides equipped with C-glycosidic aromatic nucleobases into DNA and RNA is an alluring strategy for a number of practical applications including fluorescent labelling of oligonucleotides, expansion of the genetic alphabet for the generation of aptamers and semi-synthetic organisms, or the modulation of excess electron transfer within DNA. However, the generation of C-nucleoside containing oligonucleotides relies mainly on solid-phase synthesis which is quite labor intensive and restricted to short sequences. Here, we explore the possibility of constructing biphenyl-modified DNA sequences using enzymatic synthesis. The presence of multiple biphenyl-units or biphenyl residues modified with electron donors and acceptors permits the incorporation of a single dBphMP nucleotide. Moreover, templates with multiple abasic sites enable the incorporation of up to two dBphMP nucleotides, while TdT-mediated tailing reactions produce single-stranded DNA oligonucleotides with four biphenyl residues appended at the 3′-end.

Introduction

DNA is a fascinating biopolymer that mainly serves as the repository of the genetic information. The well-known double helical structure adopted by DNA arises through specific Watson-Crick interactions and packing forces between the four canonical nucleobases A, C, G, and T. However, a combination of organic chemistry and polymerase engineering have not only permitted to broaden the horizon of applications of DNA to fields that markedly deviate from its natural role1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 but also to expand the genetic alphabet by one13., 14, 15, 16 or multiple¹⁷ orthogonal, artificial base pairs. Unnatural base pairs (UBPs) that have been used in this context mainly consist of large aromatic residues that interact via hydrophobic and packing forces,¹⁸ nucleobases with alternative hydrogen bond patterns,¹⁷ and nucleoside analogs capable of interacting through shape complementarity.19, 20 More recently, artificial metal base pairs have been proposed as alternative scaffolds for the construction of UBPs.21, 22, 23, 24., 25., 26, 27, 28 Such an expansion of the genetic alphabet has important practical repercussions since semi-synthetic organisms capable of replicating DNA containing artificial nucleotides²⁹ and even transcribing modified mRNA into proteins¹⁴ could be created and aptamers with enhanced binding properties were identified.13., 20, 30, 31 In this context, the zipper‐like interstrand stacking motif (Fig. 1A) is an alternative recognition motif based on the interaction of C-nucleotides equipped with bipyridine (Bipy) or biphenyl (dBph) moieties within a DNA duplex (Fig. 1B).32, 33., 34 The inclusion of four biphenyl base pairs in opposing positions in short DNA duplexes gave rise to important thermal stabilities (ΔT_m of up to 4.4 °C per base pair).³⁴ Interestingly, up to 7 consecutive biphenyl base pairs can be incorporated into DNA without causing any decrease in duplex stability³² and an NMR investigation concluded that stacking interactions were the main driving force behind the impressive stability of these UBPs.³⁵ The zipper-like motif thus appears to be an alluring candidate for applications such as electron transport through nucleic acids36., 37., 38 or an expansion of the genetic code.³⁹ Besides biphenyl-modified DNA, other C-nucleosides equipped with aromatic residues play central roles in numerous applications including the induction of important conformational transitions,⁴⁰ relaying electron transport through duplexes,37., 38, 41., 42, 43 assisting the homogenous assembly of aromatic carbon layers,⁴⁴ or labeling of oligonucleotides with fluorophores.45, 46, 47, 48, 49 However, solid-phase synthesis of these analogs and biphenyl-DNA particularly is quite labor intensive and often complicated by the reduced solubility and the tendency to aggregate. Therefore, alternative methods such as enzymatic synthesis of dBph containing oligonucleotides would be highly desirable.³² Here, we explored the possibility of using enzymatic synthesis to generate oligonucleotides containing multiple dBph nucleotides. To this end, we have investigated whether 1. the formation of a zipper‐like interstrand stacking motif could favor the successive incorporation of dBph nucleotides during primer extension (PEX) reactions and 2. the presence of multiple abasic sites on a template strand could be used to trigger the incorporation of multiple C-nucleotides into DNA. In this context, we demonstrate that the presence of three dBph residues on the template permits the incorporation of one modified nucleotide into DNA and that the introduction of abasic sites can be used to generate oligonucleotides containing up to two modified dBph analogs. We also show that the appendage of electron donors and acceptors on the distal ring of the biphenyl moiety can be used to improve the efficiency of enzymatic synthesis of DNA modified with aromatic C-nucleotides. Taken together, these results show that the enzymatic synthesis of oligonucleotides containing biphenyl C-nucleotide analogs is possible and could be extended to other zipper-like motifs50, 51, 52, 53, 54 and potentially for the construction of randomized libraries containing UBPs for SELEX experiments.30, 55, 56, 57