Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase.

Erika Schaudy,Mark M. Somoza,Jory Lietard

doi:10.1021/acssynbio.1c00142

Abstract

The untemplated activity of terminal deoxynucleotidyl transferase (TdT) represents its most appealing feature. Its use is well established in applications aiming for extension of a DNA initiator strand, but a more recent focus points to its potential in enzymatic de novo synthesis of DNA. Whereas its low substrate specificity for nucleoside triphosphates has been studied extensively, here we interrogate how the activity of TdT is modulated by the nature of the initiating strands, in particular their length, chemistry, and nucleotide composition. Investigation of full permutational libraries of mono- to pentamers of d-DNA, l-DNA, and 2′O-methyl-RNA of differing directionality immobilized to glass surfaces, and generated via photolithographic in situ synthesis, shows that the efficiency of extension strongly depends on the nucleobase sequence. We also show TdT being catalytically active on a non-nucleosidic substrate, hexaethylene glycol. These results offer new perspectives on constraints and strategies for de novo synthesis of DNA using TdT regarding the requirements for initiation of enzymatic generation of DNA.

Highlights

The untemplated activity of terminal deoxynucleotidyl transferase (TdT) represents its most appealing feature
A related question is whether TdT is able to extend initiator molecules other than the 3′ terminus of DNA, enabling enzymatic synthesis of chimeric nucleic acid sequences, DNA/ RNA hybrids, or even conjugates where an unnatural initiator is extended with deoxynucleoside triphosphates (dNTPs) or rNTPs
TdT was found to catalyze the extension of oligonucleotide strands with a variety of modified nucleoside triphosphates, for instance biotinylated,[11,28,29] fluorescence-tagged,[30] photo-cross-linkable[31] or light-cleavable[21] dNTPs and non-nucleosidic substrates,[32] as well as fluorescent nucleobase analogues[33] and metal basepairs,[34] showing rather low substrate specificity in contrast to other DNA polymerases, which could be further loosened by protein engineering efforts.[35]

Summary

Introduction

The untemplated activity of terminal deoxynucleotidyl transferase (TdT) represents its most appealing feature. Terminal deoxynucleotidyl transferase (TdT) is a member of the polX family of DNA polymerases first purified from calf thymus glands.[1,2] In contrast to template-dependent DNA polymerases, TdT extends DNA strands at their 3′ hydroxy terminus in the presence of divalent cation cofactors[3] and deoxynucleoside triphosphates (dNTPs), but in the absence of a template strand This activity is of major importance in the diversification of immunoglobulins and T cell receptors in the process of V(D)J recombination of the adaptive immune system via random addition of nucleotides to nicked DNA strands.[4,5] TdT’s unique ability to mediate templateindependent polymerization has made it a valuable tool in a variety of molecular biology applications including finding strand breaks,[6] modifying DNA oligomers with various NTPs,[7] and identifying DNA damage and epigenetic modifications.[8] the enzyme has proven useful for the generation of polynucleotides of high molecular weight[9] and amphiphilic structures upon extension with BODIPY-dUTP,[10] for detection of DNA and RNA on surfaces,[11,12] and immobilization of DNA on solid supports.[13] In the context of synthetic biology, template-independent DNA polymerization by TdT is, along with enzyme-based approaches,[14] a promising alternative to chemical synthesis as many of the shortcomings of the phosphoramidite approach can be potentially avoided. Whereas nucleoside triphosphate substrate specificity is rather flexible, DNA analogues in the initiating strand seem to hamper extension, for instance upon replacement of natural DNA nucleotides at the 3′ terminus with L-DNA,[37] or when using RNA initiator strands.[38,39]

Methods

Results

Conclusion