Chapter Six - Enzymatic synthesis and modification of high molecular weight DNA using terminal deoxynucleotidyl transferase
Introduction
Beyond their role as the genetic material of living things, nucleic acids (DNA and RNA) are now being used to create synthetic polymeric materials for use in nanotechnology (Bae, Kocabey, & Liedl, 2019; Liu, Jiang, Wang, & Ding, 2019; Suo et al., 2019; Zhao et al., 2019), biosensing (Abolhasan, Mehdizadeh, Rashidi, Aghebati-Maleki, & Yousefi, 2019; Mason, Tang, Li, Xie, & Li, 2018; Tjong, Yu, Hucknall, Rangarajan, & Chilkoti, 2011) and drug delivery (Liu et al., 2019; Mathur & Medintz, 2019). Nucleic acids are versatile due to their customizable sequences and specific molecular recognition. Applications of DNA and RNA nanotechnology often require chemical and enzymatic polynucleotide modifications such as the incorporation of fluorescent groups, reactive groups including azides and amines, biofunctional groups such as biotin, and sugar modifications such as 2′-O-methyl (2′-OMe) or 2′-deoxy-2′-fluoro-ribonucleotide (2′-F) to bestow RNA with nuclease stability for RNA interference (RNAi) and CRISPR-based genome editing (Yin et al., 2017).
Modifications of DNA can be introduced chemically by using solid-phase synthesis or enzymatically by using a DNA polymerase together with unnatural deoxynucleotidyl triphosphates (dNTPs). Solid-phase synthesis is widely used when large quantities are desired, but is limited by the oligonucleotide length that can be produced (~ 200 nt) and by the compatibility of functional groups with the chemical reactions used in oligonucleotide synthesis (Rothlisberger & Hollenstein, 2018). Enzymatic DNA synthesis methods such as PCR (Rittié & Perbal, 2008; Terpe, 2013; Vosberg, 1989), primer extension (Carey, Peterson, & Smale, 2013) and rolling circle amplification (RCA) (Dean, Nelson, Giesler, & Lasken, 2001) typically use polymerases that have limited tolerance for chemically modified dNTPs (Houlihan, Arangundy-Franklin, & Holliger, 2017) and that require a template to catalyze primer extension. In contrast, terminal deoxynucleotidyl transferase (TdT) is a template-independent polymerase which tolerates chemically modified dNTPs and thus is suitable for the synthesis of chemically modified, high molecular weight DNA. This mechanism is harnessed in TdT catalyzed enzymatic polymerization (TcEP) for the synthesis of single-stranded polynucleotides (Tang, Navarro Jr., Chilkoti, & Zauscher, 2017) (Fig. 1).
TdT is a member of the X family of DNA polymerases, whose members add dNTPs randomly to the 3′-hydroxyl group (3′-OH) of single-stranded DNA (Bollum, 1960). Biologically, TdT contributes to the diversity of immunoglobulins and T cell receptors (TCR) by participating in the rearrangement of variable (V), diversity (D), and joining (J) segments within immunoglobulin and TCR genes, as briefly described next.
The vertebrate adaptive immune system consists of B cells and T cells that express a vast repertoire of antigen receptors (Alberts et al., 2002; Owen, Punt, Stranford, Jones, & Kuby, 2013). Binding of these receptors to antigens on the surfaces of pathogens or foreign bodies triggers a cascade of responses that activates the immune system. Rather than encoding the huge number of antigen-binding receptors with individual genes, cells use a gene rearrangement process called V(D)J recombination. This process is orchestrated by polymerases, nucleases, and ligases. TdT participates in the second stage of the process, following a double-stranded break at a highly conserved recombination signal sequence (RSS) that is induced by the recombination-activating gene (RAG) enzyme and the endonuclease, Artemis (Jung & Alt, 2004; Nick-McElhinny et al., 2005). Cleavage at the RSS by RAG results in the formation of a hairpin structure following transesterification of the 3′-OH of a nicked strand with a phosphorous on the opposite strand. Artemis opens the hairpin and TdT adds ~ 1–10 random nucleotides (Schatz, Oettinger, & Schlissel, 1992). TdT preferentially adds dGTP and dCTP, resulting in a GC-rich region in these genes. This GC bias promotes efficient annealing of single-stranded DNA in the subsequent ligation.
As we show and discuss in more detail in Section 3, TdT catalyzed enzymatic polymerization can be harnessed for the synthesis of high molecular weight single-stranded homo- and co-polynucleotides in solution and from surfaces (Chow et al., 2005, Gu et al., 2017; Tang et al., 2014; Tang et al., 2017; Tjong et al., 2011). These polyelectrolytes hold great promis for applications ranging from drug delivery to biosensors. Very recently it was shown that TdT can be employed in the synthesis of sequenced polynucleotides, overcoming some of the limitations of current solid phase olionucleotide synthesis (Lee, Kalhor, Goela, Bolot, & Church, 2019).
Section snippets
Biochemical requirements for TdT mediated polymerization
A TdT-catalyzed enzymatic polymerization reaction consists of an oligodeoxynucleotide as the initiator, natural or unnatural dNTPs, TdT, and buffer supplemented with a metal ion cofactor (Fig. 1) (Bollum, 1960, Bollum, 2006; Kato, Goncalves, Houts, & Bollum, 1967). This section will describe each of these components in TcEP reactions in more detail.
TdT catalyzed polynucleotide synthesis in solution
Tang et al. demonstrated that TcEP proceeds by a living chain-growth polycondensation mechanism, and thus the molecular weight of the product can be tuned by changing the feed ratio of nucleotide (monomer) to oligonucleotide (initiator) (M/I ratio) (Tang et al., 2017). Taking advantage of the template-free, promiscuous polymerization ability of TdT, single stranded polynucleotides with narrow molecular weight distribution and containing various unnatural nucleotides, including amines, alkynes,
Materials
T50 and 5′ end-modified oligonucleotides can be procured from Integrated DNA Technology (IDT). Suppliers including Jena Biosciences, TriLink Biotechnologies, Sigma Aldrich and Thermo Fisher Scientific provide natural dNTPs as well as a range of unnatural dNTPs. Terminal deoxynucleotidyl transferase (TdT) and TdT buffer can be purchased from Promega. Ethylenediaminetetraacetic acid (EDTA) (0.5 M, pH 8) solution, sulfuric acid, nitric acid, sodium acetate, sodium chloride, sodium citrate, acetone,
Protocol
- 1.1
Prepare reaction solution containing 0.5 μM oligo initiator (e.g., T50), natural or unnatural dNTP (the final dNTP concentration depends on the desired length of the polynucleotide), and 1 × TdT reaction buffer (100 mM potassium cacodylate, 1 mM CoCl2, and 0.1 mM DTT, pH 6.8). Then add 1.2 U/μL TdT and pipette to mix (DO NOT VORTEX!). If the amount of TdT used is too low, the reaction will yield polydisperse polynucleotides.
- 1.2
Incubate overnight at 37 °C.
- 1.3
Heat the reaction mixture at 90 °C for 3 min or at 70
Protocol for SIEP on flat gold substrates using TdT (Chow et al., 2005)
- 1.1
Clean the gold substrate by sequential sonication in Milli-Q water, acetone, and ethanol at 50 °C with intervening rinse steps. Dry the substrate in a stream of dry N2 and store it in a clean, covered petri dish.
- 1.2
Prepare 10 μM 5′-thiol modified oligonucleotide in 1 × PBS. Drop cast the initiator solution on the gold surface (for reference: a 1 cm2 gold surface needs ~ 100 μL solution for full coverage). Cover the petri-dish and incubate the substrate at room temperature overnight.
- 1.3
Remove the solution
Summary
Template-independent terminal deoxynucleotidyl transferase catalyzed enzymatic polymerization (TcEP) provides an exciting alternative to conventional enzymatic and solid-phase DNA syntheses. Specifically, the tolerance of TdT for unnatural dNTPs can be harnessed to introduce a broad range of functional groups into polynucleotides and thus provides a means to synthesize functionalized polynucleotides for a broad range of applications. In this chapter we provided a brief background on TdT, and
Acknowledgments
This work was supported by NSF DMR-1411126, NSF DMR-1121107, and NIH 5 R21 EB 026590 (Trailblazer). We thank Loren Baugh for editorial comments.
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Contributed equally to this work.