Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity

[Abstract] DNA polymerase θ (Pol θ ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Pol θ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non- templated nucleotide addition cycles. Since human Polθ is able to modify the 3’ terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3’ terminal ends of RNA and DNA for various applications in biotechnology and biomedical research.

Indeed, in a recent study Kent et al. demonstrated that the human Polθ polymerase domain, hereinafter referred to as Polθ, exhibits robust terminal transferase activity preferentially on single-strand DNA (ssDNA) and double-strand DNA containing 3' ssDNA overhangs, referred to as partial ssDNA (pssDNA) . This study also compared the terminal transferase activities of Polθ and terminal deoxynucleotidyl transferase (TdT) using their respective optimal conditions, and found that Polθ is a more versatile enzyme for modifying the 3' terminus of nucleic acids. For example, the authors showed that Polθ is able to modify nucleic acids with a wider variety of nucleotide analogs, such as those containing large fluorophores or attachment chemistries . As a specific example, Polθ was shown to efficiently modify ssDNA with a nucleotide analog containing click chemistry applicability (i.e., a linker attached to an azide group), whereas TdT failed to use the same nucleotide as a substrate . TdT was also unable to use a Texas Red conjugated nucleotide analog that Polθ efficiently utilized to modify ssDNA . Polθ is also capable of modifying the 3' terminal ends of RNA and appears to show a significantly lower discrimination against ribonucleotides compared to TdT . Altogether, this recent report demonstrates that Polθ is a more versatile terminal transferase enzyme than TdT and therefore should be more useful for a wide range of applications in biotechnology and biomedical research that require modification of 3' terminal DNA and RNA ends . Here, we explain in detail step-by-step procedures for using Polθ as a robust terminal transferase enzyme in vitro.

Materials and Reagents
A. The following reagents are needed for modifying nucleic acids with Polθ:    3. The 2 h incubation time specified in the above procedure will give rise to multiple (i.e., 3 to > 100) terminal transfer events for most canonical nucleotides. However, in some cases only a single transfer event may occur depending on the particular nucleotide analog used. For example, certain nucleotide analogs may not be efficiently incorporated by Polθ and thus limit the enzyme to a single nucleotide transfer event. For determining the number of terminal transferase events that occur on a given substrate in the presence of particular nucleotides, we recommend visualizing the initial nucleic acid substrate and nucleic acid reaction products in a denaturing sequencing gel as described below in the Data analysis section.
B. Examples of experimental procedures for modifying the 3' terminal ends of DNA and RNA using or RNA (sequence indicated; Figure 1C) was mixed with 50 µM (Figures 1A and 1B) or 500 µM. b. Initial nucleic acid substrates and reaction products should be resolved in standard urea denaturing 10-20% polyacrylamide gels. Helpful protocols for pouring and processing urea denaturing polyacrylamide sequencing gels are referenced here (Summer et al., 2009;Flett, et al., 2013). Large sequencing gels will allow for the highest resolution (i.e., single nucleotide resolution). However, smaller gels may provide enough resolution depending on the particular application. In the case of RNA, we recommend adding 10-15% formamide to urea denaturing polyacrylamide gels to reduce RNA secondary structures that can appear as smears in the gel. Large sequencing gels are typically run at 70-80 W using a high voltage (5,000 V) power supply. The resolved nucleic acids can then be visualized using a fluorescent imager (for 5' fluorophore conjugated oligos) or using a phosphorimager or autoradiography (for 5' 32 P labeled oligos). Figure 1 shows examples of 5' 32 P radio-labeled nucleic acids that were resolved in large sequencing gels, then visualized by autoradiography.

Reproducibility
In our experience, extension of nucleic acids by Polθ is highly reproducible. However, we note that the precise amount of initial substrates extended may vary. For example, in some cases 100% of nucleic acid substrates are extended, whereas in other cases a small fraction (i.e., ~5-15%) of substrates are not extended. A 4-5 fold higher ratio of Polθ to nucleic acid substrates will usually allow for the majority of substrates to be extended. We note that the number of nucleotides transferred to the 3' terminus of nucleic acids may vary. Thus, the final length of extended nucleic acids will not be identical for all molecules. The respective structures of canonical nucleotides and nucleotide analogs will also give rise to different terminal transferase efficiencies. For example, deoxyadenosine monophosphate is most efficiently transferred by

Additional notes, technical tips and cautionary points
For optimal Polθ terminal transferase activity, we recommend storing the enzyme in buffer B (see Recipes) at concentrations ≥ 1 mg/ml in small aliquots at -80 °C and limiting freeze thaw cycles to 2-3 times. We note that oligonucleotides relatively short in length (< 10 nt) may not be extended as efficiently as those longer in length (> 10 nt). Oligos containing a high proportion of closely spaced guanosine bases, for example similar to telomere repetitive DNA sequences or those that form G quadruplexes, may exhibit a lower efficiency of extension by Polθ (Kent et al., 2016). As noted above, Polθ can also be used to modify double-stranded DNA, however, only 1-3 nucleotides are generally transferred to these substrates .