Materials and Methods

Oligonucleotides, substrate preparation enzymes and reagents

DNA and RNA oligonucleotides used in the ligase assays were purchased from Metabion International AG (Planegg/Steinkirchen Germany) in lyophilized form. A full overview of the oligos and their sequences can be found in the supporting information (Table S1). The oligos were resuspended in Tris-EDTA (TE) buffer (pH 8.0) to prepare 100 µM stock solutions. Ligation substrates were prepared by combining acceptor, donor and template oligos in a 1:1:1 ratio and then hybridizing by heating at 95 °C for 4 minutes followed by cooling to 4°C at a rate of 1°C/min. Resulting duplexes were either used immediately or stored at -20 °C. A depiction of all substrates is shown in figure 2A and supporting information table S2. The DNA ligases R2D Ligase and T4 DNA Ligase were obtained from ArcticZymes Technologies AS (Tromsø, Norway). PBCV-1 Ligase (SplintR Ligase), T7 DNA Ligase, T3 DNA Ligase, T4 RNA Ligase 1 and T4 RNA Ligase 2 were obtained from New England Biolabs (NEB, Ipswich, Massachusetts, USA). Ligases named AZ ligase 1-4 are noncommercial prototype ligase variants of ArcticZymes Technologies AS.

Nick ligation substrate specificity assay

The substrate specificity assay was adapted from Bullard and Bowater (2006), and an identical set of oligonucleotides were used for substrate production (see supporting information). The substrate set was tested in standard reaction condition unless otherwise stated (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM ATP, 10 mM DTT, 25 mM KCl, 0.9 µM nicked substrate, 0.05 mg/ml BSA). Reactions were started by adding 5 µl enzyme to 5 µl reaction mix resulting in 10 µl in total and a final concentration of 15 µM ligase. The reactions were run at 25 °C for 15 minutes and terminated by heating the reactions to 75 °C for 10 minutes. The reactions were mixed 1:1 with 95 % formamide/10 mM EDTA to stop the reactions. Reaction products were detected by electrophoresis on 20% acrylamide/7 M Urea PAGE and imaging with GelDoc XR+ (BIO-RAD, Hercules, California, USA). Data analyses were performed in ImageLab 4.1 (BIO-RAD) and Microsoft Excel.

Characterization of optimal reaction conditions

Optimization of ATP, MgCl2 and MnCl2concentrations were performed similarly to assays above, using the S1 (DNA-to-DNA, DNA-splint), S7 (RNA-to-DNA, DNA-splint) and S8 substrates (DNA-to-RNA, DNA splint) (see supporting information). With respect to the linearity of the assay in the presence of different substrate compositions, the concentrations of R2D ligase chosen were 0.4 nM, 0.2 nM and 1.4 µM for substrates S1, S7 and S8, respectively. For MnCl2 optima, 50 nM R2D ligase was used for the S1 and S7 substrate, and 8 nM for the S8 substrate. The standard reaction buffer was identical to that described above, and ATP, KCl and MgCl2 concentrations were varied according to figure legend in the results section.

Ligation of DNA to both ends of RNA in a single reaction mixture

A single reaction mixture with DNA oligos being ligated to either side of an RNA was set up using four DNA oligos and a target 5’ phosphorylated RNA (D4-D7, R4, supporting information table S2). In addition, two substrates that only ligate DNA to either end of the RNA target were prepared (S10 and S11, figure 2A). The reactions were run using 2.5 µM of T4 DNA Ligase or R2D Ligase in an altered reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM MnCl2, 0.1 mM ATP, 25 mM KCl, 10 mM DTT, 0.05 mg/ml BSA, 0.9 µM substrate) and incubated at 25 °C for 30 minutes, then inactivated at 75 °C for 10 minutes. The samples were detected by Urea PAGE using a Pharos FX (BIO-RAD, Hercules, California, USA), using two channels for FAM and TAMRA respectively.