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.