SplintR Ligase, also known as PBCV-1 DNA Ligase or Chlorella virus DNA Ligase, efficiently catalyzes the ligation of adjacent, single-stranded DNA splinted by a complementary RNA strand. This previously unreported activity may enable novel approaches for characterization of miRNAs and mRNAs, including SNPs. SplintR is ideally suited for many target enrichment workflows with applications in next-generation sequencing and molecular diagnostics. The robust activity of the enzyme and its affinity for RNA-splinted DNA substrates (apparent Km = 1 nM) enable sub-nanomolar detection of unique RNA species within a complex mixture, making SplintR ligase a superior choice for demanding RNA detection technologies.
The enzyme is active over a broad range of ATP concentrations (10 µM – 1 mM) and pH (6.5–9). Optimal activity is observed using Mg2+ > 5 mM, and a pH between 7.5 and 8.0. The activity is enhanced at higher temperatures (up to 37°C), and by supplementation with 5 mM Mn2+. The reaction is inhibited by salt concentrations in excess of 100 mM.
The enzyme tolerates all base pair combinations at the ligation junction, but is partially inhibited by dC/G and dG/C base pairs at the donor (phosphorylated) side ligation junction, particularly when the +2 base was also a C/G base pair.
Ligation of DNA splinted by RNA
(A) Outline of the ligation assay: a 5´-phosphorylated, 3´-FAM labeled DNA “donor” oligonucleotide and an unmodified DNA “acceptor” oligonucleotide are annealed to a complementary RNA splint. This substrate is reacted with a ligase to form a mixture of unreacted starting material (I), adenylylated DNA (II), and ligated product (III). These products are denatured, separated by capillary electrophoresis and detected by fluorescence. (B) Ligation of the RNA-splinted substrate in SplintR Ligase Reaction Buffer for 15 minutes at 25°C with (a) no enzyme, (b) 1 μM T4 DNA Ligase and (c) 100 nM SplintR Ligase. Indicated peaks correspond to starting pDNA (I), AppDNA (II) and ligated product (III) as determined by co-elution with synthetically prepared standards. (C) The fraction of ligated product catalyzed by either SplintR Ligase or T4 DNA Ligase was analyzed by performing sets of ligations with both ligases at concentrations between 10 pM and 10 μM for 15 minutes at 25°C. SplintR Ligase is clearly much more efficient at ligation of RNA splinted DNA than T4 DNA Ligase.
Product Source
An E. coli strain that carries a recombinant gene encoding PBCV-1 DNA Ligase.
This product is related to the following categories:
One unit is defined as the amount of enzyme needed to ligate (to 50% completion) 2 picomoles of a tripartite FAM-labeled DNA:RNA hybrid substrate in a 20 μl reaction at 25°C in 15 minutes in 1X SplintR Ligase Reaction Buffer.
Reaction Conditions
1X SplintR Ligase Reaction Buffer
Incubate at 25°C
1X SplintR Ligase Reaction Buffer
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM DTT
(pH 7.5 @ 25°C)
Storage Buffer
10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C
Krzywkowski, T., Nilsson M. (2017) Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy Nucleic Acids Res; DOI: 10.1093/nar/gkx708
Deng, Ruijie., Zhang, Kaixiang., Sun, Yupeng., Ren, Xiaojun., Li, Jinghong. (2017) Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification Chem Sci; 8, 3668-3675. PubMedID: 28580104, DOI: 10.1039/C7SC00292K
Schneider, N., Meier, M. (2016) Efficient In Situ Detection of mRNAs using the Chlorella virus DNA ligase for Padlock Probe Ligation RNA; epub ahead of print. PubMedID: 27879431
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