Abstract
Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.
Highlights
Single-stranded DNA molecules annealed to an RNA splint are notoriously poor substrates for DNA ligases
Rates of PBCV-1 DNA ligase sealing of DNA splinted by RNA under a range of buffer conditions
Initial ligation reactions were performed with 100 nM dC/phosphorylated dT (pdT) substrate in either T4 DNA ligase buffer, or a low-ATP version of this buffer (10 mM ATP rather than 1 mM) that was previously reported to facilitate ligation of RNA-splinted DNA by T4 DNA ligase [21,22]
Summary
Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. The ligation of single-stranded DNA (ssDNA) probes splinted by RNA followed by detection of ligated product by PCR or quantitative PCR (qPCR) permits the profiling and quantification of RNA species [17,18,19,20,21,22,23]. PBCV-1 DNA ligase was previously reported to efficiently ligate nicked DNA substrates as well as ligate ssRNA acceptor strands to ssDNA donors using complementary DNA splints, but was found to have no detectable activity for any RNA-splinted substrate [33]. PBCV-1 DNA ligase displays a faster maximum rate of turnover, a much lower apparent KM and a much higher proportion of direct ligation versus abortive adenylylation than T4 DNA ligase when acting on these substrates This ligation works across a range of buffer conditions and substrate sequences, and shows little inhibition at 1 mM ATP for most sequences. PBCV-1 DNA ligase’s efficient turnover of, and high affinity for, RNA-splinted DNA substrates shows great potential as a replacement for T4 DNA ligase in molecular biology applications that use these substrates to profile RNA targets
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