Further optimization of the DNA adenylation method was performed using MthRnl. MthRnl is a thermophilic ligase that catalyzes the intramolecular ligation of RNA (circularization) and is also able, with lower efficiency, to circularize ssDNA as described previously (11
). MthRnl is moderately thermostable (optimum 60–65°C), ATP dependent and requires Mg+2
for activity. The enzyme is a homodimer in solution and dimerization of MthRnl is required for ligase activity (11
). For simplicity, we use the monomer molecular weight for calculation of the substrate/enzyme molar ratios in the reaction.
ATP inhibits RNA circularization by MthRnl, yielding the intermediate 5′-adenylated product, AppRNA (11
). Competition between ATP and AppRNA for the same binding site may account for this behavior. Adenylated ligase is unable to bind AppRNA (15
The adenylation and ligation of DNA are also influenced by ATP concentration. At high concentrations of ATP adenylation of DNA is enhanced but DNA ligation is inhibited. As shown in a, when the concentration of ATP was increased to 50
µM, the AppDNA product accumulated and DNA ligation was almost completely inhibited. In the 5′-adenylation of DNA with a 3′ protected end, AppDNA formation also increased with increasing ATP concentration and reached saturation near 50
µM (b). DNA ligation is more sensitive to inhibition by ATP than ligation of RNA. The midpoint of ATP inhibition for RNA circularization is 50
Figure 2. ATP dependence of DNA adenylation by MthRnl. (a) Reaction of preadenylated MthRnl (25pmol of monomer) with 5pmol of 50-nt long 5′-phosphorylated ssDNA with unprotected 3′-end (pDNA50) at various concentrations of ATP (more ...)
It is possible that in the absence of an RNA acceptor, the ligase could use a second molecule of ATP as an alternative acceptor. This would create a 1
nt longer byproduct, pppApDNA, which could not be used for subsequent ligation. However, this does not appear to be the case because (i) the adenylated DNA product is efficiently used for ligation (), (ii) The inhibition of ligation with pre-adenylated enzyme is specific for ATP while other potential acceptor mononucleotides do not have this property. When 500
µM of ATP was substituted in reaction with 500
µM of ADP, AMP, UTP, CTP, GTP, dATP or 3′-dATP (b–d, respectively), no inhibition of circularization was observed, (iii) When radioactive γ-[32
P]ATP was used in the reaction, only a trace amount of radioactivity was incorporated into the product (data not shown).
Figure 3. Effect of mononucleotides on ssDNA adenylation and circularization by preadenylated MthRnl (a–d). All reactions except ‘input’ in (a) contained excess (25pmol) of pre-adenylated enzyme and 5pmol pDNA50, which (more ...)
A model for efficient ATP inhibition of DNA ligation is likely the same as the one described above for RNA ligation. After AppDNA dissociates from RNA ligase its rebinding will be blocked if the enzyme is also adenylated. This ATP blockage of ligation allows efficient adenylation of ssDNA even when a free 3′ OH is available for ligation. We concluded that the range of ATP concentrations 100–500
µM is sufficient for DNA adenylation even without protection of the 3′-end, and does not produce unwanted ligation products.
In the presence of Mg+2
, pH optima for both MthRnl enzyme and DNA adenylations are around pH 6.5–7.0 (@25°C) (a and b). When Mg+2
was substituted by Mn+2
, pH optima are shifted to 5.5–6.0 (@25°C) (a; data not shown for DNA adenylation). DNA adenylation reaction reached saturation with 10
at corresponding pH with similar efficiency, in contrast to Step 3 DNA ligation, where Mn+2
is more active (data not shown). In addition, at 65°C Mn+2
is less stable and more prone to oxidation. We choose 10
and pH 6.0 at 65°C (see ‘Materials and Methods’ section for calculation of pH shift with temperature) for our experiments.
Figure 4. pH dependence of adenylation of MthRnl and ssDNA. (a) Adenylation of non-adenylated MthRnl in the presence of Mg+2 or Mn+2, ATP and α-[32P]ATP with no DNA substrate. The products were analyzed by an SDS–PAGE and radioactive bands quantified (more ...)
Our initial data for DNA adenylation by MthRnl, shown in , revealed some variation in reaction efficiency depending on which substrate was used. Adenylation of the 17-nt long pDNA17c-NH2
, with 3′-amino block (panel a), was slower than the 21-nt long pDNA21-3bioTEG, with a 3′-biotinylated triethylene glycol (3bio-TEG) modification and a different sequence (panel b). To determine the substrate parameters affecting the efficiency of DNA adenylation, we analyzed a range of 5′-phosphorylated ssDNA oligonucleotides of different length, sequence and 3′-modifications. These oligos were tested at different ratios of enzyme to substrate. First, we replaced 5′-cytosine in pDNA17c-NH2
with adenine, thymine or guanine. As shown in , MthRnl is least active with 5′-cytosine, requiring an equimolar amount of enzyme to complete the reaction (panel a). Similar activity was observed with another 5′ cytosine-terminated oligonucleotide, pDNA23-ddC (panel e). However, the difference in adenylation of other three substrates was slightly better, within a dilution factor of two to four (panels b–d). Similar activity was observed with other oligonucleotides (panels f and g). DNA length, within the tested range of 17–50
nt, was not a factor in the efficiency of adenylation. Potentially a 3′-modified substrate end could be bound to an acceptor binding site and sterically affect 5′-adenylation, preventing binding of ATP or DNA donor. However, comparison of adenylation of two different 3′-modifications, amino and 3bio-TEG, with identical sequences, pDNA21-NH2
and pDNA21-3bioTEG, showed no differences in activity (panels f and g). To rule out that the differences in DNA adenylation are not due to DNA secondary structure but rather enzyme specificity, self-complementarity and hairpin formation of the oligonucleotides were calculated at the reaction conditions used for adenylation using OligoCalc and mFold calculators (16
). At low ionic strength and 65°C, no significant secondary structures were found.
Figure 5. Effect of substrate sequence and 3′-modifications on the efficiency of ssDNA adenylation. Each 1h reaction had 5pmol of the indicated substrate and serially diluted amounts of enzyme under standard reaction conditions as described (more ...)
In standard reaction condition at 65°C MthRnl was active for at least 2
h as demonstrated in h. Doubling of incubation time to 2
h using pDNA17c-NH2
with the substrate in a 2-fold excess (S/E
2) still resulted in complete adenylation of the oligo (panel h). This is the same adenylation observed in a 1
h reaction with an equal ratio of enzyme and substrate, (S/E
1) (a). After 2
h of incubation at 65°C with a substrate, activity of the enzyme gradually diminished over the next few hours (data not shown).
The adenylated oligonucleotides, produced by preparative scale MthRnl adenylation, were tested for their ability to ligate to an RNA acceptor using truncated T4 RNA ligase 2 without ATP (). T4 RNA ligase 2 (truncated) is defective in self-adenylation and readily accepts pre-adenylated substrate for ligation (18
). Adenylated DNA, AppDNA17c-NH2
, preparatively produced with MthRnl, was used for ligation with two different RNA acceptors. In both reactions, ligation products were observed (lanes 2 and 4). During ligation reaction, some deadenylation of pre-adenylated DNA occurs as expected due to reversibility of Step 2 of ligation reaction in the absence of ATP. This product runs ~1
nt faster than pre-adenylated substrate. Partial degradation of RNA22 during ligation may be responsible for smaller ligation product in lane 2. The same result was achieved with AppDNA21-NH2
(data not shown). This result demonstrates that pre-adenylated DNA, synthesized with MthRnl, is a functional substrate for truncated T4 RNA ligase 2 in the absence of ATP.
This one-step quantitative conversion of ssDNA to adenylated DNA using thermostable RNA ligase, MthRnl, greatly simplifies existing chemical and enzymatic methods. In summary, the range of DNA concentrations 0.5–30
µM was successfully tested. To achieve quantitative DNA adenylation, single turnover reaction condition with substrate to enzyme ratio 1:1 should be used. The temperature 60–65°C is optimal for activation of enzyme and its stability. pH optimum range adjusted to 65°C is 6.0–6.5. Make sure ATP is not a limiting factor in preparative adenylation. In this case and in adenylation of DNA with non-protected 3′-ends concentration of ATP should be increased to 0.5–1.0
mM. A significant benefit of this method is that high yield of the reaction and lack of a template strand eliminates the need for additional purification. The method requires only basic lab equipment and is easily scalable to micromolar level. It reduces cost and adds flexibility in designing custom adenylated DNA oligonucleotides for various applications.