A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis

Outline of the suicide cassette system

An oligonucleotide able to fold into a hairpin structure (the suicide cassette) by self-templated hybridization, thereby bringing the 5'-end and the 3'-end into proximity, can be circularized by the addition of a ligase [14] (Figure 1A–B). With the addition of primer, dNTP and polymerase, a rolling circle DNA synthesis can be initiated resulting in tandem repeats complementary to the circle (Figure 1C). Besides providing the template for the ligation reaction, the hairpin structure contains two additional features: I) It contains a recognition sequence for a nicking enzyme (a nicking enzyme binds double stranded DNA but cleaves only one strand of the DNA duplex). II) It contains a recognition sequence for the Mly I restriction enzyme (Mly I is, to our knowledge, the only commercially available enzyme which cleaves blunt end outside its binding sequence, as required here). Nicking allows the amplified rolling circle product to be turned into monomers without the addition of an extra oligonucleotide and without the gain or loss of nucleotides. Since the nick is positioned in the hairpin, the released monomers will have parts of the hairpin sequence positioned at each end. A denaturation-renaturation step can turn the monomers into open circle structures complementary to the original circle (Figure 1E–F). A second round of ligation and rolling circle DNA synthesis can again be followed by a nicking reaction producing oligonucleotides with the same polarity as the one purchased (Figure 1G–H and 1J). Alternatively, following each of the rolling circle steps, the products can be cleaved with Mly I, releasing the hairpin (the suicide cassette) from the rest of the oligonucleotide (Figure 1D and 1I). Although the amplification is only linear in each step of rolling circle DNA synthesis, combining rounds of amplification makes the amplification-level exponential. Since, in theory, an unlimited number of ligation, rolling circle DNA synthesis and nicking reactions could be performed, a massive amplification is possible. Unlike C2CA, the method presented does not require the additional production of oligonucleotides for both cleavage of the rolling circle products and ligation of linear products, and, equally important, the method presented provides free design of the ends of the DNA sequence to be amplified, since they are not defined by the chance presence of a binding/cleavage site for a restriction endonuclease. Instead, ends can be randomly chosen because the unique blunt end cleavage of the Mly I restriction enzyme recognizes and removes the suicide cassette added for amplification purposes.

Leaving the suicide cassette on the oligonucleotide provides a new class of circle probes, which, due to their ability to self-circularization, may have advantages for the detection of RNA (Stougaard et al., manuscript under revision).

Detailed description of the suicide cassette

The described suicide cassette is a hairpin-structure comprising binding and cleavage sites for both the nicking enzyme Nt. Alw I and the restriction enzyme Mly I (Figure 2). During amplification only the loop of the suicide cassette will change polarity, since each strand of the stem is complementary to the opposite strand. We have used the loop structure TTTC which is replicated to GAAA following one round of amplification. GAAA constitutes a loop with a very high melting temperature probably resulting from a pseudo-base pair between the G and the last A [15, 16]. Other loops such as AAAA, TTTT, TTATT and AATAA are also functional for our approach (data not shown). Four additional base pairs are positioned adjacent to the loop to improve ligation following nicking with Nt. Alw I (Figure 2).

Amplification of an oligonucleotide contained within a suicide cassette

To verify the potential of our amplification technique we tried to amplify a 98-mer oligonucleotide (SF-WT90) containing a suicide cassette. Sequences with the original polarity will be called (+)-strands and sequences with the complementary polarity will be called (-)-strands. The oligonucleotide was circularized using the T4 DNA ligase, creating a product with a slower migration rate than the linear substrate when separated by denaturing PAGE (Figure 3, lanes 1–2). The rolling circle DNA synthesis created a long single stranded product consisting of tandem repeats of the complementary sequence of the original oligonucleotide, which was too long to enter the gel-matrix and could therefore only be visualized as a dot in the gel slot (Figure 3, lane 3). The rolling circle product could be cut into (-)-strand monomers without the loss of nucleotides by the nicking enzyme Nt. Alw I (Figure 3, lane 4), and the suicide cassette could be released from the rest of the oligonucleotides by Mly I (Figure 3, lanes 5–6). The nicked product could again be circularized using the T4 DNA ligase, creating circles with the (-)-polarity (Figure 3, lanes 7–8). Comparing the ligation efficiencies of the chemically synthesized oligonucleotide and the enzymatically synthesized oligonucleotide, the difference was obvious; the enzymatically synthesized oligonucleotides ligate more efficiently (compare Figure 3, lanes 1–2 and lanes 7–8). Further rounds of rolling circle DNA synthesis, nicking and ligation could be performed to get the desired amount and correct polarity of the oligonucleotides (Figure 3, lanes 9–11). The purity of the HPLC-purified oligonucleotides in lane 1 is not impressive. This is possibly due to the highly structured oligonucleotide, which is difficult to purify, since dimers can be formed during the purification step. The level of amplification (of the oligonucleotide complementary to the templating circle) after rolling circle DNA synthesis and cleavage was approximately 400 fold (estimated by PAGE, data not shown). Taken together, these results showed that successive rounds of ligation, nicking and rolling circle DNA synthesis allows for the amplification of an oligonucleotide. The nicking reactions were not complete, resulting in visible oligomers in the gel. Most likely this could be avoided by further optimization of I) enzyme:DNA ratio, II) the nicking enzyme of choice and III) optimization of the suicide cassette sequence. However, for the amplification reaction per se this phenomenon was of little significance, so we were satisfied with the results as they appear here.

Solid support amplification to verify purity and ligation of the enzymatically synthesized oligonucleotides

As we did not purify the amplification products, we wanted to test if the presence of small amounts of oligonucleotides with the opposite polarity would affect the performance of an amplified SF-WT90 probe in a hybridization assay. Primers able to hybridize to either the (+)-strand (Amin-L16-Pr SF-WT90 (+)) or the (-)-strand (Amin-L16-Pr SF-WT90 (-)) of the amplified oligonucleotide were covalently linked to a solid support. After one and two rounds of amplification the nicked products were ligated (after self-templated hybridization) and hybridized onto the solid support. As a control for hybridization and ligation an un-amplified probe was used. Solid support rolling circle DNA synthesis has previously been described by Lizardi PM [5]. Working from the amplification levels estimated by gel electrophoresis, we diluted the different generations of amplification products correspondingly in an attempt to apply equimolar amounts of probe in each reaction. Following hybridization, a rolling circle DNA synthesis was performed on the support and the products were visualized by hybridization of a labeled detection probe (ID 16 or anti ID 16) to the rolling circle products (Figure 4). All products seemed reasonably clean, in that largely no signals from circles of the opposite polarity appeared, despite the application of massive amounts of probe (0.1 μM final).

To test for the ability to cleave the rolling circle product with Mly I, thereby creating e.g. a padlock probe, we cleaved the rolling circle products with Mly I after one and two rounds of amplification, releasing potential padlock probes from the suicide cassette. These padlock probes were hybridized to primers of both polarities (Amin-L16-Mly I (+) and Amin-L16-Mly I (-)), which were covalently linked to a solid support and then ligated (Figure 5). As a control for hybridization and ligation the padlock probe WT90-66 was used (WT90-66 is identical to the probe released from SF-WT90 following two rounds of amplification and cleavage with Mly I (from (+)-strand to (-)-strand and back to (+)-strand)). Following hybridization and ligation, the probes were amplified by rolling circle DNA synthesis and subsequently visualized by hybridization of a labeled detection probe (ID 16 or anti ID 16). This verified that padlock probes can indeed be synthesized using suicide cassettes and, as with the nicking enzyme, predominantly rolling circle products of the expected polarity were produced. Again, equimolar amounts of probe were applied in each reaction (0.1 μM final).

To test if the ligation efficiency was improved we compared the enzymatically produced oligonucleotide to a chemically synthesized oligonucleotide with the same expected sequence; this was done in a solid support assay as described in Figure 5. Additionally, the amplified probe was purified by PAGE after the first nicking reaction (from (+)-strand to (-)) and after Mly I cleavage (from (-)-strand to (+)) to minimize the background. Since the solid support assays in Figures 4 and 5 were used to test if any oligonucleotides of the wrong polarity would be amplified in a hybridization assay a high concentration of probes were applied (0.1 μM final). To be able to compare ligation efficiencies we now did a limiting dilution analysis of the probes. At a final concentration of 0.1 nM, only few signals were obtained using the chemically synthesized oligonucleotide (WT90-66b), whereas the enzymatically produced oligonucleotide still gave plenty of signals (Figure 6). This result is in agreement with a higher ligation efficiency of the enzymatically produced oligonucleotide.

Oligonucleotides

Amplification of DNA oligonucleotides

5'-phosphorylated oligonucleotides were ligated with T4 DNA ligase (Fermentas, Vilnius, Lithuania). Rolling circle DNA synthesis was performed for 16 hours at 37°C in a mixture containing 1× Phi29 DNA polymerase buffer (Fermentas), 1.5 nM ligated oligonucleotide, 1.5 nM primer, 250 μM dNTP, and 0.25 u/μl Phi29 DNA polymerase (Fermentas). The reactions were stopped by heat inactivation for 10 minutes at 65°C. Nicking was performed by the addition of 1 volume of a nicking mixture containing 1× NEBuffer 2 (NEB, Ipswich, MA, USA) and 1 u/μl of Nt. Alw I (NEB). Nicking reactions were incubated for 16 hours at 37°C and stopped by heat inactivation for 20 minutes at 80°C. The amount of cleavage products can be estimated by PAGE. Ligation reactions were performed by the addition of ATP and ligase to the inactivated reaction mixtures. The second round of rolling circle DNA synthesis was performed as the first one using a primer complementary to the one used in the first round. Further rounds of amplification can be performed as described above. To remove the suicide cassette, thereby terminating the amplification, cleavage of the rolling circle product was done by the addition of 1 volume of cleavage mixture containing 1× NEBuffer 4 (NEB), 0.2 μg/μl BSA (NEB) and 1 u/μl of Mly I (NEB). Cleavage reactions were incubated for 16 hours at 37°C and stopped by heat inactivation for 20 minutes at 65°C. Products can be separated and purified by PAGE.

Polyacrylamid gel electrophoresis (PAGE)

Amplified and cleaved products could be separated by PAGE. Products were separated on 10% denaturating gels and stained with SYBR Gold. Images were visualized using a Gel Doc 1000 (Biorad).

When indicated the cleaved products were purified from the gel using standard phenol/chloroform purification and the concentration estimated by photospectrometry.

Solid support amplification and detection of Nt. Alw I and Mly I cleaved rolling circle products

5'-amine coupled primers were linked to CodeLink Activated Slides (GE Healthcare) according to the manufacturers protocol. Detection after nicking: Nicked products (actual concentrations in individual experiments are given in the figure legends) were ligated in 1× ligation buffer (Fermentas), inactivated, supplemented with 0.5 M NaCl (final concentration) and hybridized to the solid support for 30 minutes at 37°C in a humidity chamber. Detection after Mly I cleavage: Cleaved products (actual concentrations in individual experiments are given in the figure legends) were ligated directly onto the coupled primes in a mixture containing 1× ligase buffer (Fermentas), 0.2 μg/μl BSA, 250 mM NaCl, and 0.05 u/μl T4 DNA ligase (Fermentas) for 30 minutes at 37°C in a humidity chamber. The slides were washed in 0.1 M Tris-HCL, 150 mM NaCl and 0.3% SDS (wash buffer 1) for 2 min at room temperature followed by a wash in 0.1 M Tris-HCL, 150 mM NaCl and 0.05% Tween-20 (wash buffer 2). Rolling circle DNA synthesis was performed for 30 minutes at 37°C in 1× Phi29 buffer, 0.2 μg/μl BSA, 250 μM dNTP, 5% glycerol, and 1u/μl Phi29 DNA polymerase. The reactions were terminated by washing 2 minutes in wash buffer 1 and 2 minutes in wash buffer 2. The rolling circle products were detected by hybridizing fluorescently labeled oligonucleotides in a buffer containing 20% formamide, 2× SSC, 5% glycerol and 0.17 μM of either ID 16 or anti ID 16 for 30 min at 37°C. Slides were washed for 2 min at room temperature in wash buffer 1 and wash buffer 2 and dehydrated through a series of ethanol and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA).

Image analysis

Solid support assays were analyzed with an epifluorescence microscope (Leica, Wetzlar, Germany) and images were recorded with a SenSys CCD-camera operated by the SmartCapture 2 version 2.0 from Digitalscientific (Cambridge, UK). A 63× objective (Leica) was used for all images. Thresholding was performed using Adobe Photoshop (Adobe Systems).