- Research article
- Open Access
Characterization of Norovirus RNA replicase for in vitro amplification of RNA
© Arai et al.; licensee BioMed Central Ltd. 2013
- Received: 30 January 2013
- Accepted: 17 September 2013
- Published: 9 October 2013
The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Qβ replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3Dpol) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3Dpolin vitro were re-evaluated in this context.
NV3Dpol, synthesized by using a cell-free translation system, represented the activities which were reported in the previous several studies and the reports were not fully consistent each other. The efficiency of the initiation of replication was dependent on the 3’-terminal structure of single-stranded RNA template, and especially, NV3Dpol preferred a self-priming small stem-loop. In the non-self-priming and primer-independent replication reaction, the presence of -CCC residues at the 3’-terminus increased the initiation efficiency and we demonstrated the one-pot isothermal RNA (even dsRNA) amplification by 16-fold. NV3Dpol also showed a weak activity of elongation-reaction from a long primer. Based on these results, we present a scheme of the primer-independent isothermal amplification of RNA with NV3Dpolin vitro.
NV3Dpol can be used as an RNA replicase in in vitro RNA + protein evolution with the RNA of special terminal sequences.
- RNA-dependent RNA polymerase
- RNA replication
- Isothermal RNA amplification
- in vitro evolution
In vitro evolution of RNA has been performed with a given replicase [1–3] or with a given RNA polymerase and a given reverse transcriptase [4–6]. In vitro evolution of RNA having a gene region encoding the replicase, that is, in vitro evolution of RNA with an evolvable replicase  is a next step approaching to synthetic biology on an origin of life. It may be one of the most fundamental experiments of in vitro evolution of a protein. In vitro evolution of a protein has to be coupled with in vitro evolution of its gene and usually is performed with a given translation system. If a synthetic biologist wishes to reconstruct the phenomenon of the origin of life, he should use primitive translation system which can evolve to high efficient system, but such a translation system is not available.
Coupling a protein evolution and its gene evolution is realized by a phenotype-genotype linking strategy in the selection process. There are four types of the strategy; ribozyme-type, virus-type, cell-type and external intelligence-type. The ribozyme-type is the strategy of phenotype-genotype linking by carrying both on an RNA [8, 9]. The virus-type is the strategy by binding a protein to its gene [10, 11]. The cell-type is the strategy by enclosing a protein and its gene in a compartment . External intelligence-type is the strategy by measuring and picking the fittest protein .
In vitro evolution experiments can be classified into two categories; artificial selection-type and natural selection-type. The former is called directed evolution, in which the fitness measure is set by the experimenter. The fitness of the latter experiment is the specific growth rate. PCR is most convenient amplification method in the former experiments, but cannot be used in the latter experiments. One-pot isothermal amplification method is convenient in the latter, because a flow reactor experiment is possible in principle and a serial transfer experiment is easily performed as an approximation of the flow reactor experiment. Examples of the one-pot isothermal amplification are Qβ replicase method , 3SR , NASBA , RNA-Z , etc.
Qβ RNA replicase is from an RNA phage Qβ of E.coli and is a hetero-tetramer, in which only β subunit is encoded in the viral genome and other three subunits (EF-Tu, EF-Ts and S1) are recruited from the host-cell translation machinery . For the effective replication of viral genome RNA, it requires a host factor Hfq, which binds to the specific site of the RNA and recruits the replicase . When the isothermal amplification of the genomic RNA was performed in a test tube, a parasitic short RNA called RQ RNA emerged and it made the amplification of genomic RNA difficult . Yomo and the co-workers demonstrated that the compartmentalization of the reaction solution into a water-in-oil emulsion inhibits the growth of the parasite .
Yomo’s compartment method is just a cell-type strategy of phenotype-genotype linking and was applied to an experiment of the coupled RNA/replicase evolution . We are planning an experiment of the coupled RNA/replicase evolution by using an isothermal RNA amplification and a virus-type strategy called in vitro virus method or mRNA display [10, 11]. This “in vitro virus” will evolve autonomously in a flow reactor. Qβ RNA replicase is not adequate for our purpose because it must recruit three proteins from the cell-free replication system and this situation makes population growth dynamics very complex in the test tube containing the cell free translation system. Norovirus RNA replicase (RNA-dependent RNA polymerase; NV3Dpol) is a candidate for our in vitro virus, because it consists of a single polypeptide chain and its molecular weight is not so large (56 kDa).
There are several preceding reports on the characteristics of various modes of RNA replication catalyzed by the NV3Dpolin vitro[17–20]. They reported on the activity of making of double-strand RNA through complementary strand polymerization on a single strand template with and without a primer, the activity of strand dissociation replication, the activity of making of a hairpin through self-priming and also the terminal nucleotidyl transferase (TNT) activity. Their reports were not consistent with each other.
Therefore we re-examined the activities of NV3Dpolin vitro in order to investigate whether we can use this enzyme as an RNA replicase in evolution experiments in vitro, instead of Qβ replicase.
Preparation of NV3Dpol
RNA amplification reaction with NV3Dpol
3’-terminal sequence dependence of initiation of ssRNA replication in a primer-independent manner
Rohayem et al. indicated the following two points. First, NV3Dpol initiated the replication on an oligo cytidine. Second, NV3Dpol added some cytidines at the 3’-terminus of the synthesized strand. From these results, they suggested that NV3Dpol prefers a C-stretch for the initiation of replication in primer-independent manner.
While Rohayem et al.  showed that NV3Dpol does not have an RNA synthesis activity on poly(A)-tail, Fukushi et al.  indicated that NV3Dpol has this activity. In order to re-examine this activity, we synthesized an RNA bearing an A-stretch of 22 nts at the 3’-terminus (named TD257-735-A22), and incubated with NV3Dpol. Our NV3Dpol showed a slight activity to poly(A)-tail and the amplified products appeared after 30 min (Additional file 6: Figure S6).
Terminal nucleotidyl transferase activity and replication of dsRNA
3’-terminal sequence of replication products under potential TNT activity
3’-terminal sequence of cRNA
Number of clones
TNT 1 nts (C)
TNT 2 nts (CC)
TNT 3nts (CCC)
TNT 2nts (AA)
TNT 2nts (GG)
Isothermal amplification of a long RNA
Primer extension replication
When a small stem-loop structure can be formed at 3’-terminus, the self-priming is more favorable initiation reaction for an ssRNA template than the primer-independent manner as seen in Figure 2B and Additional file 3: Figure S3B. For (iii), 3’-terminal -CA forms a hairpin stem with -UG- at upstream. For (iv), 3’-terminal -AC forms a hairpin stem with -GU- at upstream. As the template contains -GUU- repeat at 3’-terminal region, several hybridization sites can be expected. The fact that there was only one band of double length shows NV3Dpol recognizes small loop size (3 for (iii), 4 for (iv)).
In this context we comment the self-priming case by Belliot et al., and Wei et al.. They used an RNA template of which 3’-terminal sequence is ---GAUCCAAGCUUACGUACGCG-3’ (the sequence showed in italics is a part of cutting site of MluI restriction enzyme) and got a double length replication product. They explained it is the result of the back-priming at MluI palindrome. This explanation is not acceptable based on thermodynamic considerations. We propose another explanation that it was the result of self-priming starting from a small stem-loop (loop size = 4 nts, stem size = 2 bp) made of ---CGUACGCG-3’.
Fullerton et al. investigated the initiation reaction of RNA replication with Sapovirus (family with norovirus) replicase using 2259 nts template of which 3’-terminal sequence is ---UUGGAGCCAUUGCCCUCCAU-3’. They did not get a double length product and concluded the initiation by the primer-independent manner. In fact a potential 3’-terminal stem-loop has loop size eight (Additional file 5: Figure S5B (b)). This is not a small size and self-priming can hardly proceed. This results support our conclusion that when there is a small stem-loop (loop size 3–4, stem size 2) structure exists at 3’-terminus, the self-priming is the most preferable initiation reaction for an ssRNA template.
There were at least two amplification reaction products of the template Temp(GGG-GGG) (Figure 2 (ii); indicated with asterisk). Length of them was between 92 and 100 nts. If we consider a small stem-loop made with GG:UU stem, the length of self-priming product would be 91, 88, or 85 nts according to the loop size 5, 8, or 11 nts, respectively (Additional file 5: Figure S5A (ii)). Here we neglect loop size 2 nts, because it is very unstable because of the steric hindrance and the hairpin thermodynamics. They are probably originated from C and CCC addition to 3’-terminus with TNT activity of the enzyme. The products can make small stem-loops of GUUGGGGC and GGGGCCC which can make 94 nts and 99 nts product, respectively (Additional file 5: Figure S5A (ii)).
When there exists no small stem-loop structure at 3’-terminus and 3’-terminal sequence has C-stretch, the primer-independent initiation proceeds as shown in Figure 2B (i) and in the above mentioned case by Fullerton et al.. Rohayem et al. also pointed out that NV3Dpol prefers C-stretch at 3’-terminus for the replication initiation of the primer-independent manner. Importance of the C-stretch was confirmed also the experiment using a long template of which sequence had no relation to norovirus genome. TD257-735g734c template showed six times larger amplification than TD257-735 template. The former is a point mutant of the latter and the 3’-terminal sequence is ---UCCCC and ---UCCGC, respectively. They have no stable small stem loop at 3-terminus.
Although Rohayem et al.  indicated that NV3Dpol did not have an RNA synthesis activity on poly(A)-tail, we showed that NV3Dpol initiated replication reaction on A-stretch at the 3’-terminus of TD257-735-A22 template (Additional file 6: Figure S6). The RNA synthesis activity on A-stretch, however, was considerably lower than the case of TD257-735g734c template. Therefore, the initiation of primer-independent RNA replication reaction by NV3Dpol prefers C-stretch at the 3’-terminus rather than A-stretch or poly(A)-tail. The differences between Rohayem’s system and ours are the amino acids sequence of NV3Dpol, the length of poly(A)-tail, and enzyme concentration used. On the other hand, Fukushi et al.  already reported that NV3Dpol has the initiation activity of primer-independent RNA replication on poly(A)-tail. We supposed that the actual 3’-terminal sequence of RNA template used in Fukushi’s work was ---(A30)UGCGC.
It is reported that Qβ replicase also favours C-stretch. The initiation efficiency is high when 3’-terminal sequence is ---CCC-3’ or ---CCA-3’. The efficient parasite RQ RNA has also the sequence ---CCC-3’. By the way, we have not yet observed any parasite sequence in amplification with NV3Dpol.
In the case of a blunt-end double-stranded RNA, the breathing of terminal double helical region makes the enzyme access to CC-stretch at 3’-terminus and start to replication in the primer-independent manner. To investigate whether NV3Dpol initiates the replication to dsRNA which has a sticky-end preferably rather than the blunt-end , we prepared two kinds of RNA template, Temp(GGG-UCCCC) and Temp(GGG-UCCC) shown in Figure 6A. The secondary structure predicted by Mfold (Figure 6B) shows that the former mimics the terminal structure of dsRNA added a cytidine by TNT activity and the latter mimics the terminal structure of a blunt-end. As shown in Figure 6C, the band at about 50 bp were amplified in the case of Temp(GGG-UCCC), but not in the case of Temp(GGG-UCCCC). This indicates that NV3Dpol might initiate the replication from the blunt-end cytidines at the 3’-terminus preferably rather than from sticky-end cytidines. We investigated only about the addition of one cytidine. Although the addition of three cytidines was rare (Table 1), if dsRNA which has three cytidines overhung end were used, the initiation efficiency of replication might alter.
The initiation of the extension reaction of a long primer is not favourable for the enzyme as shown in Figure 8. Rohayem et al. reported primer-dependent replication, but Fukushi et al. reported their NV3Dpol had no activity of primer extension. Our gene was a gift of Fukushi’s group. Thus they might miss the faint band.
The self-priming reaction at a small stem-loop at 3’-terminus is very active, but the extension activity of a long primer is very poor. Thus once the enzyme dissociates from the template-polymerizing chain hybrid, it is not easy for the enzyme to bind again. On the other hand as shown in Figure 5, the native state PAGE of the amplified product (479 bp) shows a single sharp band without smear. Thus we can draw the conclusion that the processivity of NV3Dpol is very high.
NV3Dpol can perform also the chain dissociation replication smoothly as shown in Figure 5, even in the case of a template having GC-rich regions (Figure 7 and Additional file 8: Table S1(c)). The amplification products must be double stranded and amplification proceeded several times. The dissociated single stranded chain was converted to double strand in a primer-independent manner not so rapidly, because the band of ssRNA was visible on the non-denaturing PAGE (Additional file 3: Figure S3B).
Termination reaction (TNT activity)
Fukushi et al. reported that their NV3Dpol had no TNT activity. Gene of NV3Dpol of our study is a gift from the laboratory in which their study was performed. Our results rather accords qualitatively with the report by Rohayem et al.[17, 18]. They reported TNT activity of NV3Dpol added at least four cytidines, but we observed -C, -CC and -CCC addition. We also observed about half of products had no addition of C. The C-stretch (corresponding to G stretch on the template) at 3’-terminus in our replicate chain may affect the reduced number of addition. We also observe addition of A and G, which accords with the reports by Rohayem et al. and Fullerton et al. (Sapovirus 3Dpol) .
It was also reported that Moloney murine leukemia virus (MMLV) adds some deoxycytidines at the 3’-terminus of reverse transcribed DNA, and switches the template utilizing this overhung dC-stretch . If NV3Dpol has the same template-switching activity as MMLV, the band corresponding to about 100 nts, observed in the replication reaction of Temp(GGG-GGG) RNA (Figure 2 (ii)), might be a denatured form of a tandem dimer dsRNA made through this activity rather than a double length dsRNA which was the product of replication from a self-priming product. Based on the fact that initiation efficiency of primer-independent manner at 3’-terminus GGG is significantly lower than self-priming with a small stem-loop, this explanation for 100 nts band is not acceptable.
In the non-self-priming and primer-independent replication shown in Figure 3B and Figure 7, the initial template ssRNA was amplified by 16-fold and 6-fold by 240 min with NV3Dpol, respectively. In the former, the shape of amplification curve showed two phases; linear amplification phase up to 90 min and plateau phase near 240 min, because the enzymatic activity of NV3Dpol is limited by about 120 min, as indicated in previous reports . The autocatalytic replication should be realized in this case, but the exponential growth phase was not observed because the enzyme was not excess over the initial template and the linear phase can be explained by the full turnover of enzyme action. On the other hand, in the case of Figure 7, the enzyme was excess over the initial template. And the growth curve of RNA shows three phases; the initial nonlinear phase, the apparent linear phase and the plateau phase. The final phase was realized by the same reason as in the case of Figure 3B. The apparent linear phase was not originated from the full turnover of enzyme action because the enzyme was vastly excess over the existing RNA template. This apparent linear phase may be explained by attenuated exponential growth caused by deactivating replicase. The initial nonlinear phase may be explained two fold; the initial part of the exponential phase or the reaction curve of the two- (or multi-) step reaction. The isothermal amplification of RNA with Qβ replicase also showed exponential growth of RNA under the similar condition .
A possible model of molecular mechanism of the isothermal amplification of RNA with NV3Dpol
In this study, it was confirmed that NV3Dpol, prepared with cell-free protein synthesis system, was able to perform the isothermal amplification of RNA (even dsRNA) in vitro. But the initiation efficiency of replication is dependent on the 3’-terminal sequence. And we have to use the 3’-terminal sequence which does not make a small stem-loop, in order to avoid a parasitic self-priming product. Thus, NV3Dpol can be used as an RNA replicase in in vitro RNA evolution with the RNA of special terminal sequences. NV3Dpol, which is a single chain protein, is a candidate of a model protein in in vitro autonomous protein evolution in the form of an in vitro virus .
Preparation of NV replicase (NV3Dpol)
Plasmid pVL3Dwt (GenBank: AB039782 ) harbouring NV3Dpol gene was kindly provided from BML Inc. PCR was performed with KOD-plus- DNA polymerase (TOYOBO) and the PCR products were purified with QIAquick PCR Purification Kit (QIAGEN). NV3Dpol gene was amplified from pVL3Dwt by PCR using prNV3Dstart(+) 5′-ATGGGAGGTGACGACAAGGGC-3′ and prNV3Dstop(-) 5′-TTATTCGACGCCATCTTCATTCACA-3′. NV3Dpol gene was modified with the coding region for Strep-tag II sequence (WSHPQFEK)  using prNV3Dstart(+) and prNV3D-strep(-) 5′-GCATCGACTCCTTACTTTTCAAACTGCGGATGGCTCCATTCGACGCCATCTTCATTC-3′ (the sequence showed in italics indicates stop codon and Strep-tag II coding sequence) by PCR. At the downstream of the stop-codon, KpnI restriction site was added using prNV3Dstart(+) and prStrep-kpn1(-) 5′-GGGGTACCTTACTTTTCAAACTGCGGATGGCTCC-3′ (the sequence showed in italics indicates KpnI cutting site) by PCR. Then the PCR product was digested with KpnI (TaKaRa) and integrated into the pTD1 expression vector (SHIMADZU Biotech)  according to the manufacture’s instruction. The recombinant plasmid was named pTD-NV3Dpol-strep. NV3Dpol-strep expression construct DNA was amplified using prTD161-179 (5′-GCAGATTGTACTGAGAGTG-3′) and prTD845-827 (5′-GGAAACAGCTATGACCATG-3′), and transcribed in vitro with RiboMAXTM Large Scale RNA production system-T7 (Promega). The transcript, named NV3Dpol-strep mRNA was purified with NICK column (GE Healthcare). NV3Dpol-strep mRNA was translated with Transdirect insect cell cell-free protein synthesis kit (SHIMADZU Biotech)  according to the manufacture’s instruction.
The translated product was purified with Strep-tactin Superflow plus (QIAGEN). The strep-tag II modified NV3Dpol was bound onto Strep-tactin Superflow plus resin, pre-equilibrated with binding buffer (50 mM Tris–HCl [pH 8.0], 300 mM NaCl). The bound protein was washed with the binding buffer and eluted with elution buffer (50 mM Tris–HCl [pH 8.0], 300 mM NaCl, and 2.5 mM desthiobiotin (Sigma)). The eluted protein was then enriched and buffer-exchanged with buffer A (25 mM Tris–HCl [pH 8.0], 100 mM NaCl, 5 mM MgCl2, and 1 mM β-mercaptoethanol) with Microcon YM-50 column (Millipore), and stored at -80°C. In the enrichment step using Microcon YM-50 column, bovine serum albumin (BSA) (TaKaRa) was used as a carrier protein to avoid the non-specific adsorption of NV3Dpol to the column. The amount of NV3Dpol-strep was quantified on sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) referring a calibration curve with BSA.
Preparation of RNA templates
Sequences of RNA template used in this study were summarized in Additional file 9: Table S1 (a). Temp(GGG-CCC) DNA was generated by PCR using Temp(GGG-CCC) (5′-GCCAGTCGCCTGCAGTAATACGACTCACTATAGGGAATAAGATTTCACAGTTCAGAGAGACATTAAGTTGTTGTTGTTGCCC-3′) (underline indicates T7 Φ6.5 promoter sequence), prT7tempGGG + (5′-GCCAGTCGCCTGCAGTAATACGACTCACTA-3′) and prTempCCC- (5′-GGGCAACAACAACAACTTAATGTC-3′). Other DNA templates, Temp(GGG-GGG), Temp(GGG-CCA) Temp(GGG-UAC), Temp(GGG-UUC), Temp(GGG-UCC), Temp(GGG-GCCCC), Temp(GGG-UCCCC) and Temp(GGG-UCCC) DNA, were generated as same as Temp(GGG-CCC) DNA with the primer listed in Additional file 9: Table S1 (b). All PCR amplified DNAs were purified with QIAquick PCR Purification kit and in vitro transcribed with RiboMAX™ Large Scale RNA production system-T7. TD257-735 RNA were also in vitro transcribed from the PCR amplified DNA (the sequence was shown in Additional file 9: Table S1 (c)) using pTD1 , prTD161-179 and prTD735-714 (5′-GCGGATAATATTTTGAACGACG-3′). TD257-735g734c RNA was made from TD257-735 by replacing 734th guanosine to cytidine using prTD735-714g734c (5′-GGGGATAATATTTTGAACGACG-3′) in PCR. TD257-735-A22 was made from TD257-735 by adding 22 nts of adenine at the 3’-terminus using prTD757-733 (5′-TTTTTTTTTTTTTTTTTTTTTTGCG-3′) in PCR. All RNA templates were purified on denaturing PAGE and quantified by measuring absorbance at 260 nm.
RNA primer (5′-GGGCAACAACAACAAC-3′) modified with FITC at the 5’-terminus was purchased from Japan Bio Service Inc.
In vitro RNA replication or amplification with NV3Dpol-strep
RNA replication or amplification was performed with purified NV3Dpol-strep and in vitro-transcribed RNA template in a reaction buffer (50 mM Hepes-KOH [pH 7.0], 3 mM MnCl2, 4 mM DTT, 0.4 mM rNTPs, and 40 U/μL of RNasin ribonuclease inhibitor plus (Promega)) at 30°C. Reaction products were subjected to denaturing or non-denaturing PAGE followed by SYBRgreenII (Lonza) staining and visualized on Pharos Fx imager (Bio-Rad). Here, the non-denaturing PAGE was performed with 5 or 10% acrylamide, 0.625% bisacrylamide and TBE buffer at 20°C. The denaturing PAGE was performed with 8 M urea plus the same constituent of non-denaturing PAGE at 65°C.
Sequencing of 3’-terminus of amplified RNA
To detect the addition of nucleotides at the 3’-terminus of replicated RNA (RNA(-)) by TNT activity of NV3Dpol, we could not use a normal sequencing method using a primer which hybridizes 3’-terminal region of the sample RNA. Thus we sequenced the full length of RNA(-) applying the Y-ligation method , as follows (shown in Additional file 9: Figure S8). After the incubation of 240 min in the RNA amplification with NV3Dpol-strep, the reaction solution was desalted with Micro bio-spin column 30 (Bio-Rad), and ligated with 5’-phosphorylated Y-adapter (5′-CAAAGGGAATAAGATTTCACAGTTCAGAGCTTAGATAATACGACTCACTATAGGGTTAAC-3′) by Y-ligation method. The ligated product was hybridized with prT7g10SD-NV3D(-) (5′-CCTTGTCGTCACCTCCCATGGATATATCTCCTTCTTAAAGTTAACCCTATAGTGAGTCGTATTA-3′) and reverse transcribed with Avian myelobastosis virus (AMV) reverse transcriptase (Promega). The reverse transcribed product was purified on denaturing PAGE, and amplified by PCR using prTempCCC- and prSeq (5′-CCTTGTCGTCACCTCCCA-3′) as primers. TA cloning was performed with pGEM-T easy vector system (Promega). Sequencing was performed by Operon Inc. (Tokyo).
We thank BML Inc. for providing us the plasmid pVL3Dwt  harbouring NV3Dpol gene. We thank Drs. Manish Biyani and Shingo Ueno for helpful discussions.
- Haruna I, Spiegelman S: Autocatalytic synthesis of viral RNA in vitro. Science. 1965, 150: 884-886. 10.1126/science.150.3698.884.View ArticleGoogle Scholar
- Biebricher CK, Eigen M, Luce R: Product analysis of RNA generated de novo by Qβ replicase. J Mol Biol. 1981, 148: 369-390. 10.1016/0022-2836(81)90182-0.View ArticleGoogle Scholar
- Biebricher CK, Eigen M, Luce R: Kinetic analysis of template-instructed and de novo RNA synthesis by Qβ replicase. J Mol Biol. 1981, 148: 391-410. 10.1016/0022-2836(81)90183-2.View ArticleGoogle Scholar
- Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richman DD, Gingeras TR: Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modelled after retroviral replication. Proc Natl Acad Sci USA. 1990, 87: 1874-1878. 10.1073/pnas.87.5.1874.View ArticleGoogle Scholar
- Compton J: Nucleic acid sequence-based amplification. Nature. 1991, 350: 91-92. 10.1038/350091a0.View ArticleGoogle Scholar
- Breaker RR, Joyce GF: Emergence of a replicating species from an in vitro RNA evolution reaction. Proc Natl Acad Sci USA. 1994, 91: 6093-6097. 10.1073/pnas.91.13.6093.View ArticleGoogle Scholar
- Mills DR, Peterson RI, Spiegelman S: An extracellular darwinian experiment with a self-duplicating nucleic acid molecule. Proc Natl Acad Sci USA. 1967, 58: 217-224. 10.1073/pnas.58.1.217.View ArticleGoogle Scholar
- Ekland EH, Bartel DP: RNA-catalysed RNA polymerization using nucleoside triphosphates. Nature. 1996, 382: 373-376. 10.1038/382373a0.View ArticleGoogle Scholar
- Johnston WK, Unrau PJ, Lawrence MS, Glasner ME, Bartel DP: RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension. Science. 2001, 292: 1319-1325. 10.1126/science.1060786.View ArticleGoogle Scholar
- Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H: In vitro virus: Bonding of mRNA bearing puromycin at the 3’-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett. 1997, 414: 405-408. 10.1016/S0014-5793(97)01026-0.View ArticleGoogle Scholar
- Roberts RW, Szostak JW: RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci USA. 1997, 94: 12297-12302. 10.1073/pnas.94.23.12297.View ArticleGoogle Scholar
- Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D: Light-directed, spatially addressable parallel chemical synthesis. Science. 1991, 251: 767-773. 10.1126/science.1990438.View ArticleGoogle Scholar
- Blumenthal T, Carmichael GG: RNA replication: function and structure of Qβ-replicase. Annu Rev Biochem. 1979, 48: 525-548. 10.1146/annurev.bi.48.070179.002521.View ArticleGoogle Scholar
- Barrera I, Schuppli D, Sogo JM, Weber H: Different mechanisms of recognition of bacteriophage Qβ plus and minus strand RNAs by Qβ replicase. J Mol Biol. 1993, 232: 512-521. 10.1006/jmbi.1993.1407.View ArticleGoogle Scholar
- Munishkin AV, Voronin LA, Ugarov VI, Bondareva LA, Chetverina HV, Chetverin AB: Efficient templates for Qβ replicase are formed by recombination from heterologous sequences. J Mol Biol. 1991, 221: 463-472. 10.1016/0022-2836(91)80067-5.View ArticleGoogle Scholar
- Urabe H, Ichihashi N, Matsuura T, Hosoda K, Kazuta Y, Kita H, Yomo T: Compartmentalization in a water-in-oil emulsion repressed the spontaneous amplification of RNA by Qβ replicase. Biochemistry. 2010, 49: 1809-1813. 10.1021/bi901805u.View ArticleGoogle Scholar
- Rohayem J, Robel I, Jäger K, Scheffler U, Rudolph W: Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol. J Virol. 2006, 80: 7060-7069. 10.1128/JVI.02195-05.View ArticleGoogle Scholar
- Rohayem J, Jäger K, Robel I, Scheffler U, Temme A, Rudolph W: Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis. J Gen Virol. 2006, 87: 2621-2630. 10.1099/vir.0.81802-0.View ArticleGoogle Scholar
- Fukushi S, Kojima S, Takai R, Hishino BF, Oka T, Takeda N, Katayama K, Kageyama T: Poly(A)- and primer-independent RNA polymerase of norovirus. J Virol. 2004, 78: 3889-3896. 10.1128/JVI.78.8.3889-3896.2004.View ArticleGoogle Scholar
- Belliot G, Sosnovtsev SV, Chang K, Babu V, Uche U, Arnold JJ, Cameron CE, Green KY: Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase. J Virol. 2005, 79: 2393-240. 10.1128/JVI.79.4.2393-2403.2005.View ArticleGoogle Scholar
- Skerra A, Schmidt TGM: Use of the Strep- tag and streptavidin for detection and purification of recombinant proteins. Methods Enzymol. 2000, 326: 271-304.View ArticleGoogle Scholar
- Wei L, Huhn JS, Mory A, Pathak HB, Sosnovtsev SV, Green KY, Cameron CE: Proteinase-polymerase precursor as the active form of ferine calicivirus RNA-dependent RNA polymerase. J Virol. 2001, 75: 1211-1219. 10.1128/JVI.75.3.1211-1219.2001.View ArticleGoogle Scholar
- Suzuki T, Ito M, Ezure T, Kobayashi S, Shikata M, Tanimizu K, Nishimura O: Performance of expression vector, pTD1, in insect cell-free translation system. J Biosci Bioeng. 2006, 102: 69-71. 10.1263/jbb.102.69.View ArticleGoogle Scholar
- Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003, 31: 3406-3415. 10.1093/nar/gkg595.View ArticleGoogle Scholar
- Fullerton SWB, Blaschke M, Coutard B, Gebhardt J, Gorbalenya A, Canard B, Tucker PA, Rohayem J: Structural and functional characterization of sapovirus RNA-dependent RNA polymerase. J Virol. 2007, 81: 1858-1871. 10.1128/JVI.01462-06.View ArticleGoogle Scholar
- Wellenreuther R, Schupp I, Poustka A, Wiemann S, The German cDNA Consortium: SMART amplification combined cDNA size fractionation in order to obtain large full-length clones. BMC Genomics. 2004, 5: 36-10.1186/1471-2164-5-36.View ArticleGoogle Scholar
- Tabuchi I, Soramoto S, Nemoto N, Husimi Y: An in vitro DNA virus for in vitro protein evolution. FEBS Lett. 2001, 508: 309-312. 10.1016/S0014-5793(01)03075-7.View ArticleGoogle Scholar
- Ezure T, Suzuki T, Higashide S, Shintani E, Endo K, Kobayashi S, Shikata M, Ito M, Tanimizu K, Nishimura O: Cell-free protein synthesis system prepared from insect cells by freeze-thawing. Biotechnol Prog. 2006, 22: 1570-1577. 10.1021/bp060110v.View ArticleGoogle Scholar
- Nishigaki K, Taguchi K, Kinoshita Y, Aita T, Husimi Y: Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase. Mol Divers. 1998, 4: 187-190. 10.1023/A:1009644028931.View ArticleGoogle Scholar
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