In spite of the lack of an efficient reverse-genetics system for HCV study, a great deal of progress on the structures and functions of the viral proteins has been made through utilization of recombinant systems involving the expression of individual viral gene products (, ). Biochemical studies of the recombinant viral proteins have identified four different virus-specific enzymatic activities. Two distinct viral proteases are responsible for specific cleavage of the viral polyprotein. The NS2-NS3 protease is a metalloprotease that catalyzes the cleavage at the NS2-NS3 junction (, ), while NS3 is a serine protease responsible for cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (, , , , ). The NS4A protein acts as a cofactor of NS3 protease to promote proteolytic cleavage (, ). RNA helicase and NTPase activities have also been identified in the NS3 protein (, ). The N-terminal one-third of the NS3 protein functions as a protease, and the remaining two-thirds of the molecule acts as the helicase/ATPase that was speculated to be involved in HCV replication (, , ). The fourth viral enzyme, NS5B, is an RNA-dependent RNA polymerase (RdRp), a key component responsible for replication of the viral RNA genome (, , ). The RdRp activity of NS5B was first experimentally demonstrated by Behrens et al. with NS5B protein expressed from a recombinant baculovirus and a synthetic nonviral RNA as a substrate (). It was subsequently confirmed and further characterized through the use of the HCV RNA genome as a substrate (, ). Recent studies have shown that NS5B with a C-terminal 21-amino-acid truncation expressed in Escherichia coli is also active for in vitro RNA synthesis (, , ).
Accumulation of adenylated short oligo(dT)n was observed in reactions with T4 RNA ligase 1 (). T4 RNA ligase 1 was also reported to accumulate the intermediate AppRNA product when using annealed complementary blocking DNA (). Some archaeal RNA ligases accumulate AppRNA when an excess of ATP is used in the reaction i.e. (,). The substrate binding specificity of RNA ligases, in general, is more relaxed for donor substrates than acceptors () and some can efficiently ligate (circularize) ssDNA (). To our knowledge, however, no method has been demonstrated that allows scalable high yield adenylation of 5′-phosphorylated ssDNA using RNA ligase.
ATP is actually used in the synthesis of RNA ..
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 0.5–30µM 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 0.5–1.0mM. 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.
Guanine may also be used for protein synthesis
We report a simple method of enzymatic synthesis of pre-adenylated DNA linkers/adapters for next-generation sequencing using thermostable RNA ligase from Methanobacterium thermoautotrophicum (MthRnl). Using RNA ligase for the reaction instead of the existing chemical or T4 DNA ligase-based methods allows quantitative conversion of 5′-phosphorylated single-stranded DNA (ssDNA) to the adenylated form. The MthRnl adenylation reaction is specific for ATP and either ssDNA or RNA. In the presence of Mg+2, the reaction has a pH optimum of 6.0–6.5. Unlike reactions that use T4 DNA ligase, this protocol does not require synthesis of a template strand for adenylation. The high yield of the reaction simplifies isolation and purification of the adenylated product. Conducting the adenylation reaction at the elevated temperature (65°C) reduces structural constraints, while increased ATP concentrations allow quantitative adenylation of DNA with a 3′-unprotected end.
to RNA synthesis utilized ribonucleotide ..
Riboflavin is an essential vitamin for cellular metabolism, and the riboflavin carrier protein (RCP) is highly upregulated in metabolically active cells [,]. Thus, flavin mononucleotide (FMN), an endogenous RCP ligand, was used as a small molecule targeting ligand for metabolically active cancer or endothelial cells. Kiessling and co-workers synthesized FMN-coated ultrasmall superparamagnetic iron oxide nanoparticles (FLUSPIO) as MRI/optical dual probes for cancer detection . USPIO was coated with FMN through the phosphate groups of FMN, and guanosine monophosphate was added to stabilize the colloid. The hydrodynamic radius of FLUSPIO was 97 ± 3 nm, and an intense fluorescence emission band was observed at 530 nm due to FMN. cellular uptake of FLUSPIO was investigated by MRI (3T), TEM, and fluorescence microscopy of PC3 cells and HUVEC cells. Both PC3 cells and HUVEC cells showed a significantly higher R2 relaxation rate after 1 h incubation with FLUSPIO than with nontargeted USPIO. Such an uptake was considerably reduced by competitive blocking of RCP with free FMN. A strong green fluorescence in the cells was observed after FLUSPIO incubation. The perinuclear fluorescence signal suggested endosomal localization of the nanoparticles, consistent with TEM results, suggesting that FMN could serve as a versatile building block for generating tumor-targeted imaging and therapeutic modalities.