122
A mutant form of M. thermoautotrophicum RNA ligase has been developed that
lack de-adenylation activity and is ATP independent. The enzyme retains its activity
of ligating RNA or ssDNA to a pre-adenylated linker and hence is an excellent
choice for NextGen RNA sequencing. Use of RNA ligases with de-adenylation
activity can lead to activation of RNA and background production of ligation products. Since the mutant lack self-adenylation property, it does not have this problem
and there is also the added advantage of being functional at 65 °C, which minimizes
the constraints of RNA secondary structure in RNA ligation (Zhelkovsky and
McReynolds 2012).
4.10 Phosphatases and Kinases
Phosphatases are enzymes that remove a phosphate group from a substrate while kinases
are involved in the transfer of a phosphate group to a substrate. Phosphatase catalyzes
the hydrolytic cleavage of phosphate group from a substrate in an irreversible reaction.
On the basis of catalytic activity, phosphatases have been classified into two, that is, acid
phosphatase and alkaline phosphatase of which the latter is more common.
4.10.1 Alkaline Phosphatases
Alkaline phosphatase are homodimeric enzyme that catalyzes the hydrolysis of
5′-phosphate groups from nucleic acids and are most active at alkaline pH of about
10. They contain two Zn
2+
ions and one Mg
2+
ion. Alkaline phosphatases have been
isolated from different sources like microorganisms, tissues of invertebrates, fish,
and mammals, but have not been isolated from higher plants, of which the most
commonly used are those from calf intestinal mucosa and from E. coli. Alkaline
phosphatases are used in molecular biology for dephosphorylation of 5′ end of
DNA or RNA to prevent self-ligation. This is usually carried out in vectors like
plasmid or bacteriophage after restriction digestion to prevent self-ligation, thereby
increasing the ligation of desired insert. Another application is in dephosphorylation
of nucleic acid termini, enabling subsequent in vitro modification like labeling with
radioactive phosphate using T4 polynucleotide kinase which can be used for DNA
or RNA sequencing and fragment mapping.
4.10.1.1 Bacterial Alkaline Phosphatase
Bacterial alkaline phosphatase (BAP) is an 80 kDa homodimer isolated from E. coli
that shows optimum activity at 65 °C. It is a zinc and magnesium-containing protein
and is hence sensitive to chelating agents like EGTA, and low concentrations of
inorganic phosphate (Halford 1971).
4.10.1.2 Calf Intestinal Alkaline Phosphatase
Calf intestinal alkaline phosphatase (CIAP, CIP, or CAP) is a glycoprotein comprising of two identical subunits isolated from calf intestine, which catalyzes dephosphorylation of 5′ termini of DNA and RNA. This 140 kDa enzyme also contains
G. Valsala and S. Sugathan
A mutant form of M. thermoautotrophicum RNA ligase has been developed that
lack de-adenylation activity and is ATP independent. The enzyme retains its activity
of ligating RNA or ssDNA to a pre-adenylated linker and hence is an excellent
choice for NextGen RNA sequencing. Use of RNA ligases with de-adenylation
activity can lead to activation of RNA and background production of ligation products. Since the mutant lack self-adenylation property, it does not have this problem
and there is also the added advantage of being functional at 65 °C, which minimizes
the constraints of RNA secondary structure in RNA ligation (Zhelkovsky and
McReynolds 2012).
4.10 Phosphatases and Kinases
Phosphatases are enzymes that remove a phosphate group from a substrate while kinases
are involved in the transfer of a phosphate group to a substrate. Phosphatase catalyzes
the hydrolytic cleavage of phosphate group from a substrate in an irreversible reaction.
On the basis of catalytic activity, phosphatases have been classified into two, that is, acid
phosphatase and alkaline phosphatase of which the latter is more common.
4.10.1 Alkaline Phosphatases
Alkaline phosphatase are homodimeric enzyme that catalyzes the hydrolysis of
5′-phosphate groups from nucleic acids and are most active at alkaline pH of about
10. They contain two Zn
2+
ions and one Mg
2+
ion. Alkaline phosphatases have been
isolated from different sources like microorganisms, tissues of invertebrates, fish,
and mammals, but have not been isolated from higher plants, of which the most
commonly used are those from calf intestinal mucosa and from E. coli. Alkaline
phosphatases are used in molecular biology for dephosphorylation of 5′ end of
DNA or RNA to prevent self-ligation. This is usually carried out in vectors like
plasmid or bacteriophage after restriction digestion to prevent self-ligation, thereby
increasing the ligation of desired insert. Another application is in dephosphorylation
of nucleic acid termini, enabling subsequent in vitro modification like labeling with
radioactive phosphate using T4 polynucleotide kinase which can be used for DNA
or RNA sequencing and fragment mapping.
4.10.1.1 Bacterial Alkaline Phosphatase
Bacterial alkaline phosphatase (BAP) is an 80 kDa homodimer isolated from E. coli
that shows optimum activity at 65 °C. It is a zinc and magnesium-containing protein
and is hence sensitive to chelating agents like EGTA, and low concentrations of
inorganic phosphate (Halford 1971).
4.10.1.2 Calf Intestinal Alkaline Phosphatase
Calf intestinal alkaline phosphatase (CIAP, CIP, or CAP) is a glycoprotein comprising of two identical subunits isolated from calf intestine, which catalyzes dephosphorylation of 5′ termini of DNA and RNA. This 140 kDa enzyme also contains
G. Valsala and S. Sugathan