What Is a Ribonuclease?

An enzyme that can only hydrolyze the phosphodiester bond of RNA is called ribonuclease (RNase). Different RNases have different specificities. For example, bovine pancreatic ribonuclease (RNase I), its site of action is the linkage between the pyrimidine 3 'monophosphate and other nucleotides, and ribonuclease T1 ( The site of action of RNase T1) is the 5'-OH linkage between 3L guanylate and other adjacent nucleic acids. ^[1]

Ribonuclease

RNAse, white powder. A globulin-like protein. Soluble in water, 50% acetone. this

Ribonuclease, which can catalyze the degradation of RNA, can now be artificially synthesized. Medicinal ointments are used topically to treat trauma and joint pain. According to reports, ribonuclease can alter host cell metabolism, inhibit virus synthesis, inhibit influenza virus proliferation in vitro, and inhibit the formation of vaccinia and herpes viruses in chicken embryos. Clinical use of RNase intramuscular injection of 180 mg per day is beneficial for the treatment of epidemic encephalitis. ^[3]

(A) ribonuclease A

RNase A is derived from bovine pancreas. It is an endo-ribonuclease that can specifically attack the 3 'end of pyrimidine residues on RaNA, cut cytosine or uracil and adjacent nucleotides. The phosphodiester bond, the final product of the reaction is 3 'pyrimidine nucleotides and oligonucleotides with 3' pyrimidine nucleotides at the ends. In the absence of auxiliary killers and divalent cations, the effect of ribonuclease A can be inhibited by placental RNase inhibitor (RNasin) or vanadyl-ribonucloside complex (VRC). RNase A has extremely wide reaction conditions and is extremely difficult to inactivate. At low salt concentrations (0 to 100 mmol / l NaCl), RNase A cuts single- and double-stranded RNA, RNA in DNA: RNA hybrids; when the NaCl concentration is 300 mmol / l. At or above, RNase A specifically cleaves single-stranded RNA. Removal of RNase A from the reaction solution usually requires proteinase K treatment, repeated extraction of phenol and ethanol precipitation. In molecular cloning, the main uses of ribonuclease A are:

Remove unhybridized RNA regions from DNA: RNA hybrids.

Determine the location of single base mutations in RNA or DNA. In RNA: DNA or RNA: RNA hybrids, if there is a single base mismatch, it can be recognized and cleaved by RNase A. The size of the cleaved product can be analyzed by gel electrophoresis to determine the mismatch position.

RNA detection. RNase protection assay (RNase protection assay) is a hybrid technology that detects RNA developed in recent years. The basic principle is to use single-stranded RNA probes to hybridize with the RNA sample to be tested to form RNA: RNA double-stranded molecules. Because RNase can specifically degrade unhybridized single-stranded RNA, the double-stranded RNA is protected from being Degradation, gel electrophoresis can determine the length of the target RNA. This method is more sensitive than Northern hybridization and can be used for more accurate quantification. By selecting appropriate probes, you can also perform studies on gene transcription initiation sites and intron (also known as intron) splice sites. This method is several times more sensitive than the nuclease S1 protection assay.

Degradation of RNA molecules in DNA preparations. DNase-free RNase (DNase I free RNase) must be used. Commercially available general RNase A often contains DNase, which can be used at 100 retool / I Tris-CI (pH 7.5) and 15 mmol / l. The NaCI solution was heated at 100 ° C for 15 min to remove it.

(Two) ribonuclease T1

RNase T1 (RNase T1) is derived from Aspergillus orjzae. It specifically acts on the 3'-phosphate of guanine. The cleavage site is between the 3'-phosphate of guanine and the 5 'hydroxyl of adjacent nucleotides. As for the phosphodiester bond, the end product of the reaction is a 3 'guanylate and an oligonucleotide fragment with a 3' guanylate at the end. RNaseTl has a wide range of reaction conditions and is extremely difficult to inactivate. Its catalytic activity is similar to RNase A.

In RNase protection experiments, RNase T1 is used in combination with RNase A to quantify and map RNA; it can also be used to remove unhybridized RNA regions in DNA: RNA hybrids.

(Three) ribonuclease H

RNase H was first discovered in calf thymus tissue, and its coding gene has been cloned into E. coli. It can specifically degrade DNA: RNA hybrids double-stranded RNA strands to produce oligonucleotides and single nucleotides with 3'-OH and 5'-monophosphate ends. It cannot degrade single- or double-stranded DNA or RNA.

In molecular cloning, the main use of ribonuclease H is to participate in the synthesis of the second I) NA complementary strand in cDNA clones together with E. coli DNA polymerase and DNA ligase. After using the reverse transcriptase to synthesize the first strand of cDNA with mRNA as a template, the RNA in the mRNA: DNA hybrid molecule is partially digested with RNase H. The resulting RNA fragment is just like the Okazaki fragment, and serves as a primer for E. coli DNA polymerase T Use the first strand of cDNA as a template to synthesize DNA until all mRNA is replaced. Then under the action of DNA ligase, the gap is closed to form a double-stranded cDNA. ^[4]