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CN-121975799-A - Method for determining activity of RNase inhibitor

CN121975799ACN 121975799 ACN121975799 ACN 121975799ACN-121975799-A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for measuring the activity of an RNase inhibitor. The method uses DNA-RNA hybrid strand as substrate to determine RNase inhibitor activity (preferably RNase inhibitor enzyme activity), and the cost is far lower than that of the substrate cCMP of the traditional activity (enzyme activity) unit detection method (cyclic monophosphate (CMP) detection method).

Inventors

  • LI HUIZHEN
  • CHEN XINYU
  • HE XIN
  • TIAN CHAO
  • ZHAO MEIRU

Assignees

  • 吉因加生物医学科技(绍兴)有限公司
  • 长沙吉因加生物科技有限公司

Dates

Publication Date
20260505
Application Date
20251226

Claims (10)

  1. 1. DNA-RNA hybrid strand comprising a DNA strand and an RNA strand, wherein, A1 The DNA strand includes the nucleotide sequence shown in SEQ ID NO. 2, the RNA strand is substantially reverse-complementary, substantially reverse-complementary or completely reverse-complementary to the DNA strand, or A2 The RNA strand comprises the nucleotide sequence set forth in SEQ ID NO. 1, and the DNA strand is substantially reverse complementary, substantially reverse complementary or fully reverse complementary to the RNA strand.
  2. 2. The DNA-RNA hybrid strand according to claim 1, wherein, The DNA strand comprises a nucleotide sequence shown as SEQ ID NO. 2, and the RNA strand comprises a nucleotide sequence shown as SEQ ID NO. 1.
  3. 3. A reagent combination or kit comprising the DNA-RNA hybrid strand of any one of claims 1-2.
  4. 4. A kit or combination of reagents according to claim 3, wherein, The reagent combination or kit further comprises RNase; preferably, the RNase comprises an RNase capable of hydrolyzing RNA in the DNA-RNA hybrid strand, further RNaseA.
  5. 5. The kit or combination of reagents according to any one of claims 3 to 4, The reagent combination or the kit also comprises a substance for detecting the consumption or residual quantity of the DNA-RNA hybrid chain, a substance for detecting the content of the DNA-RNA hybrid chain and/or the DNA single chain, and a substance (such as picogreen dye) for detecting the content of the DNA-RNA hybrid chain; Preferably, the kit further comprises an RNase inhibitor standard.
  6. 6. A detection system includes a luminescence detection apparatus, and The DNA-RNA hybrid strand of any one of claims 1-2 or the reagent combination or kit of any one of claims 3-5.
  7. 7. The detection system of claim 6, wherein the detection system comprises a sensor, The light-emitting detection device comprises at least one of a Qubit, an enzyme-labeled instrument and a qPCR instrument, and is further the Qubit.
  8. 8. A method for determining the activity of an RNase inhibitor comprising the step of employing the DNA-RNA hybrid strand of any one of claims 1-2, the reagent combination or kit of any one of claims 3-5 or the detection system of any one of claims 6-7.
  9. 9. The method of claim 8, wherein the step of determining the position of the first electrode is performed, Mixing an RNase inhibitor, the DNA-RNA hybrid strand according to any one of claims 1 to 2, and the RNase according to any one of claims 4 to 5, reacting, detecting the consumption or residual amount of the DNA-RNA hybrid strand, and determining the activity of the RNase inhibitor according to the consumption or residual amount of the DNA-RNA hybrid strand; preferably, the final concentration of the DNA-RNA hybrid strand in the system of the reaction is 1-3uM; preferably, the final concentration of said RNase in the system of said reaction is 28-36ng/uL; preferably, the volume of the reacted system is 20-30uL.
  10. 10. The method of claim 9, wherein the step of determining the position of the substrate comprises, Obtaining the residual quantity of the DNA-RNA hybrid chain, substituting the residual quantity of the DNA-RNA hybrid chain into a standard curve of the residual quantity of the DNA-RNA hybrid chain and the RNase inhibitor activity to obtain the RNase inhibitor activity; preferably, the standard curve of the residual amount of the DNA-RNA hybrid strand and the RNase inhibitor activity is obtained by the following steps: 1) A plurality of reaction systems are configured for reaction: Each reaction system comprises an RNase inhibitor standard, a DNA-RNA hybrid strand according to any one of claims 1 to 2, an RNase according to any one of claims 4 to 5, The final concentration of the RNase inhibitor standard is different among the reaction systems, wherein the final concentration is calculated by the activity of the RNase inhibitor; 2) Obtaining the residual quantity of the DNA-RNA hybrid chain of each reaction system, and establishing a standard curve according to the corresponding relation between the residual quantity of the DNA-RNA hybrid chain and the RNase inhibitor activity.

Description

Method for determining activity of RNase inhibitor Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for measuring the activity of an RNase inhibitor. Background A conventional enzyme activity unit assay for RNase inhibitors (RNase inhibitors) is to expose cytidine 2',3' -cyclic monophosphate sodium salt (cCMP) to RNaseA in the presence and absence of the RNase inhibitor, hydrolyze the cCMP to increase the OD285, and the activity of the RNase inhibitor can be analyzed by detecting the OD285 using an ultraviolet spectrophotometer (CN 112898402A). Although this assay demonstrated the effect of RNase inhibitors on RNase a inhibition, the shift in absorbance peak wavelength (285 nm-296 nm) after cmp was hydrolyzed resulted in larger detection errors, lower flux as measured by spectrophotometry, and higher cost for cmp. In addition, some manufacturers detect the activity of RNase inhibitors by functionally testing the exposure of radiolabeled RNA to a variety of rnases in the presence and absence of RNase inhibitors. The results were analyzed on polyacrylamide gel to determine the extent to which RNA degradation was inhibited. This functional assay can directly reflect information on the ability of inhibitors to block RNA degradation. However, this detection method cannot quantitatively analyze the activity of the RNase inhibitor, and is complicated in operation and has radioactive contamination. Disclosure of Invention The object of the first aspect of the present invention is to provide a DNA-RNA hybrid strand. The object of the second aspect of the present invention is to provide a reagent combination or kit. A third aspect of the present invention is directed to a detection system. The object of the fourth aspect of the present invention is to provide a method for determining the activity of an RNase inhibitor. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: in a first aspect of the invention, there is provided a DNA-RNA hybrid strand comprising a DNA strand and an RNA strand, wherein, A1 The DNA strand includes the nucleotide sequence shown in SEQ ID NO. 2, the RNA strand is substantially reverse-complementary, substantially reverse-complementary or completely reverse-complementary to the DNA strand, or A2 The RNA strand comprises the nucleotide sequence set forth in SEQ ID NO. 1, and the DNA strand is substantially reverse complementary, substantially reverse complementary or fully reverse complementary to the RNA strand. In some embodiments, the DNA strand comprises the nucleotide sequence set forth in SEQ ID NO. 2 and the RNA strand comprises the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the nucleotide sequence of the DNA strand is shown in SEQ ID NO. 2 and the nucleotide sequence of the RNA strand is shown in SEQ ID NO. 1. In some embodiments, the DNA-RNA hybrid strand is used to determine an RNase (RNase, ribonuclease) activity (preferably RNase enzyme activity) and/or an RNase inhibitor (RNase inhibitor, ribonuclease inhibitor) activity (preferably RNase inhibitor enzyme activity), and further to determine an RNase inhibitor activity (preferably RNase inhibitor enzyme activity). In some embodiments, the RNase is an RNase capable of hydrolyzing RNA in the DNA-RNA hybrid strand, further an RNaseA. In a second aspect of the invention there is provided a reagent combination or kit comprising the DNA-RNA hybrid strand of the first aspect of the invention. In some embodiments, the reagent combination or kit is used to determine an RNase (ribonuclease) activity (preferably an RNase enzyme activity) and/or an RNase inhibitor (RNase inhibitor, ribonuclease inhibitor) activity (preferably an RNase inhibitor enzyme activity), and further to determine an RNase inhibitor activity (preferably an RNase inhibitor enzyme activity). In some embodiments, the reagent combination or kit further comprises RNase. In some embodiments, the RNase comprises an RNase capable of hydrolyzing RNA in the DNA-RNA hybrid strand, further RNaseA. In some embodiments, the reagent combination or kit further comprises a substance for detecting the consumption or residual amount of the DNA-RNA hybrid strand, a substance for detecting the content of the DNA-RNA hybrid strand and/or the DNA single strand, and a substance for detecting the content of the DNA-RNA hybrid strand (e.g., picogreen dye). In some embodiments, the substance that detects the consumption or residual amount of the DNA-RNA hybrid strand comprises the Qubit DNA HS detection kit (Invitrogen, Q32854). In some embodiments, the reagent combination or kit further comprises an RNase inhibitor standard. In some embodiments, the reagent combination or kit is used to determine RNase inhibitor activity (preferably RNase inhibitor enzyme activity). In a third aspect of the present invention, there is provided a detection system comprising a lumines