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CN-121975811-A - Mirror image aptamer 5' -Biotin L-PEA1-20 combined with myoglobin and application thereof

CN121975811ACN 121975811 ACN121975811 ACN 121975811ACN-121975811-A

Abstract

The invention provides a mirror image nucleic acid aptamer 5' -Biotin L-PEA1-20 combined with myoglobin, which shows excellent combination capacity in myoglobin combination, and a unique mirror image structure of the mirror image nucleic acid aptamer also endows the mirror image nucleic acid aptamer with extremely high digestion degradation resistance, and the mirror image nucleic acid aptamer can keep high stability under the existence of DNase I and EXO I. The invention has important significance in myoglobin related research, can be widely applied to a plurality of fields such as medical diagnosis, drug development, biotechnology and the like, and has obvious advantages in the accurate detection and targeting research of myoglobin in complex physiological environment.

Inventors

  • LI ZILONG
  • HAN DA
  • HE LEI
  • XIE YUANYUAN

Assignees

  • 浙江工业大学
  • 中国科学院杭州医学研究所

Dates

Publication Date
20260505
Application Date
20251223

Claims (8)

  1. 1. The myoglobin-binding mirror image aptamer 5'-Biotin L-PEA1-20 is characterized in that the aptamer 5' -Biotin L-PEA1-20 has a nucleotide sequence shown in SEQ ID.1.
  2. 2. A method for preparing myoglobin-binding mirror nucleic acid aptamer 5'-Biotin L-PEA1-20 according to claim 1, wherein the method is characterized in that the myoglobin-binding mirror nucleic acid aptamer 5' -Biotin L-PEA1-20 is obtained by a DNA solid phase phosphoramide triester synthesis technology according to a nucleotide sequence shown in SEQ ID 1 and is purified by HPLC.
  3. 3. Use of the myoglobin-binding mirror aptamer 5' -Biotin L-PEA1-20 of claim 1 for detecting myoglobin concentration.
  4. 4. The use according to claim 3, wherein the detection method is: Mixing the myoglobin-bound mirror image aptamer 5' -Biotin L-PEA1-20 with a first buffer solution in a SA modified 96-well plate, performing first incubation for 45-60 minutes, removing liquid after the incubation is completed, adding a second phosphate buffer solution for cleaning, adding a myoglobin standard substance solution, performing second incubation for 45-75 minutes at room temperature, removing liquid, adding a third phosphate buffer solution, continuously adding a luminol chemiluminescent substrate, detecting the chemiluminescent signal intensity after 0-5 minutes, and obtaining a standard curve of the signal intensity changing with the concentration of myoglobin, wherein the volume ratio of the third phosphate buffer solution to the luminescent substrate is 2:1; Mixing the myoglobin-bound mirror image aptamer 5' -Biotin L-PEA1-20 with a fourth buffer solution in a SA modified 96-well plate, performing third incubation for 45-60 minutes, removing liquid after the incubation is completed, adding a fifth phosphate buffer solution for cleaning, adding a sample solution to be detected, performing fourth incubation for 45-75 minutes at room temperature, removing liquid again, adding a sixth phosphate buffer solution, continuously adding a luminol chemiluminescent substrate, detecting the intensity of a chemiluminescent signal after 0-5 minutes, comparing the intensity of the chemiluminescent signal with a standard curve of the change of the signal intensity along with the concentration of myoglobin, and determining the concentration of myoglobin in the sample to be detected, wherein the volume ratio of the sixth phosphate buffer solution to the luminescent substrate is 2:1.
  5. 5. The use according to claim 4, wherein the myoglobin-binding mirror aptamer L-PEA1-20 is subjected to a heat treatment at 95℃for 10 minutes followed by a cooling to 25℃over 30 minutes before use.
  6. 6. The use of claim 4, wherein said first phosphate buffer is 10mm in concentration and has a ph of 6.5, said second buffer is DPBS containing 0.2% Tween 20, said third buffer is DPBS containing 0.2% Tween 20, said fourth phosphate buffer is the same as said first phosphate buffer, said fifth phosphate buffer is the same as said second phosphate buffer, and said sixth phosphate buffer is the same as said third phosphate buffer.
  7. 7. The use of claim 4, wherein the first incubation time is 60 minutes, the second incubation time is 60 minutes, the third incubation time is 60 minutes, and the fourth incubation time is 60 minutes.
  8. 8. The use according to claim 4, wherein the method detects myoglobin in a linear range of 10 pM to 10 nM with a limit of detection of 62.96pM.

Description

Mirror image aptamer 5' -Biotin L-PEA1-20 combined with myoglobin and application thereof Technical Field The invention relates to a mirror image nucleic acid aptamer L-PEA1-20 capable of binding myoglobin (Myoglobin) and enhancing peroxidase activity thereof. Background Nucleic acid aptamers are a class of artificially screened and synthesized nucleic acid molecules that are capable of specifically binding to a variety of targets, including proteins, small molecules, and cells, and of modulating their function. By virtue of the characteristics of high specificity, good stability and easy modification, the aptamer has great application potential in the fields of diagnosis, treatment, biotechnology and the like. Myoglobin is a monomeric globular protein containing heme and plays an important role in storing and transporting oxygen in muscle tissue. Furthermore, myoglobin may also exhibit peroxidase-like activity under certain conditions. Also, the massive release of myoglobin is one of the key hallmarks of rhabdomyolysis and can lead to acute kidney injury. Therefore, the development of the aptamer capable of efficiently combining myoglobin has important significance and wide application prospect in the application of myoglobin in the fields of medical research, drug development and biotechnology. Currently, the aptamer for myoglobin binding is a natural right-handed DNA sequence obtained by in vitro screening techniques. These aptamers are susceptible to nuclease degradation in complex physiological environments, resulting in structural disruption, and thus loss of the ability to bind myoglobin, which severely limits their use in biomedical fields to some extent. Mirror image nucleic acid is an artificial nucleic acid composed of chiral nucleic acid molecules, and the structure of the mirror image nucleic acid is a mirror image form of natural nucleic acid, so that the mirror image nucleic acid has extremely high stability and degradation resistance. Mirror nucleic acids, being unrecognized by natural enzymes, have demonstrated great potential for use in biomedical fields such as drug development and molecular diagnostics. Disclosure of Invention The invention provides a myoglobin mirror image nucleic acid aptamer (namely left chirality) 5' -Biotin L-PEA1-20, which is proved to be capable of binding myoglobin with high affinity, has similar binding characteristics with reported right chirality myoglobin nucleic acid aptamer D-PEA1-20, can enhance peroxidase activity of myoglobin, has obviously enhanced cleavage resistance, and has the characteristic of indicating wider application potential of the aptamer in complex physiological environments. The technical scheme adopted by the invention is as follows: the invention provides a mirror image nucleic acid aptamer 5'-Biotin L-PEA1-20 combined with myoglobin, wherein the nucleic acid aptamer 5' -Biotin L-PEA1-20 has a nucleotide sequence shown as SEQ ID.1. Furthermore, according to the nucleotide sequence shown in SEQ ID.1, the mirrored nucleic acid aptamer 5' -Biotin L-PEA1-20 combined with myoglobin is obtained through a DNA solid phase phosphoramide triester synthesis technology and HPLC purification. The invention also provides application of the mirror image aptamer 5' -Biotin L-PEA1-20 combined with myoglobin in detecting myoglobin concentration. Further, the detection method comprises the following steps: Mixing the myoglobin-bound mirror image aptamer 5' -Biotin L-PEA1-20 with a first buffer solution, mixing the mixture with a SA modified 96-well plate, performing first incubation for 45-60 minutes (fixing the 5' -Biotin L-PEA1-20 in the SA modified 96-well plate), removing liquid after incubation, adding a second phosphate buffer solution for cleaning, adding a myoglobin standard substance solution, performing second incubation for 45-75 minutes (preferably 60 minutes) at room temperature (capturing MB by the 5' -Biotin L-PEA1-20 to form an aptamer-myoglobin complex), adding a third phosphate buffer solution, continuously adding a luminol chemiluminescent substrate, detecting the intensity of a chemiluminescent signal after 0-5 minutes to obtain a standard curve of the change of the signal intensity along with the concentration of myoglobin, and the volume ratio of the third phosphate buffer solution to the luminescent substrate is 2:1; Mixing the myoglobin-bound mirror image aptamer 5' -Biotin L-PEA1-20 with a fourth buffer solution in a SA modified 96-well plate, performing third incubation for 45-60 minutes (fixing the 5' -Biotin L-PEA1-20 in the SA modified 96-well plate), removing liquid after the incubation is finished, adding a fifth phosphate buffer solution for cleaning, adding a sample solution to be tested, performing fourth incubation for 45-75 minutes (preferably 60 minutes) at room temperature, capturing MB by the 5' -Biotin L-PEA1-20 to form an aptamer-myoglobin complex, adding a sixth phosphate buffer solution again, continuously adding