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Single-stranded nucleic acid aptamers and their applications

A single-stranded nucleic acid and aptamer technology, applied in the fields of biomedicine and clinical medicine, can solve the problems of decreased antibody detection ability, inability to identify irregular antibody specificity, and disappearance of degradation, so as to avoid damage and prevent neonatal hemolysis disease, and the effect of avoiding transfusion reactions

Inactive Publication Date: 2017-08-29
SHENZHEN BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of irregular antibodies by spectrum red blood cells is currently the only method for detecting irregular antibodies, but this method has the following disadvantages: ① The storage time of red blood cells is short, and with the prolongation of the storage period, some blood group antigens will degrade and disappear, ②Due to the skewed distribution of most red blood cell antigens in the population, it is difficult to find some antigen-negative red blood cells, so it is impossible to identify and distinguish some irregular antibodies; ③For the presence of multiple antibodies or autoantibodies In the case of erythrocytes, the specificity of irregular antibodies cannot be identified; ④The current method of using erythrocytes to identify antibodies cannot be automated and standardized, and quantitative detection of antibodies cannot be achieved
In order to alleviate the above-mentioned crisis, the blood transfusion rules allow Rh-positive blood to be given to Rh-negative emergency patients, but only to RhD-negative patients who have not produced RhD antibodies; for RhD-negative patients who have produced RhD antibodies, they can still only wait for Rh-negative blood

Method used

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  • Single-stranded nucleic acid aptamers and their applications
  • Single-stranded nucleic acid aptamers and their applications
  • Single-stranded nucleic acid aptamers and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] This example provides a method for preparing a ssDNA secondary library, the preparation process is shown in figure 1 , including the following steps:

[0034] Step 1: Use biotin-labeled upstream and downstream primers to perform PCR amplification on the ssDNA library to obtain biotin-carrying dsDNA to provide sufficient templates for asymmetric PCR.

[0035] The PCR reaction system is as follows:

[0036]

[0037] The Taq enzyme is selected from the kit of model M1665S of Promega Company.

[0038] The biotin-labeled upstream primer is 5'-biotin-AGAGACGGACACAGGATGAGC-3';

[0039] The biotin-labeled downstream primer is 5'-biotin-TGGATGCTGTCTTGGGGAAGG-3';

[0040] Wherein, biotin is biotin.

[0041] The ssDNA library is composed of 82 bp, the middle of the library is a random sequence of 40 bp, and the two sides are primer binding regions composed of 21 bp respectively: 5'-AGAGACGGACACAGGATGAGC-N40-CCTTCCCCAAAGACAGCATCCA-3'.

[0042] The above primers and ssDNA li...

Embodiment 2

[0079] This example provides a method for screening single-stranded nucleic acid aptamers capable of specifically neutralizing RhD antibodies through the ssDNA secondary library of Example 1: Step 1: Sequencing of the screened ssDNA sequence library

[0080] The ssDNA secondary library obtained in Example 1 was sequenced and analyzed using Ion Torrent semiconductor sequencing technology (the sequencing service was completed by Life Technologies).

[0081] according to Figure 5 It can be seen from the ssDNA single-strand sequence library length classification statistics chart that the expected sequencing target length meets the requirements, which is about 82bp; see Figure 6 .

[0082]According to the sequencing results of the ssDNA sequence library, we took the sequence with the target sequence in the middle, adapters at both ends, and the adapters completely matched as the key verification object. At the same time, considering that the sequencing has no directionality, th...

Embodiment 3

[0156] This embodiment provides ssDNA, a kit or a chip for detecting RhD antibodies, which contain the single-stranded nucleic acid aptamer SEQ ID NO: 19 described in Example 2.

[0157] This embodiment also provides a therapeutic agent for neonatal hemolytic disease, the therapeutic agent contains the single-stranded nucleic acid aptamers SEQ ID NO: 14 and SEQ ID NO: 20 described in Example 2.

[0158] This embodiment further provides an adjuvant for blood transfusion of RhD-negative patients, the adjuvant containing the single-stranded nucleic acid aptamers SEQ ID NO: 14 and SEQ ID NO: 20 described in Example 2.

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

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Abstract

The invention discloses single-stranded aptamer. A general formula of the single-stranded aptamer is 5'-AGAGACGGACACAGGATGAGC-NCCTTCCCCAAGACAGCATCCA-3', with N referring to a random sequence 35 to 45 bp in length; the single-stranded aptamer can specifically neutralize RhD antibody; N is SEQ ID NO.2, SEQ ID NO.7 or SEQ ID NO.8. The single-stranded aptamer can specifically neutralize the RhD antibody and can be made into a composition, a kit or a chip for detection of the RhD antibody; therapeutic agent made from the single-stranded aptamer is applicable to treatment of hemolytic disease of the newborn, and auxiliary made from the single-stranded aptamer is suitable for emergency blood transfusion of patients with negative RhD.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and clinical medicine. Specifically, the present invention relates to single-stranded nucleic acid aptamers and applications thereof. Background technique [0002] Nucleic acid aptamer (Aptamer) screening technology is an emerging multidisciplinary research technology in recent years. Nucleic acid aptamers have strong specificity and high affinity for the recognition of target molecules. Interaction, new anti-infection drug development, refractory viral infectious diseases (such as AIDS, HBV, HSV, Rabies virus, etc.) and cancer treatment have shown broad application prospects. Although no nucleic acid aptamers have been used in the research of the Rh blood group system, similar studies have shown that peptides, bacteria, virus particles, mammalian cells, nucleic acids, etc. can be screened for high-affinity nucleic acid aptamers by SELEX technology. Nucleic acid aptamers can be obtained in l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/68A61K31/7088A61P7/00
Inventor 张印则吴凡庄乃保徐华周华友苏宇清梁延连李大成周丹
Owner SHENZHEN BLOOD CENT
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