RNA sensor for detecting thiamine pyrophosphate and application thereof

A technology of pyrophosphate and thiamine, applied in the field of RNA sensors, can solve the problems of cumbersome, high operation requirements, and false positive signals, and achieve the effects of short detection cycle, improved detection limit, and convenient sensitivity

Active Publication Date: 2018-07-24
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many kinds of methods used to detect TPP, all of them have certain limitations and shortcomings.
For example, the detection method based on liquid chromatography-mass spectrometry, although the detection results of this technology are more accurate and reproducible, but it requires professional operation, and the equipment is sophisticated and expensive, and most importantly, it cannot achieve clinical high-throughput detection
Although intracellular fluorescent biosensors use the principle of riboswitches and have good specificity, they require high and cumbersome operations. In addition, this method requires a high level of background concentration (micromol / L, umol / L). Therefore, this method is not suitable for the detection of TPP content in human biological samples (such as in blood: nmol / liter, nmol / L)
Nanogold-based detection methods can achieve lower detection limits, but the specificity of this detection method is not high, such as binding other small molecules through electrostatic interactions, resulting in false positive signals
Although the detection methods based on electrochemical method and chemiluminescence method are relatively convenient and fast, they all have the disadvantage of low specificity for TPP recognition.

Method used

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  • RNA sensor for detecting thiamine pyrophosphate and application thereof
  • RNA sensor for detecting thiamine pyrophosphate and application thereof
  • RNA sensor for detecting thiamine pyrophosphate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The construction of embodiment 1 DNA library

[0055] DNA library construction, such as figure 2 As shown, the DNA sequence was synthesized, purified by 8% denaturing polyacrylamide gel electrophoresis (Polyacrylamide Gel Electrophoresis, PAGE), and the target sequence was recovered with a fluorescence imager and a rubber tapping instrument. Due to technical limitations, the yield of in vitro synthesis of DNA double strands exceeding 100bp is not high, so the present invention uses two segments of single-stranded DNA to extend double-stranded DNA templates in vitro through Polymerase Chain Reaction (PCR), namely Primer 1 (SEQ ID NO: 9) and Primer 2 (SEQ ID NO: 10) are paired with complementary sequences at the ends, and are mutually extended as templates to establish a double-stranded DNA library (random bases are represented by N).

[0056] PCR reaction system:

[0057]

[0058] PCR reaction conditions:

[0059] Pre-denaturation at 95°C for 30s, followed by each...

Embodiment 2

[0064] The construction of embodiment 2 RNA library

[0065] RNA library construction, such as figure 1 As indicated, the above DNA library was transcribed in vitro and incubated at 37°C for 2-3 hours. The transcribed RNA was separated by 8% denaturing PAGE to obtain a sequence that has no cleavage activity in the absence of thiamine pyrophosphate, and the background signal was removed. That is, the RNA library required for screening is constructed, containing 10 random bases.

[0066] Transcription system:

[0067]

Embodiment 3

[0068] Example 3 In vitro screening

[0069] In vitro screening, such as figure 2 shown. The invention adopts the ligand system evolution technology of exponential enrichment, and is divided into steps of pre-screening, screening, reverse transcription, PCR amplification, transcription and the like in vitro. Clones were sequenced after 12 rounds of selection. Specific screening steps include:

[0070] 1. Pre-screening

[0071] Recover the target RNA separated by electrophoresis after transcription, add 50 μl of buffer 1, heat to 70°C for 3-5 minutes, then return to room temperature, add an equal volume of buffer 2 to provide Mg necessary for the enzyme digestion reaction 2+ , allowing the cleavage of sequences that can self-cleavage in the absence of TPP. Perform 8% denaturing PAGE, recover the RNA that has no self-cleavage activity in the absence of TPP, that is, the complete sequence fragment, and elute overnight.

[0072]

[0073] 2. Screening

[0074] After the ...

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PUM

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Abstract

The invention discloses an RNA sensor for detecting thiamine pyrophosphate. The sensor comprises an RNA aptamer for specifically sensing thiamine pyrophosphate, linkage segment RNA and hammerhead ribozyme, wherein the RNA aptamer is connected with the hammerhead ribozyme by virtue of the linkage segment RNA; when the RNA aptamer senses the thiamine pyrophosphate, self-cleavage of the hammerhead ribozyme is triggered. The invention further discloses a kit comprising the RNA sensor disclosed by the invention and a method for detecting the thiamine pyrophosphate. The RNA sensor disclosed by the invention is high in sensitivity of detecting the thiamine pyrophosphate and capable of performing qualitative and quantitative detection on the thiamine pyrophosphate.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an RNA sensor for rapidly and sensitively detecting thiamine pyrophosphate (TPP) and an application thereof. Background technique [0002] Alzheimer's disease (AD) is a degenerative disease of the nervous system. Clinically, it is characterized by generalized dementia such as memory impairment, aphasia, agnosia, executive dysfunction, and personality and behavior changes. The most prominent pathological features of AD include deposition of β-amyloid protein in the brain, hyperphosphorylation of tau-like protein, disturbance of glucose metabolism in the brain, and loss of neurons and their synapses. TPP is the coenzyme factor of the pyruvate dehydrogenase complex in the glucose metabolism pathway, the α-ketoglutarate dehydrogenase complex and the transketolase in the pentose phosphate pathway. The lack of TPP will lead to the decrease of the activity of the above three enzymes, the di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/113C12Q1/6844C12Q1/34G01N33/53
CPCG01N33/5308C12N15/113C12N15/115C12Q1/34C12Q1/6844G01N2333/9005G01N2800/2821C12N2310/121C12N2310/16C12Q2531/119C12Q2563/103
Inventor 顾宏周杜鑫雨钟春玖程小芹
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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