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Method and Application of Monitoring mRNA Poly(A) Tail Length

A tail reaction and reaction technology, applied in the field of monitoring the tail length of mRNAPoly, can solve the problems of high cost and time-consuming, and achieve the effects of intuitive results, low detection cost, and simple and efficient detection.

Active Publication Date: 2021-04-13
LIVERNA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional method is to detect the change of mRNA molecular weight before and after tailing by electrophoresis to reflect the tailing length, which is time-consuming and expensive
There is currently no simple, fast, and real-time monitoring method to control tailing length during mRNA production

Method used

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  • Method and Application of Monitoring mRNA Poly(A) Tail Length
  • Method and Application of Monitoring mRNA Poly(A) Tail Length
  • Method and Application of Monitoring mRNA Poly(A) Tail Length

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Using bioluminescence to detect ATP consumption and control tail length

[0101] 1. Standard tailing reaction

[0102] S1. Prepare ATP detection working solution

[0103] Prepare an appropriate amount of ATP detection working solution according to the ratio of 100 μl ATP detection working solution required for each sample. Take an appropriate amount of ATP detection reagent and dilute the ATP detection reagent with ATP detection reagent diluent in a ratio of 1:9. For example, 100 μl of ATP detection reagent is added to 900 μl of ATP detection reagent diluent to prepare 1 ml of ATP detection working solution. The detection working solution should be placed at room temperature for 3-5 minutes before use.

[0104] S2, build a standard tailing reaction

[0105] 500 μM ATP was added to the tailing reaction with a constant concentration of 0.6 μM mRNA for the reaction. When the reaction time was 0, 5, 10, 15, 20, 25, 30, and 35 min, 10 μl was sampled and added t...

experiment example 1

[0119]In this experimental example, Bioanalyzer was used to sample the samples at the 15th, 17th and 20th minutes in the above-mentioned pilot-scale reaction, and then analyzed, and the detection results of the two methods were compared. The results are shown in Table 2 and Figure 10 shown, from Figure 10 It can be seen that the values ​​obtained by the two detection methods provided in Example 1 and Comparative Example 1 are very close. As can be seen from Table 2, among the detection methods provided in the comparative example, pretreatment is required before sample detection, and the techniques to be used include capillary electrophoresis, etc. These techniques require a relatively complex sample preparation process, and the instruments are relatively expensive. The cost is high, and the entire inspection process takes a long time, which cannot achieve the purpose of real-time quality feedback, thereby controlling product quality. However, the detection method provided i...

Embodiment 2

[0122] Example 2 The effect of tail length on stable expression of intracellular proteins

[0123] This example evaluates the effect of tail length on stable expression of intracellular proteins and its potential impact on the efficacy of mRNA-based therapies. Specifically, the protein expression levels of mRNAs with different tail lengths in cells were evaluated. mRNA was synthesized from a plasmid DNA template encoding the gene by in vitro transcription, with the addition of a 5' cap structure and Poly(A) tails of various lengths.

[0124] HEK293 cells were plated at a density of 8 × 10 5 pcs / well, at 37°C, 5.0% CO 2 overnight in the incubator. Then the medium was replaced, the cell plate was taken out and the medium was sucked off, then fresh medium was added, and placed at 37°C, 5.0% CO. 2 incubator. Solution A: Mix 2 μg mRNA in 100 μl Opti-MEM medium, Solution B: Mix 3 μl Lipo MessengerMax with 100 μl Opti-MEM medium, Mix A and B evenly, and let stand at room tempera...

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Abstract

The present invention provides a method and application for monitoring mRNA Poly(A) tail length, and relates to the field of biotechnology. The method for monitoring mRNA Poly(A) tail length provided by the invention includes measuring ATP consumption in mRNA tailing reaction Quantity and / or PPi generation yields mRNA Poly(A) tailed length. This method is applicable to any type of mRNA, has wide applicability, and can effectively replace complex detection methods such as capillary electrophoresis used in the prior art to quickly and effectively detect the tail length of mRNA, making the detection simpler and more efficient. Moreover, the relationship between ATP consumption and mRNA tail length is used to monitor the mRNA Poly(A) tail length, and the result is more intuitive and accurate. At the same time, the detection of ATP consumption does not require expensive instruments, and the detection cost is low.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for monitoring the length of mRNA Poly(A) tails. Background technique [0002] Messenger RNA (mRNA) is a promising new therapeutic option with broad prospects in fields such as immunology, oncology, and vaccines. mRNA contains instructions for cells to make proteins and deliver them to various parts of the body, so mRNA can be used as a drug to direct intracellular protein expression or extracellular protein secretion. Effective mRNA therapy requires efficient delivery of mRNA into a patient and efficient production of the protein encoded by the mRNA in the patient. To prevent mRNA degradation and enhance its stability, appropriate tailing is usually required at the 3' end of the mRNA. Therefore, accurately reflecting the length of the Poly(A) tail is very important for the quality control of mRNA in the production process. [0003] The traditional method ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/00C12Q1/68
CPCC12Q1/008C12Q1/68C12Q2525/173
Inventor 苏晓晔李爽彭育才刘隽向晟楠刘琪
Owner LIVERNA THERAPEUTICS INC
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