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KR-102961084-B1 - Biosensor for simultaneous detection of cell-free DNA and microRNA using CRISPR-Cas12a and CRISPR-Cas13a

KR102961084B1KR 102961084 B1KR102961084 B1KR 102961084B1KR-102961084-B1

Abstract

The present invention relates to a biosensor for the simultaneous multiplex detection of target nucleic acid molecules comprising gold nanorods immobilized with ssDNA and ssRNA, and a composition for cancer diagnosis using said biosensor. By enabling rapid simultaneous multiplex detection of target nucleic acid molecules, such as cancer-related cfDNA and miRNA, with high accuracy and sensitivity, it can be usefully employed in the field of cancer diagnosis.

Inventors

  • 최진하
  • 최민유

Assignees

  • 전북대학교산학협력단

Dates

Publication Date
20260507
Application Date
20250116
Priority Date
20241226

Claims (19)

  1. As a biosensor for the simultaneous multiplex detection of target nucleic acid molecules comprising gold nanorods immobilized with ssDNA (single-stranded DNA) and ssRNA (single-stranded RNA), The above ssDNA and ssRNA each have a fluorescent substance attached to one end, and The above target nucleic acid molecules are target DNA and RNA, and The above ssDNA is nonspecifically cleaved by the activation of the CRISPR-Cas12a complex when target DNA is present, generating a fluorescent signal, and A biosensor for the simultaneous multiplex detection of target nucleic acid molecules, wherein the above ssRNA is nonspecifically cleaved by the activation of the CRISPR-Cas13a complex to generate a fluorescent signal when target RNA is present.
  2. In Article 1, A biosensor in which the above cleavage is a non-specific cleavage by a trans -cleavage reaction.
  3. delete
  4. In Article 1, The above fluorescent substances are FAM (6-carboxyfluorescein), Texas Red, fluorescein, TET (tetrachlorofluorescein), HEX (hexachloro-6-carboxyfluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), ROX (6-Carboxyl-X-Rhodamine), FITC (fluorescein isothiocyanate), Oregon Green, Alexa Fluor, and JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein). A biosensor selected from the group consisting of VIC(2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein), NED(N-(1-naphthyl) ethylenediamine), Cy5(cyanine-5) and Cy3(cyanine-3).
  5. In Article 1, A biosensor in which the above ssDNA and ssRNA have a length of 10 to 50 nucleotides (nt).
  6. In Article 5, A biosensor in which the above ssDNA comprises the nucleic acid sequence of SEQ ID NO. 1.
  7. In Article 1, A biosensor in which the above ssRNA comprises the nucleic acid sequence of SEQ ID NO. 2.
  8. In Article 1, A biosensor in which the above target nucleic acid molecules are cancer-related cfDNA and miRNA.
  9. In Article 8, A biosensor in which the above cancer-related cfDNA is selected from the group consisting of PTEN, TMPRSS2-ERG fusion, FGFR3 mutation, EGFR, KRAS, TP53, CTNNB1 (β-catenin), and BRCA1.
  10. In Article 8, A biosensor in which the above cancer-related miRNA is selected from the group consisting of miR-141, miR-574-3p, miR-155, miR-21, miR-34a, miR-200 family, miR-122, and miR-10b.
  11. delete
  12. In Article 1, A biosensor in which the above-mentioned CRISPR-Cas12a complex comprises a Cas12a nuclease and a CRISPR RNA (crRNA) specific to target cfDNA.
  13. In Article 1, A biosensor in which the above-mentioned CRISPR-Cas13a complex comprises a Cas13a nucleus and a CRISPR RNA (crRNA) specific to a target miRNA.
  14. A biosensor for simultaneous multiplex detection of target nucleic acid molecules according to any one of claims 1, 2, 4 to 10, 12 and 13; CRISPR-Cas12a complex; and A cancer diagnostic composition comprising a CRISPR-Cas13a complex.
  15. A cancer diagnostic kit comprising a cancer diagnostic composition according to claim 14.
  16. (a) reacting a biological sample separated from an analysis subject with a cancer diagnostic composition according to claim 14; and (b) A method for simultaneous multiplex detection of target nucleic acid molecules comprising the step of obtaining an upper layer and measuring a fluorescence signal, The above target nucleic acid molecules are target DNA and RNA, and The above target DNA and RNA are simultaneously confirmed to be detected, and When target DNA is present, ssDNA is nonspecifically cleaved by the activation of the CRISPR-Cas12a complex to generate a fluorescent signal, and A method for the simultaneous multiplex detection of target nucleic acid molecules, wherein ssRNA is nonspecifically cleaved by the activation of the CRISPR-Cas13a complex to generate a fluorescent signal when target RNA is present.
  17. In Article 16, A method in which the biological sample is selected from the group consisting of blood, serum, plasma, saliva, and urine.
  18. In Article 16, A method in which the target nucleic acid molecules are cancer-related cfDNA and miRNA.
  19. delete

Description

Biosensor for simultaneous detection of cell-free DNA and microRNA using CRISPR-Cas12a and CRISPR-Cas13a The present invention relates to a biosensor for the simultaneous multiplex detection of target nucleic acid molecules comprising gold nanorods immobilized with ssDNA and ssRNA, and a composition for cancer diagnosis using said biosensor. Cell-free DNA (cfDNA) is released from advanced cancer cells as a byproduct generated during apoptosis, necrosis, or active cellular secretion processes. However, the clinical implementation of cfDNA detection is known to be challenging due to low concentrations in body fluids such as blood (Nano Lett 2021, 21, 693-699). Similarly, microRNA (miRNA), a type of noncoding RNA composed of 19 to 25 nucleotides, is helpful in understanding disease progression and is considered a promising biomarker for early diagnosis (ACS Appl Mater Interfaces 2019, 11, 18923-18929; Sens Actuators B Chem 2020, 325, 128799; and Sens Actuators B Chem 2023, 393, 134280). Since both cfDNA and miRNA exist at low concentrations in body fluids, a high-sensitivity system capable of accurately distinguishing trace amounts of target molecules is required for early diagnosis. Furthermore, while both are being studied as biomarkers that provide disease information, a single biomarker alone is insufficient to accurately identify a specific disease. For example, EGFR is a well-studied DNA biomarker associated with various cancers, including head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, and esophageal cancer (Eur J Cancer 2001, 37, 9-15). Similarly, it is difficult to definitively diagnose that the abnormal expression of a specific miRNA implies only one specific disease (Sens Actuators B Chem 2023, 393, 134280; and Biosens Bioelectron 2019, 124-125, 199-204). Therefore, it is important to detect multiple biomarkers simultaneously, as this contributes to effectively reducing false positive or false negative results. Multiple biomarker analysis is highly preferred as it not only improves diagnostic accuracy but also reduces sample usage, analysis time, and costs (Angewandte Chemie 2019, 131, 13342-13353; and Biosens Bioelectron 2024, 253, 116172). Existing nucleic acid detection technologies include quantitative reverse transcription polymerase chain reaction (qRT-PCR) along with isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) (Biosensors (Basel) 2024, 14, 197). Among these, qRT-PCR has garnered attention for its high sensitivity, and its importance was highlighted during the COVID-19 pandemic. However, the application of qRT-PCR is limited due to its long analysis time, complex procedures, reliance on temperature-controlled equipment, specialized primers, and skilled operators (Sens Actuators B Chem 2023, 393, 134280; Anal Chem 2021, 93, 11899-11909; and Microchemical Journal 2024, 197, 109792). In addition, while LAMP and RPA exhibit similar sensitivity and rapid reaction speeds compared to qRT-PCR, they also have limitations in that they require specialized equipment, expensive facilities, and significant reagent consumption (Nano Lett 2021, 21, 693-699; Biosens Bioelectron 2020, 166, 112445; and ACS Nano 2022, 16, 1381-1394). Furthermore, since these methods necessarily require a nucleic acid amplification step, there is a possibility of resulting in false positive diagnoses (Angewandte Chemie International Edition 2023, 62). A technology attracting attention as an alternative nucleic acid detection method is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. The CRISPR system enables nucleic acid detection with very high specificity and accuracy by utilizing the complementary binding of a target nucleic acid sequence with CRISPR RNA (crRNA) (Nano Today 2021, 40, 101268; and Sens Actuators B Chem 2022, 371, 132537). Among CRISPR-Cas, CRISPR-Cas12a, after being activated by complementary target DNA, induces a non-specific trans -cleavage reaction on adjacent single-stranded DNA (ssDNA) (Nano Lett 2021, 21, 693-699; and Biosens Bioelectron 2020, 166, 112445). Similarly, when CRISPR-Cas13a is activated by target RNA, it exhibits a trans -cleavage response to nearby single-stranded RNA (ssRNA) (Sens Actuators B Chem 2020, 325, 128799; Anal Chem 2021, 93, 2038-2044; Biosens Bioelectron 2022, 201, 113960; Advanced Materials 2019, 31; Biosens Bioelectron 2022, 217, 114712; and Nat Commun 2020, 11, 4906). These CRISPR systems can distinguish even single nucleotide differences in target sequences and do not require an amplification step, enabling time-saving and simplified procedures (Nano Lett 2021, 21, 693-699; and ACS Nano 2021, 15, 13475-13485). However, to further enhance sensitivity, a nucleic acid pretreatment amplification step of the target is often used prior to the CRISPR reaction (Science (1979) 2018, 360, 439-444; and Anal Chem 2021, 93, 4967-4974). Accordingl