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EP-4735627-A1 - MOLECULES RESISTANT TO NUCLEASE DIGESTION

EP4735627A1EP 4735627 A1EP4735627 A1EP 4735627A1EP-4735627-A1

Abstract

Disclosed herein are dual-expression polynucleotide vectors including a first polynucleotide sequence including, in the 5' to 3' direction, a bacteriophage promoter sequence operatively linked to a sequence encoding a plurality of first RNA hairpin structures, a multiple cloning site sequence, a sequence encoding a plurality of second RNA hairpin structures, and a transcription terminator sequence; and a second polynucleotide sequence including, in the 5' to 3' direction, a bacteriophage promoter sequence operatively linked to a ribosome binding site sequence, a viral coat protein sequence, and the transcription terminator sequence. RNA detection and/or quantification standards or controls produced from these dual-expression vectors, along with methods for producing the RNA detection and/or quantification standards or controls, and methods of detecting the presence or quantity of nucleic acid using the RNA detection and/or quantification standards, are also disclosed.

Inventors

  • BLANCHARD, DANIEL

Assignees

  • Life Technologies Corporation

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A dual-expression polynucleotide vector comprising: a first polynucleotide sequence that comprises, in the 5’ to 3’ direction, a bacteriophage promoter sequence operatively linked to a sequence encoding a plurality of first RNA hairpin structures, a multiple cloning site sequence, a sequence encoding a plurality of second RNA hairpin structures, and a transcription terminator sequence; and a second polynucleotide sequence that comprises, in the 5’ to 3’ direction, a bacteriophage promoter sequence operatively linked to a ribosome binding site sequence, a viral coat protein sequence, and the transcription terminator sequence.
  2. 2. The vector of claim 1 , further comprising a sequence encoding a viral maturase protein.
  3. 3. The vector of claim 2, wherein the viral maturase protein is a MS2 viral maturase protein.
  4. 4. The vector of any of claims 1-3, further comprising a sequence encoding a gene that confers antibiotic resistance.
  5. 5. The vector of claim 4, wherein the gene that confers antibiotic resistance is selected from the group consisting of ampicillin, kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, streptomycin, tetracycline, and chloramphenicol.
  6. 6. The vector of any of claims 1-5, wherein the bacteriophage promoter sequence is an inducible promoter sequence.
  7. 7. The vector of any of claims 1-5, wherein the bacteriophage promoter sequence is one selected from a T7 promoter sequence, a pTAC promoter sequence, and a pBAD promoter sequence.
  8. 8. The vector of any of claims 1-7, wherein the viral coat protein is a MS2 viral coat protein.
  9. 9. The vector of any of claims 1-7, wherein the viral coat protein is a modified viral coat protein comprising a viral coat protein having an exogenous polypeptide sequence conjugated to an exterior of said viral coat protein.
  10. 10. The vector of claim 9, wherein the exogenous polypeptide sequence is a targeting ligand that specifically binds to a cell surface protein or is an antigen.
  11. 11. The vector of claim 9, wherein the exogenous polypeptide sequence is a detectable tag.
  12. 12. The vector of any of claims 1-7, wherein the viral coat protein is a modified viral coat protein comprising a viral coat protein having an exogenous polypeptide sequence conjugated to the interior of said viral coat protein.
  13. 13. The vector of any of claims 1-12, wherein the plurality of the first RNA hairpin structures are identical.
  14. 14. The vector of any of claims 1-12, wherein the plurality of the second RNA hairpin structures are identical.
  15. 15. The vector of any of claims 1-14, wherein at least one of the plurality of first RNA hairpin structures is identical to at least one of the second RNA hairpin structures.
  16. 16. The vector of any of claims 1-15, wherein at least one of the plurality of first and second RNA hairpin structures includes SEQ ID NO: 1.
  17. 17. The vector of any of claims 1-16, wherein the sequence encoding the plurality of first RNA hairpin structures encodes two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve RNA hairpin structures.
  18. 18. The vector of any of claims 1-17, wherein the sequence encoding the plurality of second RNA hairpin structures encodes two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve RNA hairpin structures.
  19. 19. The vector of any of claims 1-18, wherein the multiple cloning site sequence further comprises a non-bacteriophage sequence.
  20. 20. The vector of claim 19, wherein the non-bacteriophage sequence is greater than 3 kb in length.

Description

MOLECULES RESISTANT TO NUCLEASE DIGESTION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. §1 19(e) to U.S. Provisional Patent Application No. 63/51 1 ,127, filed June 29, 2023, which is herein incorporated by reference in its entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created, June 26, 2024, is named “TP386985WO1.xml” and is 2,160 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. FIELD [0003] This disclosure relates to various aspects and uses of nuclease resistant nucleic acids. The disclosure also provides various methods of producing, or synthesizing, such nuclease resistant nucleic acids, use of viral-like systems to produce large amounts of nucleic acid and the use of nuclease resistant nucleic acids in various diagnostic assays. The disclosure provides methods of assaying for the presence and/or quantity of RNA. BACKGROUND [0004] Naked RNA standards, single stranded RNA molecules and double stranded RNA molecules are highly susceptible to ribonuclease digestion. Due to the prevalence of ribonucleases in the environment, the use of naked RNA standards in assays designed to detect the presence of an RNA virus in a sample has been challenging. For example, a method typically employed to detect the presence of an RNA virus is fluorescent RT-PCR. An inherent problem in diagnostic PCR assays is the presence of amplification inhibitors which may cause false-negative results. Therefore, the addition of an amplifiable nucleic acid in the PCR assay serves as an internal control (IC). These internal controls are typically naked RNA molecules and can be readily degraded by any ribonuclease at the site of sample preparation and/or the site where the fluorescent RT-PCR method is performed. Since effectively monitoring whether ribonuclease contamination occurs during the nucleic acid extraction process of the sample and/or during the setup of the detection method cannot be easily performed, and the accuracy of the detection method is difficult to ensure, the development of quality control samples and standard substances that are stable, have no infectivity and can be used for detecting viral nucleic acid sequence is of great significance to the clinical detection of viral species. [0005] Bacteriophages that possess RNA as their genetic material (i.e. , RNA bacteriophages) have developed a means to protect their RNA genetic material from ribonuclease digestion. RNA bacteriophages have long been used as model systems to study the mechanisms of RNA replication and translation. The RNA genome within RNA bacteriophages is resistant to ribonuclease digestion due to the protein coat produced by the bacteriophage. Bacteriophages are simple to grow and purify, and genomic RNA can be purified from the bacteriophages. Bacteriophages can be classified into subgroups based on their serotyping (e.g., group 1, 11, III or IV); however, genetically speaking, bacteriophages are divided into two major subclasses, A and B (Stockley, 1994; Witherell, 1991). Bacteriophage MS2/R17 (serological group I) has been well- studied, in addition to other RNA bacteriophages such as GA (group II), Q-beta (group III), and SP (group IV). These RNA bacteriophages infect the male strains of Escherichia coli, that is, those which harbor the F' plasmid and produce an F pilus for conjugation. [0006] The MS2 bacteriophage is an icosahedral structure, 275 A in diameter, and lacks a tail or any other obvious surface appendage (Stockley, 1994). This bacteriophage has large holes at both the 5- and 3-fold axes which might be the exit points of the RNA during bacterial infection. The MS2 bacteriophage consists of 180 units of the bacteriophage coat protein ("14 kDa) which encapsidate the bacteriophage genome (Stockley, 1994; Witherell, 1991). The MS2 RNA genome is a single strand encoding the (+) sense of 3569 nucleotides. The genes of the RNA genome are organized from the 5' end as follows: the maturase or A protein, the bacteriophage coat protein, a 75 amino acid lysis protein, and a Replicase subunit. The lysis gene overlaps the coat protein gene and the Replicase gene and is translated in the +1 reading frame of the coat protein. Each bacteriophage particle has a single copy of maturase which is required for interacting with the F pilus and thus mediating bacterial infection. [0007] The MS2 bacteriophage has been used to create protected RNA molecules. Pasloske et al. (J. Clin. Microbiol. 1998, 3590-3594) produced a plasmid-driven packaging system possessing DNA that encoded the MS2 coat protein, a target RNA sequence, and the MS2 operator downstream of an inducible lac promoter. The recombinant packaging vector was t