Trna-based methods and related compositions
Methods for determining tRNA acylation and preparing peptides through RNA manipulation improve the assessment of tRNA acylation efficiency, enabling precise peptide preparation and RNA manipulation.
Patent Information
- Authority / Receiving Office
- HK · HK
- Patent Type
- Applications
- Current Assignee / Owner
- UNITED KINGDOM RESEARCH AND INNOVATION
- Filing Date
- 2026-05-27
- Publication Date
- 2026-07-10
AI Technical Summary
Current methods for determining the effect of reagents on tRNA acylation and assessing acylation efficiency are inadequate, and there is a need for improved methods to prepare peptides and manipulate tRNA molecules, particularly through splitting and fusing RNA molecules.
The development of methods for determining tRNA acylation and acylation efficiency, including the preparation of peptides, splitting and fusing RNA molecules, and the use of labeled tRNA moieties, as well as nucleic acid constructs encoding circularly rearranged transcripts, proteins, and enzymes.
Enables precise determination of tRNA acylation effects and efficiency, facilitating the preparation of peptides and manipulation of RNA molecules, thereby enhancing the understanding and application of tRNA in biological processes.
Smart Images
Figure 00000000_0000_ABST
Abstract
Description
This invention relates to methods for determining the effect of reagents on tRNA acylation, methods for determining the acylation state or acylation efficiency of tRNA, and methods for preparing peptides. The invention also relates to splitting tRNA, fusing RNA molecules, and labeled tRNA or tRNA moieties. The invention further relates to nucleic acid constructs comprising nucleic acids encoding circularly rearranged transcripts. Furthermore, the invention relates to proteins, enzymes, and synthases obtained by the methods disclosed herein or using the products disclosed herein. Abstract
Claims
CLAIMS 1. A method of determining the effect of a polypeptide-of-interest or nucleic-acid-of-interest on acylation of a tRNA or acylation status of a tRNA, the method comprising: i) incubating the polypeptide-of-interest or nucleic-acid-of-interest, the tRNA, and a substrate with which the tRNA could be acylated, under conditions conducive to acylation of the tRNA, wherein the tRNA is split into at least two portions, and one of said tRNA portions is present as part of a fusion RNA molecule also comprising a sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest; ii) exposing the tRNA to conditions capable of labelling tRNAs that have been acylated; and iii) identifying whether the polypeptide-of-interest or nucleic-acid-of-interest is associated with a labelled tRNA.
2. The method of claim 1, wherein the tRNA does not comprise an anticodon.
3. The method of claim 1 or claim 2, wherein the tRNA is split at the location of an anticodon in a parental tRNA from which the tRNA portion is derived.
4. The method of any preceding claim, wherein the tRNA is a tRNAPyl, tRNALeu, tRNAAla, or tRNASer.
5. The method of any preceding claim, wherein the tRNA is split into a first tRNA chain and a second tRNA chain, the first tRNA chain comprises a tRNA portion from the 5’ side of the location of anticodon in a parental tRNA from which the tRNA portion is derived, and the second tRNA chain comprises a tRNA portion from the 3’ side of the location of anticodon in a parental tRNA from which the tRNA portion is derived.
6. The method of claim 5, wherein: a) the first tRNA chain comprises a sequence encoded by SEQ ID NO: 2 and the second tRNA chain comprises a sequence encoded by SEQ ID NO: 1; b) the first tRNA chain comprises a sequence encoded by SEQ ID NO: 24 and the second tRNA chain comprises a sequence encoded by SEQ ID NO: 23; c) the first tRNA chain comprises a sequence encoded by SEQ ID NO: 26 and the second tRNA chain comprises a sequence encoded by SEQ ID NO: 25; d) the first tRNA chain comprises a sequence encoded by SEQ ID NO: 28 and the second tRNA chain comprises a sequence encoded by SEQ ID NO: 27; or e) the first tRNA chain comprises a sequence encoded by SEQ ID NO: 47 and the second tRNA chain comprises a sequence encoded by SEQ ID NO:
46.
7. The method of claim 1, wherein the tRNA is split in the D loop, in the anticodon loop and to the 5’ side of the anticodon, in the variable loop, or in the T loop.
8. The method of claim 1 or claim 7, wherein tRNA is split between residues D15 & D16, D15 & D17, D15 & D18, D16 & D17, D16 & D18, D17 & D18, A31 & A32, A31 & A33, A32 & A33, V45 & V46, V45 & V47, V45 & V48, V46 & V47, V4640 & V48, V47 & V48, T56 & T57, T56 & T58, or T57 & T58.
9. The method of claim 8, wherein tRNA is split between residues D15 & D17, D15 & D18, D16 & D17, A31 & A33, A32 & A33, V45 & V48, V46 & V48, V47 & V48, T56 & T57, or T56 & T58.
10. The method of any one of claims 7-9, wherein the tRNA is a tRNATrpor tRNAPro.
11. The method of any one of claims 1 or 7-10, wherein the tRNA is split into a first tRNA chain and a second tRNA chain, the first tRNA chain comprises a tRNA portion from the 5’ side of the split in a parental tRNA from which the tRNA portion is derived, and the second tRNA chain comprises a tRNA portion from the 3’ side of the location of the split in a parental tRNA from which the tRNA portion is derived.
12. The method of claim 11, wherein the first tRNA chain and the second tRNA chain comprise, respectively, sequences encoded by: SEQ ID NO: 49 & SEQ ID NO: 48, SEQ ID NO: 51 & SEQ ID NO: 50, SEQ ID NO: 53 & SEQ ID NO: 52, SEQ ID NO: 55 & SEQ ID NO: 54, SEQ ID NO: 57 & SEQ ID NO: 56, SEQ ID NO: 59 & SEQ ID NO: 58, SEQ ID NO: 61 & SEQ ID NO: 60, or SEQ ID NO: 63 & SEQ ID NO:
62.
13. The method of claim 5 or claim 6, wherein the 3’ end of the first tRNA chain is connected to a first stem region, wherein the first stem region comprises 8-14, 8-12, or 10-12 nucleotides, and the 5’ end of the second tRNA chain is connected to a second stem region, wherein the second stem region comprises 8-14, 8-12, or 10-12 nucleotides and is complementary to the first stem region.
14. The method of claim 13, wherein the first stem region and the second stem region each consist of a region that is 10 nucleotides in length.
15. The method of claim 13, wherein: a) the first stem region is encoded by the sequence SEQ ID NO: 3 and the second stem region is encoded by SEQ ID NO: 4; or b) the first stem region is encoded by the sequence SEQ ID NO: 5 and the second stem region is encoded by SEQ ID NO: 6; c) the first stem region is encoded by the sequence SEQ ID NO: 7 and the second stem region is encoded by SEQ ID NO: 8; or d) the first stem region is encoded by the sequence SEQ ID NO: 64 and the second stem region is encoded by SEQ ID NO:
65.
16. The method of any preceding claim, wherein the tRNA is split into a first tRNA chain and a second tRNA chain, the first tRNA chain comprises a tRNA portion from the 5’ side of the split, and the second tRNA chain comprises a tRNA portion from the 3’ side of the split.
17. The method of claim 16, wherein the 3’ end of the first tRNA chain is connected to a first stem region and the 5’ end of the second tRNA chain is connected to a second stem region.
18. The method of any one of claims 5, 6, 11, 12, or 13-17, wherein the second tRNA chain is a part of the fusion RNA molecule also comprising the sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest.
19. The method of claim 18, wherein the connection between the second tRNA chain and the sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest is via the second stem region.
20. The method of any preceding claim, wherein the tRNA is split into a first tRNA chain and a second tRNA chain, and the fusion RNA molecule is expressed by a nucleic acid construct that comprises from 5’ to 3’: the sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest, a sequence encoding the second tRNA chain, and a sequence encoding the first tRNA chain.
21. The method of claim 20, wherein the tRNA is split into a first tRNA chain and a second tRNA chain, the first tRNA chain comprises a first stem region, and the second tRNA chain comprises a second stem region, the first stem region and the second stem region are complementary, wherein the nucleic acid construct comprises, from 5’ to 3’, the sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest, optionally a linker, a sequence encoding the second stem region, the sequence encoding the second tRNA chain, a loop, the sequence encoding the first tRNA chain, and a sequence encoding the first stem region.
22. The method of claim 21, wherein the loop is cleaved, removed, or spliced during RNA processing.
23. The method of claim 21 or claim 22, wherein expression of the nucleic acid construct results in: the fusion RNA molecule comprising, from 5’ to 3’, the sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest, optionally the linker, the second stem region, and the second tRNA chain; and a separate RNA molecule comprising, from 5’ to 3’, the first tRNA chain and the first stem region.
24. The method of any preceding claim, wherein the conditions capable of labelling tRNAs that have been acylated are conditions that comprise the following steps: a) exposure to conditions that block the 3’ end of free tRNAs but do not block the 3’ end of acylated tRNAs, b) exposure to conditions suitable to remove substrates that have acylated tRNAs; and c) exposure to conditions that attach a label to 3’ ends of tRNAs that are not blocked.
25. The method of claim 24, wherein step c) comprises conditions that lead to the addition of nucleotides to the 3’ ends of tRNAs that are not blocked, wherein at least one nucleotide comprises a label.
26. The method of any preceding claim, wherein the method comprises a step of capturing the fusion RNA molecule if it is associated with a labelled tRNA.
27. The method of any preceding claim, wherein the method comprises a step of sequencing a barcode associated with any labelled or captured fusion RNA molecule.
28. The method of any preceding claim, wherein the method comprises a step of obtaining sequence information for at least one sequence fused to any labelled or captured tRNA portion.
29. The method of any preceding claim, wherein the method comprises a step of obtaining sequence information for the sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest.
30. The method of any preceding claim, wherein the polypeptide-of-interest is an acyl-tRNA synthetase.
31. The method of any one of claims 1 to 29, wherein step i) comprises incubating the tRNA and the substrate under conditions conducive to acylation of the tRNA, and incubating the acylated tRNA with the polypeptide-of-interest or nucleic-acid-of-interest.
32. The method of claim 31, wherein, prior to step ii), the method comprises exposure to conditions suitable for conditionally deacylating acylated tRNAs.
33. The method of any one of claims 1-29, 31, and 32, wherein the polypeptide-of-interest or nucleic-acid-of- interest is capable of chemically altering the substrate or wherein the method comprises determining whether the polypeptide-of-interest or nucleic-acid-of-interest is capable of chemically altering the substrate.
34. A split tRNA comprising a first chain and a second chain, wherein:the first chain comprises a first tRNA portion and comprises a first stem region, and the second chain comprises a second tRNA portion and comprises a second stem region.
35. A split tRNA comprising a first chain and a second chain, wherein: the first chain comprises a first tRNA portion and comprises a first stem region, and the second chain comprises a second tRNA portion and comprises a second stem region; the first tRNA portion corresponds to the 5’ portion of a parental tRNA split at the anticodon and the second tRNA portion corresponds to the 3’ portion of the parental tRNA split at the anticodon; the first stem region is located at the 3’ end of the first tRNA portion and the second stem region is located at the 5’ end of the second tRNA portion; and the first stem region and the second stem region are complementary.
36. The method of claim 35, wherein a) the first tRNA portion comprises a sequence encoded by SEQ ID NO: 2 and the second tRNA portion comprises a sequence encoded by SEQ ID NO: 1; b) the first tRNA portion comprises a sequence encoded by SEQ ID NO: 24 and the second tRNA portion comprises a sequence encoded by SEQ ID NO: 23; c) the first tRNA portion comprises a sequence encoded by SEQ ID NO: 26 and the second tRNA portion comprises a sequence encoded by SEQ ID NO: 25; d) the first tRNA portion comprises a sequence encoded by SEQ ID NO: 28 and the second tRNA portion comprises a sequence encoded by SEQ ID NO: 27; or e) the first tRNA portion comprises a sequence encoded by SEQ ID NO: 47 and the second tRNA portion comprises a sequence encoded by SEQ ID NO:
46.
37. The split tRNA of any one of claims 34 to 36, wherein the first and the second stem region are 8-14, 8-12, or 10-12 nucleotides in length.
38. The split tRNA of any one of claims 34 to 37, wherein: a) the first stem region is encoded by the sequence SEQ ID NO: 3 and the second stem region is encoded by SEQ ID NO: 4; or b) the first stem region is encoded by the sequence SEQ ID NO: 5 and the second stem region is encoded by SEQ ID NO: 6; c) the first stem region is encoded by the sequence SEQ ID NO: 7 and the second stem region is encoded by SEQ ID NO: 8; or d) the first stem region is encoded by the sequence SEQ ID NO: 64 and the second stem region is encoded by SEQ ID NO:
65.
39. The split tRNA of any one of claims 34 to 38, wherein the first and second tRNA portion are from a tRNAPyl, tRNALeu, tRNAAla, or tRNASer.
40. The split tRNA of claim 34, wherein the first tRNA portion corresponds to the 5’ portion of a parental tRNA split at a split site and the second tRNA portion corresponds to the 3’ portion of the parental tRNA split at a split site; and the split site is in the D loop, in the anticodon loop and to the 5’ side of the anticodon, in the variable loop, or in the T loop.
41. The split tRNA of claim 40, wherein the split site is between residues D15 & D16, D15 & D17, D15 & D18, D16 & D17, D16 & D18, D17 & D18, A31 & A32, A31 & A33, A32 & A33, V45 & V46, V45 & V47, V45 & V48, V46 & V47, V4640 & V48, V47 & V48, T56 & T57, T56 & T58, or T57 & T58.
42. The split tRNA of claim 41, wherein the split site is between residues D15 & D17, D15 & D18, D16 & D17, A31 & A33, A32 & A33, V45 & V48, V46 & V48, V47 & V48, T56 & T57, or T56 & T58.
43. The split tRNA of any one of claims 40 to 42, wherein the first and second tRNA portion are from a tRNATrpor tRNAPro.
44. The split tRNA of any one of claims 34 to 43, wherein the second chain comprises a sequence, located 5’ to the second stem region and the second tRNA portion, encoding a polypeptide-of-interest or nucleic-acid-of- interest.
45. An RNA molecule comprising a sequence encoding a polypeptide-of-interest or nucleic-acid-of-interest and comprising a portion of a tRNA.
46. The RNA molecule of claim 45, wherein the portion of a tRNA corresponds to the 3’ portion of a parental tRNA split: at the anticodon; in the D loop, in the anticodon loop and to the 5’ side of the anticodon, in the variable loop, or in the T loop; between residues D15 & D16, D15 & D17, D15 & D18, D16 & D17, D16 & D18, D17 & D18, A31 & A32, A31 & A33, A32 & A33, V45 & V46, V45 & V47, V45 & V48, V46 & V47, V4640 & V48, V47 & V48, T56 & T57, T56 & T58, or T57 & T58; or between residues D15 & D17, D15 & D18, D16 & D17, A31 & A33, A32 & A33, V45 & V48, V46 & V48, V47 & V48, T56 & T57, or T56 & T58.
47. The RNA molecule of claim 45 or claim 46, wherein the portion of a tRNA is from a tRNAPyl, tRNALeu, tRNAAla, tRNASer, tRNATrp,or tRNAPro.
48. The RNA molecule of any one of claims 45 to 47, wherein the portion of a tRNA comprises a sequence encoded by any one of SEQ ID NOs: 1, 23, 25, 27, 46, 48, 50, 52, 54, 56, 58, 60, or 62.
49. The RNA molecule of any one of claims 45 to 48, wherein the RNA molecule comprises a stem region located at the 5’ end of the portion of a tRNA, and wherein the sequence encoding the polypeptide-of-interest or nucleic-acid-of-interest is located 5’ to the stem region.
50. The RNA molecule of claim 49, wherein the stem region is: 8-14, 8-12, or 10-12 nucleotides in length; or 8-25, 10-23, 12-22, 13-21, 14-20, 15-19, or 16-18 nucleotides in length.
51. The RNA molecule of claim 49 or claim 50, wherein the stem region is encoded by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 64, or SEQ ID NO:
66.
52. A method of determining the acylation status of a tRNA or efficiency of acylation of a tRNA, the method comprising: i) incubating the tRNA and a substrate with which the tRNA could be acylated, under conditions conducive to acylation of the tRNA; ii) exposing the tRNA to conditions capable of blocking the 3’ end of free tRNAs; and iii) exposing the tRNA to conditions that lead to the addition of nucleotides to the 3’ end of tRNAs that are not blocked, wherein at least one nucleotide comprises a label.
53. The method according to claim 52, wherein, prior to step iii), the method comprises exposing the tRNA to conditions capable of removing a substrate that has acylated a tRNA.
54. The method according to claim 52 or claim 53, wherein the label is, or is capable of being linked to, an optically detectable moiety or a physically detectable moiety.
55. The method according to claim 54, wherein the optically detectable moiety is a fluorescent moiety.
56. The method according to claim 54, wherein the physically detectable moiety is a magnetic moiety or is a ligand or receptor of a ligand-receptor pair.
57. The method according to any one of claims 52 to 56, wherein the method comprises capturing the tRNA if it has been labelled.
58. The method according to any one of claims 52 to 57, wherein the tRNA is a split tRNA according to any one of claims 34 to 44 or comprises an RNA molecule of any one of claims 45 to 51.
59. The method according to any one of claims 52 to 58, wherein the method is performed in a cell and the tRNA does not comprise an anticodon.
60. A tRNA comprising additional nucleotides at the 3’ end, wherein at least one nucleotide comprises a label.
61. The tRNA of claim 60, wherein the label is, or is capable of being linked to, an optically detectable moiety or a physically detectable label.
62. The tRNA of claim 61, wherein the optically detectable moiety is a fluorescent moiety.
63. The tRNA of claim 61, wherein the physically detectable moiety is a magnetic moiety or a ligand or receptor of a ligand-receptor pair.
64. The tRNA according to any of one claims 60 to 63, wherein the tRNA is a split tRNA according to any one of claims 34 to 44 or comprises an RNA molecule of any one of claims 45 to 51.
65. A nucleic acid encoding the split tRNA of any one of claims 34 to 44 or the RNA molecule of any one of claims 45 to 51.
66. A nucleic acid comprising, from 5’ to 3’: a second stem-region-encoding sequence, a second tRNA-portion-encoding sequence, a sequence encoding a loop, a first tRNA-portion-encoding sequence, and a first stem-region-encoding sequence, optionally wherein the first stem-region-encoding sequence and second stem-region-encoding sequence encode complementary stem region sequences.
67. The nucleic acid of claim 66, wherein the nucleic acid comprises, from 5’ to 3’: a sequence encoding a polypeptide-of-interest or nucleic-acid-of-interest, optionally a sequence encoding a linker, the second stem-region-encoding sequence, the second tRNA-portion-encoding sequence,the sequence encoding a loop, the first tRNA-portion-encoding sequence, and the first stem-region-encoding sequence.
68. The nucleic acid of claim 66 or claim 67, wherein: the second tRNA-portion-encoding sequence encodes a second tRNA portion corresponding to the 3’ portion of a parental tRNA split at the anticodon and the first tRNA-portion-encoding sequence encodes a first tRNA portion corresponding to the 5’ portion of the parental tRNA split at the anticodon; or the second tRNA-portion-encoding sequence encodes a second tRNA portion corresponding to the 3’ portion of a parental tRNA split at a split site and the first tRNA-portion-encoding sequence encodes a first tRNA portion corresponding to the 5’ portion of the parental tRNA split at a split site, wherein the split site is: in the D loop, in the anticodon loop and to the 5’ side of the anticodon, in the variable loop, or in the T loop; between residues D15 & D16, D15 & D17, D15 & D18, D16 & D17, D16 & D18, D17 & D18, A31 & A32, A31 & A33, A32 & A33, V45 & V46, V45 & V47, V45 & V48, V46 & V47, V46 40 & V48, V47 & V48, T56 & T57, T56 & T58, or T57 & T58; or between residues D15 & D17, D15 & D18, D16 & D17, A31 & A33, A32 & A33, V45 & V48, V46 & V48, V47 & V48, T56 & T57, or T56 & T58.
69. The nucleic acid of any one of claims 66 to 68, wherein the sequence encoding the loop comprises a sequence that is removed, spliced, or cleaved during RNA processing.
70. The nucleic acid of any one of claims 66 to 69, wherein the sequence encoding the loop comprises or is according to any one of SEQ ID NOs: 15 to 22 or TCGTCCT.
71. The nucleic acid of any one of claims 66 to 70, wherein the sequence encoding the loop comprises or is according to SEQ ID NO: 17 or SEQ ID NO:
19.
72. A method of making a polypeptide or nucleic acid, wherein the method comprises: i) providing a library comprising a plurality of sequences encoding polypeptides-of-interest or nucleic- acids-of-interest, wherein each sequence within the library is linked to a portion of a tRNA; ii) incubating, under conditions conducive to acylation, each polypeptide-of-interest or nucleic-acid-of- interest with a tRNA comprising the tRNA portion linked to the sequence encoding the respective polypeptide-of- interest or nucleic-acid-of-interest, and wherein the incubation includes a substrate with which the tRNAs could be acylated; iii) exposing the tRNAs to conditions capable of labelling tRNAs that have been acylated; iv) identifying whether each polypeptide-of-interest or nucleic-acid-of-interest is associated with a labelled tRNA; and v) making a polypeptide according to the sequence of an identified polypeptide-of-interest or nucleic-acid- of-interest.
73. A method of making a polypeptide, wherein the method comprises: i) providing a sequence of a polypeptide-of-interest identified by the screening method of any one of claims 1 to 33 or claims 52 to 59, and ii) producing a polypeptide according to said sequence.
74. The method of claim 72 or claim 73, wherein the polypeptide is an acyl-tRNA synthetase.