TREM composition and method of use thereof

Abstract

The present invention relates to tRNA-based effector molecules having generally non-natural modifications and related methods. This disclosure features modified tRNA-based effector molecules (TREMs, e.g., TREM or TREM fragments), as well as related compositions and uses. TREMs are complex molecules capable of mediating various cellular processes. For example, the TREMs described herein may have: (i) the ability to assist in protein synthesis, (ii) the ability to be loaded by tRNA synthetases, (iii) the ability to be bound by elongation factors, (iv) the ability to introduce amino acids into peptide chains, (v) the ability to assist in protein elongation, or (vi) the ability to assist in initiating protein synthesis, for example, in cells.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to U.S. Provisional Application No. 63 / 458,891, filed on April 12, 2023; U.S. Provisional Application No. 63 / 458,894, filed on April 12, 2023; and U.S. Provisional Application No. 63 / 458,911, filed on April 12, 2023. The entire contents of each of the above applications are incorporated herein by reference. [Background technology]

[0002] Transfer RNA (tRNA) is a complex, naturally occurring RNA molecule that has several functions, including protein initiation and elongation. [Overview of the project] [Means for solving the problem]

[0003] This disclosure features modified tRNA effector molecules (TREMs, e.g., TREM or TREM fragments), as well as related compositions and uses thereof. TREMs are complex molecules capable of mediating various cellular processes. For example, the TREMs described herein may have: (i) the ability to assist in protein synthesis, (ii) the ability to be loaded by tRNA synthetases, (iii) the ability to be bound by elongation factors, (iv) the ability to introduce amino acids into peptide chains, (v) the ability to assist in protein elongation, or (vi) the ability to assist in initiating protein synthesis, for example, in cells.

[0004] This specification describes TREMs and related methods for optimizing the nucleotide sequence of TREMs, for example, to improve the functional parameters of TREMs, for example, to enhance the stability of TREMs. For example, the methods provided demonstrate that the functional parameters of TREMs can be regulated by modifying the nucleotide sequence of a TREM, for example, by substituting one nucleotide with another, thereby achieving, for example, an improvement in the functional parameters of a TREM. In some embodiments, the TREMs disclosed herein include, for example, at least one chemical modification (e.g., a non-natural modification) on the constituent nucleotides (e.g., nucleic acid bases or sugars) or in the internucleotide region, for example, within the TREM skeleton. In other embodiments, the TREMs disclosed herein do not include, for example, chemical modifications (e.g., non-natural modifications) on the constituent nucleotides (e.g., nucleic acid bases or sugars) or in the internucleotide region (e.g., the TREM skeleton). This disclosure provides methods for regulating the functional parameters of TREMs by optimizing the nucleotide sequence and demonstrates that nucleotide sequence substitutions (e.g., or further including non-natural modifications) can regulate the functional parameters of TREMs.

[0005] In one embodiment, a TREM is provided comprising the sequence of formula (A): [L1]x-[ASt domain 1]-[L2]x-[DH domain]-[L3]x-[ACH domain]-[VL domain]-[TH domain]-[L4]x-[ASt domain 2]-[L5]x, where [L1] and [VL domain] are independently arbitrary; x is independently 0 or 1 for each occurrence; and one of [L1], [ASt domain 1], [L2]-[DH domain], [L3], [ACH domain], [VL domain], [TH domain], [L4], and [ASt domain 2] comprises a nucleotide substitution relative to a reference sequence (e.g., a second TREM), where the TREM comprises nucleotide substitutions (e.g., nucleotide mutations) within the TREM that can modulate the functional parameters of the TREM. In one embodiment, the TREM described herein includes a plurality of nucleotide substitutions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80) relative to the reference sequence.

[0006] In another embodiment, a TREM is provided comprising the sequence of formula (A): [L1]x-[ASt domain 1]-[L2]x-[DH domain]-[L3]x-[ACH domain]-[VL domain]-[TH domain]-[L4]x-[ASt domain 2]-[L5]x, where [L1] and [VL domain] are independently arbitrary; x is independently 0 or 1 for each occurrence; and one of [L1], [ASt domain 1], [L2]-[DH domain], [L3], [ACH domain], [VL domain], [TH domain], [L4], and [ASt domain 2] contains a nucleotide with an unnatural modification. In one embodiment, the TREM described herein comprises at least X consecutive nucleotides (where X is greater than 3, 4, 5, 6, 7, 8, 9, or 10) that do not contain unnatural modifications; comprises at least three, but not all, nucleotides of one type (e.g., A, T, C, G, or U) that contain the same unnatural modification; comprises at least X nucleotides of one type (e.g., A, T, C, G, or U) that do not contain unnatural modifications, where X = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80; and one type (e.g., A, T, C, G, or U) that contains unnatural modifications. For example, containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80 or fewer nucleotides of the nucleotides A, T, C, G, or U; and / or not It contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80 or fewer nucleotides of one type (e.g., A, T, C, G, or U) that does not contain natural modifications.

[0007] In certain embodiments, a TREM domain comprising a non-natural modification has a function, e.g., a domain function as described herein.

[0008] In certain aspects, herein, the sequence of Formula B: [L1]-[ASt domain 1] x -[L2] y -[DH domain] y -[L3] y -[ACH domain] x -[VL domain] y -[TH domain] y -[L4] y -[ASt domain 2] x is provided, a TREM core fragment comprising where x = 1 and y = 0 or 1; and one of [ASt domain 1], [ACH domain], and [ASt domain 2] comprises nucleotides having a non-natural modification.

[0009]

[0010] In certain embodiments, TREM has the ability to assist protein synthesis. In certain embodiments, TREM has the ability to be altered by synthetase. In certain embodiments, TREM has the ability to be bound by an elongation factor. In certain embodiments, TREM has the ability to introduce an amino acid into a peptide chain. In certain embodiments, TREM has the ability to assist elongation. In certain embodiments, TREM has the ability to assist initiation.

[0011] In certain embodiments, [ACH domain] comprising a non-natural modification has the ability to mediate base pairing with a codon.

[0012] ​In one embodiment, y=1 for one, two, three, four, five, six, all, or any combination of [L1], [L2], [DH domain], [L3], [VL domain], [TH domain], and [L4].

[0013] In one embodiment, y=0 for one, two, three, four, five, six, all, or any combination of [L1], [L2], [DH domain], [L3], [VL domain], [TH domain], and [L4].

[0014] In one embodiment, y=1 for linker[L1], and L1 contains a nucleotide having unnatural modifications.

[0015] In one embodiment, y=1 for linker [L2], and L2 contains a nucleotide having unnatural modifications.

[0016] In one embodiment, y=1 for the [DH domain (DHD)], and the DHD contains a nucleotide with unnatural modifications. In one embodiment, the DHD containing unnatural modifications has the ability to mediate recognition by aminoacyl-tRNA synthetase.

[0017] In one embodiment, y=1 for linker[L3], and L3 contains a nucleotide having unnatural modifications.

[0018] In one embodiment, y=1 for the [VL domain (VLD)], and the VLD contains a nucleotide having a non-natural modification.

[0019] In one embodiment, y=1 for the [TH domain (THD)], and the THD contains nucleotides with unnatural modifications. In one embodiment, the THD containing unnatural modifications has the ability to mediate ribosome recognition.

[0020] In one embodiment, y=1 for linker [L4], and L4 contains a nucleotide having unnatural modifications.

[0021] In another embodiment, the present disclosure relates to a TREM fragment comprising a portion of TREM, wherein TREM is an array of formula A: The TREM fragment includes [L1]-[ASt domain 1]-[L2]-[DH domain]-[L3]-[ACH domain]-[VL domain]-[TH domain]-[L4]-[ASt domain 2], and provides a TREM fragment that includes unnatural modifications.

[0022] In one embodiment, the TREM fragment includes one, two, three, or all or any combination of the following: (a) a TREM half (e.g., derived from a cleavage in the anticodon sequence, e.g., a 5' half or a 3' half); (b) a 5' fragment (e.g., derived from a cleavage in the DH domain or ACH domain, e.g., a fragment containing the 5' end); (c) a 3' fragment (e.g., derived from a cleavage in the TH domain, e.g., a fragment containing the 3' end); or (d) an internal fragment (e.g., derived from a cleavage in any one of the ACH domain, DH domain or TH domain).

[0023] In one embodiment, the TREM fragment comprises (a) a TREM half containing a nucleotide having an unnatural modification.

[0024] In one embodiment, the TREM fragment includes (b) a 5' fragment containing a nucleotide having an unnatural modification.

[0025] In one embodiment, the TREM fragment includes a 3' fragment containing a nucleotide having an unnatural modification (c).

[0026] In one embodiment, the TREM fragment includes an internal fragment comprising (d) a nucleotide having an unnatural modification.

[0027] In some embodiments of the TREM, TREM core fragment, or TREM fragment disclosed herein, the TREM domain comprises a plurality of nucleotides, each having an unnatural modification. In some embodiments, the unnatural modification includes nucleic acid base modification, sugar (e.g., ribose) modification, or skeletal modification. In some embodiments, the unnatural modification is sugar (e.g., ribose) modification. In some embodiments, the unnatural modification is 2'-ribose modification, e.g., 2'-OMe, 2'-halo (e.g., 2'-F), 2'-MOE, or 2'-deoxy modification. In some embodiments, the unnatural modification is skeletal modification, e.g., phosphorothioate modification.

[0028] In some embodiments of the TREM, TREM core fragment, or TREM fragment disclosed herein, the TREM sequence includes a CCA sequence at a terminal, e.g., the 3' terminal. In some embodiments, the TREM sequence does not include a CCA sequence at a terminal, e.g., the 3' terminal.

[0029] In some embodiments of the TREM, TREM core fragment, or TREM fragment disclosed herein, the non-natural modification is a modification of the nucleotide base or backbone, for example, a modification selected from any one of Tables 5, 6, 7, 8, or 9.

[0030] In some embodiments of the TREM, TREM core fragment, or TREM fragment disclosed herein, the non-natural modification is a base modification selected from the modifications listed in Table 5.

[0031] In some embodiments of the TREM, TREM core fragment, or TREM fragment disclosed herein, the TREM, TREM core fragment, or TREM fragment is coded by one of the sequences listed in Table 1, for example, sequence numbers 1 to 451.

[0032] In some embodiments of the TREM, TREM core fragment, or TREM fragment disclosed herein, the TREM, TREM core fragment, or TREM fragment is coded by a consensus sequence selected from any one of sequence numbers 562 to 621.

[0033] In some embodiments of any TREM, TREM core fragment, or TREM fragment disclosed herein, the TREM, TREM core fragment, or TREM fragment is encoded by one of the sequences shown in Figure 3, for example, SEQ ID NOs. 625-1151. In some embodiments, the TREM, TREM core fragment, or TREM fragment includes a TREM having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with one of the TREMs shown in Figure 3, for example, SEQ ID NOs. 625-1151. In some embodiments, the TREM, TREM core fragment, or TREM fragment includes a sequence that differs by only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from one of the TREMs shown in Figure 3, for example, SEQ ID NOs. 625-1151. In one embodiment, the TREM, TREM core fragment, or TREM fragment includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional non-natural modifications (e.g., 2'-ribose modifications or internucleotide modifications, e.g., 2'OMe, 2'-halo, 2'-MOE, 2'-deoxy, or phosphorothioate modifications) compared to any one of the TREM, TREM core fragment, or TREM fragments shown in Figure 3, e.g., SEQ ID NOs: 625-1151.

[0034] In any embodiment of a TREM, TREM core fragment, or TREM fragment disclosed herein, the TREM, TREM core fragment, or TREM fragment is one of the TREMs shown in Figure 3, for example, SEQ ID NOs. 625 to 1151. In some embodiments, the TREM, TREM core fragment, or TREM fragment includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional non-natural modifications (e.g., 2'-ribose modifications or internucleotide modifications, for example, 2'OMe, 2'-halo, 2'-MOE, 2'-deoxy, or phosphorothioate modifications) compared to one of the TREMs shown in Figure 3, for example, SEQ ID NOs. 625 to 1151.

[0035] In another embodiment, the present disclosure provides a pharmaceutical composition comprising a TREM, a TREM core fragment, or a TREM fragment as disclosed herein.

[0036] In another embodiment, TREM or related compositions may be used, in particular, to modulate functional parameters (e.g., expression parameters and / or signaling parameters) of RNA corresponding to a nucleic acid sequence containing an endogenous open reading frame (ORF) having an immature stop codon (PTC), or of a polypeptide encoded by such a nucleic acid sequence.

[0037] In another embodiment, a method is provided herein for modulating a functional parameter of mRNA corresponding to an endogenous open reading frame (ORF) in a subject, or a polypeptide encoded by such an endogenous open reading frame (ORF), comprising contacting the subject with a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment disclosed herein, in an amount and / or for a sufficient amount of time to modulate the functional parameter of the mRNA or polypeptide, thereby modulating the functional parameter in the subject, wherein the TREM, TREM core fragment, or TREM fragment has an anticodon that pairs with a codon having a first sequence. In one embodiment, the functional parameter includes, for example, a signaling parameter and / or an expression parameter as described herein.

[0038] In another embodiment, a method for regulating the expression of a protein in a cell is provided, wherein the protein is encoded by a nucleic acid comprising an endogenous open reading frame (ORF) containing an immature stop codon (PTC), and the method comprises contacting a cell with a TREM, TREM core fragment, or TREM composition comprising a TREM fragment disclosed herein in an amount and / or for a sufficient amount of time to regulate the expression of the encoded protein, thereby regulating the expression of the protein in the cell, wherein the TREM, TREM core fragment, or TREM fragment has an anticodon that pairs with a codon having a first sequence. In some embodiments, the PTC comprises UAA, UGA, or UAG.

[0039] In another embodiment, a method for regulating the expression of a protein in a subject is provided herein, wherein the protein is encoded by a nucleic acid comprising an endogenous open reading frame (ORF) comprising an immature stop codon (PTC), and the method comprises contacting the subject with a TREM composition comprising: (i) an anticodon that pairs with the PTC, (ii) an aminoacyl-tRNA synthetase specific to Trp, Tyr, Cys, Glu, Lys, Gln, Ser, Leu, Arg, or Gly, and (iii) a sequence of formula A, or (iv) an unnatural modification, in an amount and / or for a sufficient amount of time to regulate the expression of the encoded protein. In some embodiments, the PTC comprises UAA, UGA, or UAG. In some embodiments, the TREM composition comprises (i). In some embodiments, the TREM composition comprises (ii). In some embodiments, the TREM composition comprises (iii). In some embodiments, the TREM composition comprises (iv). In some embodiments, the TREM composition comprises two of (i) to (iv). In one embodiment, the TREM composition comprises three of (i) to (iv). In another embodiment, the TREM composition comprises each of (i) to (iv).

[0040] In another embodiment, the Disclosure provides a method for treating a subject having an endogenous open reading frame (ORF) containing an immature stop codon (PTC), the method comprising: providing a TREM, a TREM core fragment, or a TREM composition comprising a TREM fragment (where the TREM comprises an anticodon that pairs with a PTC in the ORF); and contacting the subject with the TREM, a TREM core fragment, or a composition comprising a TREM fragment in an amount sufficient to treat the subject and / or for a sufficient amount of time, thereby treating the subject. In some embodiments, the PTC comprises a UAA, a UGA, or a UAG.

[0041] In another embodiment, the Disclosure provides a method for treating a subject having a disease or disorder associated with an immature stop codon (PTC), the method comprising: providing a TREM, a TREM core fragment, or a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment as disclosed herein; and treating the subject by contacting the subject with a TREM, a TREM core fragment, or a composition comprising a TREM, in an amount sufficient to treat the subject and / or for a sufficient amount of time. In some embodiments, the PTC includes UAA, UGA, or UAG. In some embodiments, the disease or disorder associated with a PTC is a disease or disorder as described herein, for example, cancer or a monogenetic disorder.

[0042] In some embodiment of any of the methods disclosed herein, the codon having the first sequence includes a mutation (e.g., a point mutation, e.g., a nonsense mutation) which results in an immature stop codon (PTC) selected from UAA, UGA, or UAG. In some embodiments, the codon or PTC having the first sequence includes a UAA mutation. In some embodiments, the codon or PTC having the first sequence includes a UGA mutation. In some embodiments, the codon or PTC having the first sequence includes a UAG mutation.

[0043] In another aspect, the present disclosure provides a method for producing a TREM, a TREM core fragment, or a TREM fragment as disclosed herein, comprising ligating a first nucleotide to a second nucleotide to form a TREM.

[0044] In one embodiment, the TREM, TREM core fragment, or TREM fragment is non-natural (e.g., synthetic).

[0045] In one embodiment, TREM, TREM core fragment, or TREM fragment is prepared by cell-free solid-phase synthesis.

[0046] In another aspect, the Disclosure provides a method for regulating the intracellular tRNA pool, comprising providing a TREM, a TREM core fragment, or a TREM fragment as disclosed herein, and a method for contacting a cell with a TREM, a TREM core fragment, or a TREM fragment, thereby regulating the intracellular tRNA pool.

[0047] In some embodiments, the Disclosure provides a method for contacting cells, tissues, or subjects with a TREM, a TREM core fragment, or a TREM fragment as disclosed herein, comprising contacting cells, tissues, or subjects with a TREM, a TREM core fragment, or a TREM fragment, thereby bringing cells, tissues, or subjects into contact with a TREM, a TREM core fragment, or a TREM fragment.

[0048] In another aspect, the Disclosure provides a method for delivering a TREM, a TREM core fragment, or a TREM fragment to a cell, tissue, or subject, comprising providing the cell, tissue, or subject, and bringing the cell, tissue, or subject into contact with the TREM, a TREM core fragment, or a TREM fragment disclosed herein.

[0049] In one embodiment, the present disclosure is a method for regulating an intracellular tRNA pool comprising an endogenous open reading frame (ORF) having a codon having a first sequence, By choice, to acquire knowledge about the abundance of one or both of (i) and (ii), for example, to acquire knowledge about the relative amounts of (i) and (ii) within a cell, where (i) is a tRNA portion having an anticodon that pairs with a codon of an ORF having a first sequence (first tRNA portion), and (ii) is an isoacceptor tRNA portion having an anticodon that pairs with a codon other than the codon having the first sequence within the cell (second tRNA portion); Contacting a cell with a TREM, TREM core fragment, or TREM fragment disclosed herein for a sufficient amount and / or for a sufficient amount of time to regulate the relative amounts of a first tRNA portion and a second tRNA portion within the cell, wherein the TREM, TREM core fragment, or TREM fragment has a codon having a first sequence; or an anticodon that pairs with a codon other than the codon having a first sequence; This allows for the regulation of the intracellular tRNA pool. This provides a method that includes [something].

[0050] In another aspect, the present disclosure is a method for modulating the tRNA pool in an object having an ORF containing a codon having a first sequence, By choice, to acquire knowledge about the abundance of one or both of (i) and (ii), for example, to acquire knowledge about the relative amounts of (i) and (ii) in the subject, where (i) is a tRNA portion having an anticodon that pairs with a codon of an ORF having a first sequence (first tRNA portion), and (ii) is an isoacceptor tRNA portion having an anticodon that pairs with a codon other than the codon having the first sequence in the subject (second tRNA portion); Contacting a subject with a TREM, TREM core fragment, or TREM fragment disclosed herein for a sufficient amount and / or for a sufficient amount of time to adjust the relative amounts of the first tRNA portion and the second tRNA portion in the subject, wherein the TREM, TREM core fragment, or TREM fragment has a codon having the first sequence; or an anticodon that pairs with a codon other than the codon having the first sequence; This allows for the regulation of the tRNA pool in the target. This provides a method that includes [something].

[0051] In some embodiments, the Disclosure provides a method for regulating a tRNA pool, for example, a tRNA pool in an intracellular cell having an endogenous ORF or in a subject, comprising a composition comprising a TREM, a TREM core fragment, or a TREM fragment as disclosed herein, wherein the TREM, the TREM core fragment, or the TREM fragment comprises nucleotide sequence modifications and / or unnatural modifications; and a method comprising contacting the subject with the composition in an amount and / or for a sufficient amount of time to regulate the tRNA pool in the subject, thereby regulating the tRNA pool in the subject.

[0052] In one embodiment, the present disclosure is a method for regulating the expression of a protein in a cell, wherein the protein is encoded by a nucleic acid comprising an ORF, the ORF comprising a mutated codon, and the method is Cells are brought into contact with a TREM, TREM core fragment, or composition comprising a TREM fragment as disclosed herein, in an amount and / or for a sufficient amount of time to modulate the expression of the encoded protein. Here, TREM, TREM core fragment, or TREM fragment has an anticodon that pairs with a mutated codon, This provides a method that includes regulating the expression of proteins within cells.

[0053] In another aspect, the present disclosure relates to a method for regulating the expression of a protein in a subject, wherein the protein is encoded by a nucleic acid comprising an endogenous ORF, the ORF comprising a mutated codon, and the method is The subject is brought into contact with a TREM, TREM core fragment, or composition comprising a TREM fragment as disclosed herein, in an amount and / or for a sufficient amount of time to modulate the expression of the encoded protein. Here, TREM, TREM core fragment, or TREM fragment has an anticodon that pairs with a mutated codon, This provides a method that includes regulating the expression of proteins within cells.

[0054] In some embodiment of any of the methods disclosed herein, the mutation in the ORF is a nonsense mutation resulting in an immature stop codon selected from, for example, UAA, UGA, or UAG. In some embodiment, the stop codon is UAA. In some embodiment, the stop codon is UGA. In some embodiment, the stop codon is UAG.

[0055] In one embodiment of any of the methods disclosed herein, the TREM includes an anticodon that pairs with a stop codon.

[0056] The TREMs described herein include TREM, TREM core fragments, and TREM fragments. TREM, TREM core fragments, or TREM fragments may be modified with unnatural modifications, for example, to increase the level and / or activity (e.g., stability) of the TREM. For example, pharmaceutical TREM compositions containing TREM having unnatural modifications can be administered to cells, tissues, or subjects, for example, in vitro or in vivo, to modulate their functions. This specification discloses TREM having unnatural modifications, TREM core fragments, or TREM fragments, TREM compositions, preparations, methods for preparing TREM compositions and preparations, and methods for using them.

[0057] In one embodiment, the TREM, TREM core fragment, and TREM fragment include unnatural modifications that improve the stability of the TREM, TREM core fragment, or TREM fragment, or enhance its activity.

[0058] Any additional features of the aforementioned TREM, TREM core fragments, TREM fragments, TREM compositions, preparations, methods for producing TREM compositions and preparations, and methods for using TREM compositions and preparations include one or more features described in the listed embodiments, drawings, descriptions, examples, or claims.

[0059] Those skilled in the art will be able to recognize or confirm, by conventional experimentation alone, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be included in the embodiments, drawings, description, examples, or claims listed below. [Brief explanation of the drawing]

[0060] [Figure 1] Figure 1 is a graph showing the read-through activity of TREM for immature stop codons (PTCs) incorporated into the NanoLuc reporter protein to produce functional NanoLuc protein in cell lines that stably express the NanoLuc reporter protein after transduction with a TREM expression construct. Figure 2 is a graph comparing the log2 PTC read-through activity of TREM composed of nucleotide sequence scaffolds listed in Table 7, modified with each of the chemical modification patterns listed in Table 6. [Figure 2] Figure 1 is a graph showing the read-through activity of TREM for immature stop codons (PTCs) incorporated into the NanoLuc reporter protein to produce functional NanoLuc protein in cell lines that stably express the NanoLuc reporter protein after transduction with a TREM expression construct. Figure 2 is a graph comparing the log2 PTC read-through activity of TREM composed of nucleotide sequence scaffolds listed in Table 7, modified with each of the chemical modification patterns listed in Table 6. [Figure 3-1] This is a table listing exemplary TREMs described herein. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above. [Figure 3-6] Same as above. [Figure 3-7] Same as above. [Figure 3-8] Same as above. [Figure 3-9] Same as above. [Figure 3-10] Same as above. [Figure 3-11] Same as above. [Figure 3-12] Same as above. [Figure 3-13] Same as above. [Figure 3-14] Same as above. [Figure 3-15] Same as above. [Figure 3-16] Same as above. [Figure 3-17] Same as above. [Figure 3-18] Same as above. [Figure 3-19] Same as above. [Figure 3-20] Same as above. [Figure 3-21] Same as above. [Figure 3-22] Same as above. [Figure 3-23] Same as above. [Figure 3-24] Same as above. [Figure 3-25] Same as above. [Figure 3-26] Same as above. [Figure 3-27] Same as above. [Figure 3-28] Same as above. [Figure 3-29] Same as above. [Figure 3-30] Same as above. [Figure 3-31] Same as above. [Figure 3-32] Same as above. [Figure 3-33] Same as above. [Figure 3-34] Same as above. [Figure 3-35] Same as above. [Figure 3-36] Same as above. [Figure 3-37] Same as above. [Figure 3-38] Same as above. [Figure 3-39] Same as above. [Figure 3-40] Same as above. [Figure 3-41] Same as above. [Figure 3-42] Same as above. [Figure 3-43] Same as above. [Figure 3-44] Same as above. [Figure 3-45] Same as above. [Figure 3-46] Same as above. [Figure 3-47] Same as above. [Figure 3-48] Same as above. [Figure 3-49] Same as above. [Figure 3-50] Same as above. [Figure 3-51] Same as above. [Figure 3-52] Same as above. [Figure 3-53] Same as above. [Figure 4] This shows the frequency of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening for PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence numbers indicate a reference TREM, from which a pool of TREMs with nucleotide substitutions is generated for screening. Figure 4A is a schematic diagram of a TREM in which nucleotide substitutions typically result in regions that lead to increased PTC readthrough activity (e.g., typically positive), regions that typically lead to decreased PTC readthrough activity (e.g., typically negative), or regions that lead to increased PTC readthrough activity for a particular scaffold (e.g., scaffold-specific positive). Figure 4B shows the normalized hit abundance of nucleotide substitutions at each position in various TREM scaffolds, e.g., the frequency with which each position in the TREM contains a nucleotide substitution within a pool of hits obtained from screening for increased PTC readthrough activity. [Figure 5]This heatmap shows the frequency with which each position in a TREM contains a nucleotide substitution, e.g., a mutation site enrichment, at various TREM scaffold positions within a pool of hits obtained from a pooled screening of TREMs containing nucleotide substitutions for PTC readthrough activity, e.g., for increased PTC readthrough activity. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. The framed region indicates the position where the nucleotide substitution results in increased TREM activity. [Figure 6]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 7]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 8]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 9]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 10]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 11]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 12]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 13]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 14]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 15]This graph shows the abundance of nucleotide substitutions at each position in various TREM scaffolds within a pool of hits obtained from pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence number indicates a reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. Figure 6 shows the normalized hit abundance of nucleotide substitutions at each position in the Arg-TGA reference scaffold (sequence number 1638). Figure 7 shows the normalized hit abundance of nucleotide substitutions at each position in the Gln-TAG reference scaffold (sequence number 1835). Figure 8 shows the normalized hit abundance of nucleotide substitutions at each position in two Gln-TAG reference scaffolds (sequence numbers 1660 and 1654) obtained from the first round of pooled screening shown in Figure 7. Figure 9 shows the total normalized hit abundance of nucleotide substitutions at each position of three Glu-TAG reference scaffolds (SEQ ID NOs. 1867, 2000, and 2001). Figure 10 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 1867). Figure 11 shows the normalized hit abundance of nucleotide substitutions at each position of the Glu-TAG reference scaffold (SEQ ID NOs. 2000). Figure 12 shows the normalized hit abundance of two Leu-TAG reference scaffolds (SEQ ID NOs. 2016 and 2017). Figure 13 shows the normalized hit abundance of nucleotide substitutions at each position in two Tyr-TAG reference scaffolds (SEQ ID NOs. 2022 and 2023). Figure 14 shows the normalized hit abundance of nucleotide substitutions at each position in two Ser-TAG reference scaffolds (SEQ ID NOs. 2020 and 2021). Figure 15 shows the normalized hit abundance of nucleotide substitutions at each position in two Lys-TAG reference scaffolds (SEQ ID NOs. 2018 and 2019). [Figure 16]Figure 16 is a heatmap showing enrichment of disrupted base pairing at various positions in different TREM scaffolds obtained from a pooled screening of PTC readthrough activity of TREMs containing nucleotide substitutions. The sequence numbers indicate the reference TREM, from which a pool of TREMs containing nucleotide substitutions is generated for screening. The framed regions indicate the positions where nucleotide substitutions disrupting base pairing result in increased TREM activity. [Figure 17] This is a scatter plot showing the enrichment scores of TREM in Gln-TAG pooled screening. Candidates are shown in gray, negative control TREMs in black, and positive controls, such as human-derived tRNAs with anticodons similar to TAG PTCs, in magenta. The hit threshold is shown by vertical and horizontal dashed lines, with candidates in the upper right quadrant designated as hits. [Figure 18] Figure 17 shows a graph illustrating the luciferase reporter assay performed on the candidates selected for hit validation. The start sequence from which the mutant was generated is shown in magenta, and the activity of all tested sequences was evaluated at two concentrations. [Figure 19] Figure 19A shows the enrichment scores for TREMs in Glu-TAG pooled screening. In Figure 19A, candidate TREMs are shown in light gray, negative control TREMs in dark gray, and human-derived tRNAs with anticodons similar to those of TAG PTCs are shown in magenta as positive controls. The hit threshold is indicated by vertical and horizontal dashed lines, with candidates in the upper right quadrant designated as hits. Figure 19B shows the Glu-TAG TREM hits obtained from the screening. [Figure 20] Figure 19 shows the luciferase reporter assays performed on the candidates selected for hit validation. [Figure 21] Figure 20 shows a schematic diagram of the TREM obtained from the hit shown. Figure 21A shows the parent TREM. Figure 21B shows the Glu-TAG TREM hit. Figure 21C shows the Glu-TAG TREM hit. [Figure 22]This scatter plot shows the results of luciferase reporter assays performed on candidate TREMs selected for hit validation as both synthetic oligonucleotides and lentiviral particles. Parent sequences are shown in magenta, and hits are shown in gray. [Figure 23-1] This is a table listing exemplary TREMs described herein. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above. [Figure 23-5] Same as above. [Figure 23-6] Same as above. [Figure 23-7] Same as above. [Figure 23-8] Same as above. [Figure 23-9] Same as above. [Figure 23-10] Same as above. [Figure 23-11] Same as above. [Figure 23-12] Same as above. [Figure 23-13] Same as above. [Figure 23-14] Same as above. [Figure 23-15] Same as above. [Figure 23-16] Same as above. [Figure 23-17] Same as above. [Figure 23-18] Same as above. [Figure 23-19] Same as above. [Figure 23-20] Same as above. [Figure 23-21] Same as above. [Figure 23-22] Same as above. [Figure 23-23] Same as above. [Figure 23-24] Same as above. [Figure 23-25] Same as above. [Figure 23-26] Same as above. [Figure 23-27] Same as above. [Figure 23-28] Same as above. [Figure 23-29] Same as above. [Figure 23-30] Same as above. [Figure 23-31] Same as above. [Figure 23-32] Same as above. [Figure 23-33] Same as above. [Figure 24-1] Figure 23 is a table listing the effects of nucleotide substitutions on the PTC readthrough activity of TREM. "Sub." indicates substitution; "compens." indicates compensatory effect; and "rel." indicates relative effect. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above. [Figure 24-5] Same as above. [Figure 24-6] Same as above. [Figure 24-7] Same as above. [Figure 24-8] Same as above. [Figure 24-9] Same as above. [Figure 24-10] Same as above. [Figure 25-1] This table compares the PTC readthrough activity of hits from a pooled screening of TREMs containing nucleotide substitutions, measured by validation as synthetic oligonucleotides or by lentiviral delivery. [Figure 25-2] Same as above. [Modes for carrying out the invention]

[0061] This disclosure relates to tRNA-based effector molecules (TREMs), compositions, and related methods useful for optimizing the functional parameters of TREMs, for example, by introducing nucleotide sequence modifications, non-natural chemical modifications, or both, into TREM sequences. As disclosed herein, TREMs are complex molecules capable of mediating various cellular processes. The inventors have found that the nucleotide sequences of TREMs can be optimized to modulate their functional parameters; the methods described herein illustrate how multiple sequences (e.g., tens, hundreds, or thousands) can be screened to select TREM sequences having different functional readouts. For example, by analyzing TREM sequences from a pooled screening of TREM sequences in which each nucleotide position of the TREM is substituted with another nucleotide sequence, it is possible to select TREMs containing specific nucleotide substitutions that can enhance the TREM for a particular function, such as improving the readthrough of immature stop codons (PTCs) in transcripts.

[0062] Pharmaceutical TREM compositions, such as the TREMs described herein, can be administered to cells, tissues, or subjects to modulate specific cellular functions. Also disclosed herein are methods for modulating protein expression in a subject or cell, wherein the protein is encoded by a nucleic acid comprising a first sequence, e.g., a mutation, e.g., an endogenous open reading frame (ORF) containing an immature stop codon (PTC), and methods for treating subjects having an endogenous open reading frame (ORF) containing an immature stop codon (PTC). Furthermore, TREMs comprising unnatural modifications, methods for producing the same, and compositions thereof are disclosed.

[0063] definition "To acquire" or "to obtain," as the terms are used herein, means to obtain ownership of a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" a physical entity or value. "Directly acquiring" means to obtain a value by performing a process (e.g., performing an analytical method). "Indirectly acquiring" means to receive a value from another party or source (e.g., a third-party laboratory that directly acquired the value).

[0064] "PTC-related disease or disorder," as used herein, includes, but is not limited to, a disease or disorder in which cells express or have previously expressed a polypeptide encoded by an ORF containing a PTC. In some embodiments, the PTC-related disease is selected from proliferative disorders (e.g., cancer), genetic disorders, metabolic disorders, immunological disorders, inflammatory diseases, or neurological disorders. Exemplary PTC-related diseases or disorders are provided in any one of Tables 15, 16, and 17. In some embodiments, the PTC-related disease is cancer. In some embodiments, the PTC-related disease is a monogenic disorder.

[0065] When the term "isoreceptor" is used herein, it refers to a plurality of molecules, each containing a different native anticodon sequence, and each of the plurality of molecules mediating the incorporation of the same amino acid, which is an amino acid that naturally corresponds to a plurality of anticodons.

[0066] When the term “modification” is used herein in relation to TREM, it may refer to sequence modification or chemical modification of TREM. When used in relation to sequence modification, modification may include nucleotide addition, deletion, or substitution. When the term is used in relation to chemical modification, modification may include modification of the chemical structure of the nucleotide in question, such as covalent modification. Chemical modification may be natural or unnatural. In some embodiments, the modification is unnatural. In some embodiments, the modification is natural. In some embodiments, the modification is synthetic. In some embodiments, the modification is one of the modifications shown in Table 5.

[0067] "Naturally occurring nucleotides," as the term is used herein, refer to nucleotides that do not contain unnatural modifications. In some embodiments, they include natural modifications.

[0068] When the term is used herein in relation to nucleotides, “unnatural modification” means (a) a modification that a cell, e.g., a human cell, does not produce on endogenous tRNA; or (b) a modification that a cell, e.g., a human cell, may produce on endogenous tRNA, but such a modification is located at a position where it does not occur on endogenous tRNA, e.g., the modification is in a domain, linker or arm, or in a domain, linker or arm where such a modification is not present in the nucleotide and / or in nature. In either case, the modification is added by synthesis, e.g., in a cell-free reaction, e.g., a solid-phase or liquid-phase synthesis reaction. In some embodiments, an unnatural modification is a modification that does not exist (in identity, location or position) when the TREM sequence is expressed in mammalian cells, e.g., the HEK293 cell line. Exemplary unnatural modifications are found in Table 5.

[0069] When the term is used herein, “unnaturally modified nucleotide” refers to a nucleotide that includes unnatural modifications on or to a sugar, nucleic acid base, or phosphate moiety.

[0070] When the term “nucleotide” is used herein, it refers to an entity comprising a sugar, typically a pentameric sugar; a nucleic acid base; and a phosphate-binding group. In some embodiments, the nucleotides include naturally occurring nucleotides (e.g., those naturally present in human cells), such as adenine, thymine, guanine, cytosine, or uracil nucleotides.

[0071] "Immature stop codon" or "PTC" refers, as used herein, to a stop codon present in an open reading frame (ORF) of DNA or mRNA. In some embodiments, the PTC is located upstream of a native stop codon in the ORF. In some embodiments, for example, a PTC located upstream of a native stop codon in an ORF results in the regulation of the production parameters of the polypeptide encoded by the corresponding mRNA or ORF. In some embodiments, the PTC may differ from (or result from) a point mutation, such as a nonsense mutation, from the pre-mutation sequence. In some embodiments, the PTC may differ from (or result from) a gene alteration, such as an abnormality other than a point mutation, such as a frameshift, deletion, insertion, rearrangement, inversion, translocation, duplication, or transversion, from the pre-mutation sequence. In some embodiments, the PTC results in the production of a cleaved protein that lacks native activity or is associated with a mutant, disease, or other undesirable phenotype. In some embodiments, the ORF containing the PTC is an ORF derived from a tumor suppressor gene. In one embodiment, the mutation that causes PTC is a driver mutation, for example, a mutation that gives tumor cells a proliferative advantage.

[0072] "Functional parameters" refer to expression parameters and / or signal transduction parameters. In some embodiments, functional parameters are expression parameters. Expression parameters include expression parameters of polypeptides or proteins encoded by endogenous ORFs having a first sequence or PTC; or expression parameters of RNA encoded by endogenous ORFs having a first sequence or PTC, such as messenger RNA. In some embodiments, expression parameters are (a) Protein translation; (b) Expression level (e.g., of polypeptides, proteins, or mRNA); (c) Post-translational modification of polypeptides or proteins; (d) Folding (for example, of polypeptides or proteins, or mRNA), (e) Structure (e.g., of polypeptides or proteins, or mRNA), (f) Transfer (e.g., of polypeptides or proteins), (g) Computation (e.g., of polypeptides or proteins, or mRNA), (h) Incorporation into supramolecular structures (e.g., polypeptides or proteins, or mRNA), e.g., incorporation into membranes, proteasomes, or ribosomes, (i) Incorporation into a multimer polypeptide, e.g., homodimer or heterodimer, and / or (j) Stability It may include.

[0073] In one embodiment, the functional parameter is a signal transduction parameter. The signal transduction parameter is (1) Regulation of signaling pathways, such as cellular signaling pathways, downstream or upstream of proteins encoded by endogenous ORFs having a first sequence or PTC; (2) Regulation of cell fate; (3) Regulation of ribosome occupation; (4) Regulation of protein translation; (5) mRNA stability regulation; (6) Protein folding and structural regulation; (7) Protein transduction or compartmentalization control; and / or (8) Regulation of protein stability It may include.

[0074] When used herein, “ORF having a PTC” refers to an open reading frame (ORF) containing an immature stop codon (PTC). In some embodiments, an ORF having a PTC is associated with a disease or disorder related to the PTC, for example, one of the diseases or disorders described herein, such as those listed in Tables 15, 16, and 17. In some embodiments, an ORF having a PTC is not associated with a disease or disorder related to the PTC.

[0075] The term "stop codon" refers to a sequence of three nucleotides within messenger RNA that designates the termination of translation. For example, UAG, UAA, UGA (in RNA) and TAG, TAA, TGA (in DNA) are stop codons. Stop codons are also known as amber (UAG), ochre (UAA), and opal (UGA).

[0076] When the term is used herein, “tRNA-based effector molecule” or “TREM” refers to an RNA molecule having the structure or properties of (a) to (v) below, which may be recombinant TREM, synthetic TREM, or TREM expressed from heterologous cells. The TREMs described herein are synthetic molecules and are prepared, for example, in cell-free reactions, e.g., solid-phase or liquid-phase synthesis reactions. TREMs are chemically different from endogenous tRNA molecules prepared in cells, e.g., mammalian cells, e.g., human cells, in terms of modifications, e.g., primary sequence, type, or position. TREMs may have several of the structures and functions of (a) to (v) (e.g., 2, 3, 4, 5, 6, 7, 8, 9).

[0077] In some embodiments, TREM is non-natural, as can be assessed by its structure or the method by which it is manufactured.

[0078] In one embodiment, the TREM includes one or more of the following structures or characteristics: (a') Any selected linker region of the consensus sequence provided in the “consensus sequence” interval, e.g., linker 1 region; (a) an amino acid-binding domain, e.g., an acceptor stem domain (AStD) (an AStD, for example, when present in other wild-type tRNA, contains an RNA sequence sufficient to mediate the acceptance of an amino acid, e.g., a cognate or non-cognate amino acid, and the transfer of an amino acid (AA) in polypeptide chain initiation or elongation). Typically, an AStD contains a 3'-terminal adenosine (CCA) for acceptor stem loading, which is part of synthetase recognition. In some embodiments, the AStD has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring AStD, e.g., an AStD encoded by the nucleic acid in Table 1. In some embodiments, the TREM may contain a fragment or analogue of an AStD, e.g., an AStD encoded by the nucleic acid in Table 1, the fragment having AStD activity in one embodiment and not having AStD activity in another embodiment. (A person skilled in the art can appropriately determine the sequence corresponding to any of the domains, stems, loops, or other sequence features described herein that are derived from the nucleic acid-encoded sequences in Table 1. For example, a person skilled in the art can determine the sequence corresponding to the AStD derived from the nucleic acid-encoded tRNA sequence in Table 1.)

[0079] In one embodiment, AStD is either below the corresponding sequence of the consensus sequence provided in the “consensus sequence” interval, or differs from the consensus sequence in 1, 2, 5, or 10 or fewer locations; In one embodiment, AStD is formula I ZZZ The residues R1-R2-R3-R4-R5-R6-R7 and residue R 65 -R 66 -R 67 -R 68 -R 69 -R 70 -R 71 It contains, and ZZZ represents one of the 20 amino acids; In one embodiment, AStD is Equation II ZZZ The residues R1-R2-R3-R4-R5-R6-R7 and residue R 65 -R 66 -R67 -R 68 -R 69 -R 70 -R 71 It contains, and ZZZ represents one of the 20 amino acids; In one embodiment, AStD is Equation III ZZZ The residues R1-R2-R3-R4-R5-R6-R7 and residue R 65 -R 66 -R 67 -R 68 -R 69 -R 70 -R 71 It contains, and ZZZ represents one of the 20 amino acids; (a'-1) A linker containing residues R8-R9 of the consensus sequence provided in the "consensus sequence" interval, e.g., linker 2 region; (b) Dihydrouridine hairpin domain (DHD) (DHD contains an RNA sequence sufficient to act as a recognition site for aminoacyl-tRNA synthetase for amino acid loading of TREM, for example, when present in other wild-type tRNAs, mediating recognition by aminoacyl-tRNA synthetase). In embodiments, DHD mediates the stabilization of the tertiary structure of TREM. In some embodiments, DHD has at least 75, 80, 85, 85, 90, 95, or 100% identity with naturally occurring DHD, e.g., DHD encoded by nucleic acids in Table 1. In some embodiments, TREM may contain a fragment or analogue of DHD, e.g., DHD encoded by nucleic acids in Table 1, the fragment having DHD activity in one embodiment and not having DHD activity in another embodiment.

[0080] In one embodiment, the DHD is either below the corresponding sequence of the consensus sequence provided in the “consensus sequence” interval, or differs from the consensus sequence in 1, 2, 5, or 10 or fewer locations; In one embodiment, DHD is given by formula I ZZZ residue R 10 -R 11 -R 12 -R 13 -R 14-R 15 -R 16 -R 17 -R 18 -R 19 -R 20 -R 21 -R 22 -R 23 -R 24 -R 25 -R 26 -R 27 -R 28 comprising, wherein ZZZ represents any one of 20 amino acids; In certain embodiments, DHD is of formula II ZZZ residue R of 10 -R 11 -R 12 -R 13 -R 14 -R 15 -R 16 -R 17 -R 18 -R 19 -R 20 -R 21 -R 22 -R 23 -R 24 -R 25 -R 26 -R 27 -R 28 comprising, wherein ZZZ represents any one of 20 amino acids; In certain embodiments, DHD is of formula III ZZZ residue R of 10 -R 11 -R 12 -R 13 -R 14 -R 15 -R 16 -R 17 -R 18 -R 19 -R 20 -R 21 -R 22 -R 23 -R 24 -R 25 -R 26 -R 27 -R 28 comprising, wherein ZZZ represents any one of 20 amino acids; (b’-1) Residue R of the consensus sequence provided in the "consensus sequence" region29 A linker, for example, linker 3 region; (c) An anticodon that binds to each codon in the mRNA, for example, an anticodon hairpin domain (ACHD) (ACHD, when present in other wild-type tRNAs, for example, contains a sequence sufficient to mediate (with or without wobbling) base pairing with a codon, for example, an anticodon triplet; in certain embodiments, ACHD has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring ACHD, for example, an ACHD encoded by the nucleic acids in Table 1). In certain embodiments, TREM may include an ACHD, for example, a fragment or analog of an ACHD encoded by the nucleic acids in Table 1, where the fragment in some embodiments has ACHD activity and in other embodiments does not have ACHD activity.

[0081] In certain embodiments, ACHD is below the corresponding sequence of the consensus sequence provided in the "consensus sequence" section or differs from the consensus sequence at 1, 2, 5, or 10 or fewer positions. In certain embodiments, ACHD is of formula I ZZZ residue -R 30 -R 31 -R 32 -R 33 -R 34 -R 35 -R 36 -R 37 -R 38 -R 39 -R 40 -R 41 -R 42 -R 43 -R 44 -R 45 -R 46 and contains, where ZZZ represents any of the 20 amino acids; In certain embodiments, ACHD is of formula II ZZZ residue -R 30 -R 31 -R 32 -R 33 -R 34 -R 35 -R 36 -R37 -R 38 -R 39 -R 40 -R 41 -R 42 -R 43 -R 44 -R 45 -R 46 It contains, and ZZZ represents one of the 20 amino acids; In one embodiment, ACHD is Equation III ZZZ The residue -R 30 -R 31 -R 32 -R 33 -R 34 -R 35 -R 36 -R 37 -R 38 -R 39 -R 40 -R 41 -R 42 -R 43 -R 44 -R 45 -R 46 It contains, and ZZZ represents one of the 20 amino acids; (d) Variable loop domain (VLD) (The VLD contains an RNA sequence sufficient to act as a recognition site for aminoacyl-tRNA synthetase for aminoacyl-tRNA loading of TREM, for example, when present in other wild-type tRNAs, mediating the recognition of aminoacyl-tRNA synthetase). In embodiments, the VLD mediates the stabilization of the tertiary structure of TREM. In some embodiments, the VLD modulates, for example, the specificity of TREM with respect to its homologous amino acids, for example, increasing it, for example, the VLD modulates the homologous adapter function of TREM. In some embodiments, the VLD has at least 75, 80, 85, 85, 90, 95, or 100% identity with naturally occurring VLDs, for example, VLDs encoded by nucleic acids in Table 1. In some embodiments, TREM may include a fragment or analogue of a VLD, for example, a VLD encoded by nucleic acids in Table 1, the fragment having VLD activity in one embodiment and not having VLD activity in another embodiment.

[0082] In one embodiment, the VLD is located below the corresponding sequence of the consensus sequence provided in the “consensus sequence” interval.

[0083] In one embodiment, the VLD is a residue of the consensus sequence provided in the "consensus sequence" interval -[R 47 ] X Including x=1~271 (for example, x=1~250, x=1~225, x=1~200, x=1~175, x=1~150, x=1~125, x=1~100, x=1~75, x=1~50, x=1~40, x=1~30, x=1~29, x=1~28, x=1~27, x=1~26, x=1~25, x=1~24, x=1~23, x =1~22, x=1~21, x=1~20, x=1~19, x=1~18, x=1~17, x=1~16, x=1~15, x=1~14, x=1~13, x=1~12 , x=1~11, x=1~10, x=10~271, x=20~271, x=30~271, x=40~271, x=50~271, x=60~271, x=70~27 1, x=80~271, x=100~271, x=125~271, x=150~271, x=175~271, x=200~271, x=225~271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18 x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271); (e) Thymine hairpin domain (THD) (THD comprises an RNA sequence sufficient to mediate ribosome recognition when present in other wild-type tRNAs, for example, to act as a recognition site for ribosomes to form a TREM-ribosome complex during translation). In some embodiments, THD has at least 75, 80, 85, 85, 90, 95, or 100% identity with naturally occurring THD, for example, THD encoded by the nucleic acids in Table 1. In some embodiments, TREM may comprise a fragment or analogue of THD, for example, THD encoded by the nucleic acids in Table 1, the fragment having THD activity in one embodiment and not having THD activity in another embodiment.

[0084] In one embodiment, the THD is either below the corresponding sequence of the consensus sequence provided in the “consensus sequence” interval, or differs from the consensus sequence in 1, 2, 5, or 10 or fewer locations; In one embodiment, THD is formula I ZZZ The residue -R 48 -R 49 -R 50 -R 51 -R 52 -R 53 -R 54 -R 55 -R 56 -R 57 -R 58 -R 59 -R 60 -R 61 -R 62 -R 63 -R 64 It contains, and ZZZ represents one of the 20 amino acids; In one embodiment, THD is given by formula II ZZZ The residue -R 48 -R 49 -R 50 -R 51 -R 52 -R 53 -R 54 -R 55 -R 56 -R 57 -R 58 -R 59 -R60 -R 61 -R 62 -R 63 -R 64 It contains, and ZZZ represents one of the 20 amino acids; In one embodiment, THD is given by Equation III ZZZ The residue -R 48 -R 49 -R 50 -R 51 -R 52 -R 53 -R 54 -R 55 -R 56 -R 57 -R 58 -R 59 -R 60 -R 61 -R 62 -R 63 -R 64 It contains, and ZZZ represents one of the 20 amino acids; (e'1) The residue R of the consensus sequence provided in the "consensus sequence" interval 72 A linker that includes, for example, linker 4 region; (f) Under physiological conditions, it comprises a stem structure and one or more loop structures, e.g., one, two, or three loops. The loops may comprise domains described herein, e.g., domains selected from (a) to (e). The loops may comprise one or more domains. In some embodiments, the stem or loop structure has at least 75, 80, 85, 85, 90, 95, or 100% identity with naturally occurring stem or loop structures, e.g., stem or loop structures encoded by nucleic acids in Table 1. In some embodiments, TREM may comprise fragments or analogues of stem or loop structures, e.g., stem or loop structures encoded by nucleic acids in Table 1, wherein the fragments in one embodiment have the activity of the stem or loop structure, while in other embodiments they do not; (g) Tertiary structure, e.g., L-shaped tertiary structure; (h) Adapter function, i.e., TREM mediates the acceptance of amino acids, such as its congener amino acids, and mediates the transfer of AAs in the initiation or elongation of polypeptide chains; (i) Homogeneic adapter function (TREM mediates the acceptance and incorporation of amino acids (e.g., homogeneic amino acids) that are naturally bound to the anticodon of the TREM that initiates or extends the polypeptide chain); (j) Non-homogeneic adapter function (TREM mediates the acceptance and incorporation of amino acids other than those naturally bound to the TREM anticodon (e.g., non-homogeneic amino acids) during polypeptide chain initiation or elongation); (k) Regulatory functions, such as epigenetic functions (e.g., gene silencing functions or signaling pathway regulation functions), cell fate regulation functions, mRNA stability regulation functions, protein stability regulation functions, protein transduction regulation functions, or protein compartmentalization functions; (l) Structures that enable ribosome binding; (m) Post-transcriptional modifications, e.g., naturally occurring post-transcriptional modifications; (n) Functional properties of tRNA, for example, the ability to inhibit any of the properties (h) to (k) possessed by tRNA; (o) The ability to regulate cell fate; (p) Ability to regulate ribosome occupation; (q) The ability to regulate protein translation; (r) Ability to regulate mRNA stability; (s) Ability to regulate protein folding and structure; (t) Ability to regulate protein transduction or compartmentalization; (u) the ability to regulate protein stability; or (v) The ability to regulate signaling pathways, such as cellular signaling pathways.

[0085] In one embodiment, TREM comprises a full-length tRNA molecule or a fragment thereof.

[0086] In one embodiment, TREM includes the following characteristics: (a) to (e).

[0087] In one embodiment, TREM includes the following characteristics: (a) and (c).

[0088] In one embodiment, TREM includes the following characteristics: (a), (c), and (h).

[0089] In one embodiment, TREM includes the following characteristics: (a), (c), (h), and (b).

[0090] In one embodiment, TREM includes the following characteristics: (a), (c), (h), and (e).

[0091] In one embodiment, TREM includes the following characteristics: (a), (c), (h), (b), and (e).

[0092] In one embodiment, TREM includes the following characteristics: (a), (c), (h), (b), (e), and (g).

[0093] In one embodiment, TREM includes the following characteristics: (a), (c), (h), and (m).

[0094] In one embodiment, TREM includes the following characteristics: (a), (c), (h), (m), and (g).

[0095] In one embodiment, TREM includes the following characteristics: (a), (c), (h), (m), and (b).

[0096] In one embodiment, TREM includes the following characteristics: (a), (c), (h), (m), and (e).

[0097] In one embodiment, TREM includes the following characteristics: (a), (c), (h), (m), (g), (b), and (e).

[0098] In one embodiment, TREM includes the following characteristics: (a), (c), (h), (m), (g), (b), (e), and (q).

[0099] In one embodiment, TREM is (i) an amino acid-binding domain that binds to an amino acid (e.g., AStD as described in (a) of this specification); and (ii) comprising an anticodon that binds to each codon in the mRNA (e.g., ACHD as described in (c) herein).

[0100] In one embodiment, the TREM includes a mobile RNA linker that provides the covalent bonds of (i) and (ii).

[0101] In one embodiment, TREM mediates protein translation.

[0102] In some embodiments, the TREM includes a linker, e.g., an RNA linker, e.g., a mobile RNA linker, that provides a covalent bond between the first and second structures or domains. In some embodiments, the RNA linker includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ribonucleotides. The TREM may include one or more linkers; for example, in embodiments, the TREM comprising (a), (b), (c), (d), and (e) may have a first linker between the first and second domains, and a second linker between the third domain and another domain.

[0103] For a certain purpose, TREM contains the sequence of formula A: [L1]-[ASt domain 1]-[L2]-[DH domain]-[L3]-[ACH domain]-[VL domain]-[TH domain]-[L4]-[ASt domain 2].

[0104] In one embodiment, TREM comprises an RNA sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to an RNA sequence encoded by a DNA sequence listed in Table 1, or a fragment or functional fragment thereof, or differs from it by 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 or fewer ribonucleotides. In one embodiment, TREM comprises an RNA sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to an RNA sequence listed in Table 1, or a fragment or functional fragment thereof. In some embodiments, the TREM includes a TREM domain, for example, a domain described herein, which is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to an RNA encoded by a DNA sequence listed in Table 1, or a fragment or functional fragment thereof, or differs from it by 1, 2, 3, 4, 5, 10, or 15 or fewer ribonucleotides. In some embodiments, the TREM includes a TREM domain, for example, a domain described herein, which includes an RNA sequence encoded by a DNA sequence listed in Table 1, or a fragment or functional fragment thereof. In some embodiments, the TREM includes a TREM domain, for example, a domain described herein, which includes an RNA sequence encoded by a DNA sequence that is at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to a DNA sequence listed in Table 1, or a fragment or functional fragment thereof.

[0105] In one embodiment, the TREM is 76 to 90 nucleotides long. In the embodiment, the TREM or its fragment or functional fragment is 10 to 90 nucleotides, 10 to 80 nucleotides, 10 to 70 nucleotides, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 90 nucleotides, 20 to 80 nucleotides, 20 to 70 nucleotides, 20 to 60 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 30 to 90 nucleotides, 30 to 80 nucleotides, 30 to 70 nucleotides, 30 to 60 nucleotides, or 30 to 50 nucleotides.

[0106] In one embodiment, TREM is aminoacylated, for example, by loading amino acids with aminoacyl-tRNA synthetase.

[0107] In one embodiment, the TREM is unloaded with amino acids, for example, an unloaded TREM (uTREM).

[0108] In some embodiments, the TREM contains tRNA that is shorter than the full length. In embodiments, the TREM may correspond to a naturally occurring or non-naturally occurring fragment of tRNA. Exemplary fragments include the TREM half (e.g., derived from a cleavage in the ACHD, e.g., an anticodon sequence, e.g., a 5' half or a 3' half); the 5' fragment (e.g., derived from a cleavage in the DHD or ACHD, e.g., a fragment containing the 5' end); the 3' fragment (e.g., derived from a cleavage in the THD, e.g., a fragment containing the 3' end); or the internal fragment (e.g., derived from a cleavage in one or more of the ACHD, DHD, or THD).

[0109] When the term "TREM core fragment" is used herein, the sequence of formula B:[L1] y -[ASt Domain 1] x -[L2] y -[DH domain] y -[L3] y -[ACH domain] x-[VL domain] y -[TH domain] y -[L4] y -[ASt Domain 2] x This refers to the part where x=1 and y=0 or 1 in the equation.

[0110] As used herein, "TREM fragment" refers to the portion of TREM, where TREM comprises the sequence of formula A: [L1]-[ASt domain 1]-[L2]-[DH domain]-[L3]-[ACH domain]-[VL domain]-[TH domain]-[L4]-[ASt domain 2].

[0111] When the term is used herein, "congenital adapter function TREM" refers to a TREM that mediates initiation or extension by AA (congenital AA) that naturally binds to the anticodon of the TREM.

[0112] When the term is used herein, “reduced expression” refers to a reduction compared to a reference, for example, if modification of a control region or addition of a drug results in a reduction in the expression of the product in question, then that reduction is compared to other similar cells without modification or addition.

[0113] When the term is used herein, “external nucleic acid” refers to a nucleic acid sequence that is not present in the nearest sequence in a reference cell, e.g., the cell into which the external nucleic acid is introduced, or that differs from it by at least one nucleotide. In some embodiments, the external nucleic acid includes a nucleic acid encoding TREM.

[0114] "Foreign TREM" is used in this specification, (a) The reference cell, for example, the cell into which the foreign nucleic acid is introduced, has at least one nucleotide or one post-transcriptional modification that is different from the nearest sequence tRNA; (b) Whether it has been introduced into cells other than the transcribed cell; (c) Whether it is present in cells other than those that exist naturally; or (d) Refers to a TREM that has a non-wild-type expression profile, e.g., a level or distribution (e.g., it is expressed at a higher level than the wild type). In some embodiments, the expression profile may be mediated by a change introduced into the nucleic acid that modulates expression or by the addition of a drug that modulates the expression of an RNA molecule. In some embodiments, an exogenous TREM includes one, two, three, or four of characteristics (a) to (d).

[0115] Where used herein, “GMP-grade composition” refers to a composition that conforms to current Good Manufacturing Practices (cGMP) guidelines or other similar requirements. In some embodiments, a GMP-grade composition may be used as a pharmaceutical product.

[0116] As used herein, the terms “increase” and “decrease” refer to adjustments that result in an increase or decrease in the amount of a particular indicator of function, expression, or activity relative to a reference, respectively. For example, after administration of TREM to cells, tissues, or subjects as described herein, the amount of the indicator markers (e.g., protein translation, mRNA stability, protein folding) as described herein may increase or decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, 2×, 3×, 5×, or 10×, relative to the amount of the marker before administration or relative to the effect of a negative control drug. The indicator may be measured after administration at the time when the administration has had the listed effects, for example, at least 12 hours, 24 hours, 1 week, 1 month, 3 months, or 6 months after the start of treatment.

[0117] When the term is used herein, “increased expression” means an increase compared to a reference, for example, if modification of a control region or addition of a drug results in increased expression of the product of interest, it is considered increased compared to otherwise similar cells without modification or addition.

[0118] "Non-homogeneous adapter functional TREM" refers, as used herein, to a TREM that mediates initiation or extension by AAs other than AAs that naturally bind to the TREM's anticodon (non-homogeneous AAs). In some embodiments, a non-homogeneous adapter functional TREM is also called a misloaded TREM (mTREM).

[0119] "Non-natural sequence," as used herein, refers to a sequence in which adenine is replaced by a residue other than an adenine analog, cytosine is replaced by a residue other than a cytosine analog, guanine is replaced by a residue other than a guanine analog, and uracil is replaced by a residue other than a uracil analog. An analog refers to any possible derivative of ribonucleotide A, G, C, or U. In some embodiments, a sequence having a derivative of any one of ribonucleotides A, G, C, or U is a non-natural sequence.

[0120] When the term is used herein, “pharmaceutical TREM composition” refers to a TREM composition suitable for pharmaceutical use. Typically, a pharmaceutical TREM composition contains pharmaceutical excipients. In some embodiments, TREM may be the sole active ingredient in the pharmaceutical TREM composition. In embodiments, the pharmaceutical TREM composition may contain, substantially no, or pharmaceutically acceptable amounts of, host cell proteins, DNA, e.g., host cell DNA, endotoxins, and bacteria.

[0121] "Post-transcriptional processing," as used herein with respect to the target molecule, e.g., TREM, RNA, or tRNA, refers to covalent modification of the target molecule. In some embodiments, the covalent modification occurs after transcription. In some embodiments, the covalent modification occurs concurrently with transcription. In some embodiments, the modification is performed in vivo, for example, in the cells used to produce TREM. In some embodiments, the modification is performed ex vivo, for example, on TREM isolated from or obtained from the cells that produced TREM. In some embodiments, the post-transcriptional modification is selected from the post-transcriptional modifications listed in Table 2.

[0122] When the term "tRNA" is used herein, it refers to transtransition ribonucleic acid that is naturally present in its natural state.

[0123] When the term "TREM composition" is used herein, it refers to a composition comprising multiple TREMs, multiple TREM core fragments, and / or multiple TREM fragments. A TREM composition may comprise one or more species of TREMs, TREM core fragments, or TREM fragments. In some embodiments, the composition comprises only a single species of TREM, TREM core fragment, or TREM fragment. In some embodiments, the TREM composition comprises a first TREM, TREM core fragment, or TREM fragment species; and a second TREM, TREM core fragment, or TREM fragment species. In some embodiments, the TREM composition comprises X TREMs, TREM core fragments, or TREM fragment species (X = 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the TREMs, TREM core fragments, or TREM fragments have at least 70, 75, 80, 85, 90, or 95, or 100%, identity with the sequences encoded by nucleic acids in Table 1. A TREM composition may comprise one or more species of TREMs, TREM core fragments, or TREM fragments. In some embodiments, the TREM composition is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% dry weight TREM (for liquid compositions, dry weight refers to the weight after substantially all liquid has been removed, e.g., after freeze-drying). In some embodiments, the composition is liquid. In some embodiments, the composition is dry, e.g., freeze-dried material. In some embodiments, the composition is frozen composition. In some embodiments, the composition is sterile. In some embodiments, the composition contains at least 0.5 g, 1.0 g, 5.0 g, 10 g, 15 g, 25 g, 50 g, 100 g, 200 g, 400 g, or 500 g of TREM (e.g., measured by dry weight). In some embodiments, at least X% of the TREM in the TREM composition has non-natural modifications at selected positions, where X is 80, 90, 95, 96, 97, 98, 99, or 99.5.

[0124] In one embodiment, at least X% of the TREM in the TREM composition has a non-natural modification at a first position and a non-natural modification at a second position, where X is independently 80, 90, 95, 96, 97, 98, 99, or 99.5. In the embodiment, the modifications at the first and second positions are the same. In the embodiment, the modifications at the first and second positions are different. In the embodiment, the nucleotides at the first and second positions are the same, for example, both being adenine. In the embodiment, the nucleotides at the first and second positions are different, for example, one being adenine and the other being thymine.

[0125] In one embodiment, at least X% of the TREM in the TREM composition has a non-natural modification at the first position, and less than Y% has a non-natural modification at the second position, where X is 80, 90, 95, 96, 97, 98, 99, or 99.5, and Y is 20, 20, 5, 2, 1, .1, or .01. In several embodiments, the nucleotides at the first and second positions are the same, for example, both being adenine. In several embodiments, the nucleotides at the first and second positions are different, for example, one being adenine and the other being thymine.

[0126] TREM, TREM core fragments, and TREM fragments A “tRNA-based effector molecule” or “TREM” refers to an RNA molecule having one or more of the characteristics described herein. A TREM may include, for example, unnatural modifications as shown in Table 5. A TREM may further include nucleotide modifications to a second TREM, such as nucleotide substitutions, nucleotide deletions, or nucleotide additions.

[0127] In one embodiment, the TREM includes a TREM containing an array of formula A; a TREM core fragment containing an array of formula B; or a TREM fragment containing a portion of the TREM containing an array of formula A.

[0128] In one embodiment, TREM includes the sequence of formula A: [L1]-[ASt domain 1]-[L2]-[DH domain]-[L3]-[ACH domain]-[VL domain]-[TH domain]-[L4]-[ASt domain 2]. In one embodiment, [VL domain] is optional. In one embodiment, [L1] is optional.

[0129] In one embodiment, the TREM core fragment is an array of formula B:[L1] y -[ASt Domain 1] x -[L2] y -[DH domain] y -[L3] y -[ACH domain] x -[VL domain] y -[TH domain] y -[L4] y -[ASt Domain 2] x The equation includes x=1 and y=0 or 1. In one embodiment, y=0. In another embodiment, y=1.

[0130] In one embodiment, the TREM fragment comprises a portion of TREM, where TREM comprises the sequence of formula A:[L1]-[ASt domain 1]-[L2]-[DH domain]-[L3]-[ACH domain]-[VL domain]-[TH domain]-[L4]-[ASt domain 2], and the TREM fragment comprises one, two, three or all or any combination of the following: a TREM half (e.g., derived from a cleavage in the anticodon sequence, e.g., a 5' half or a 3' half); a 5' fragment (e.g., derived from a cleavage in the DH domain or ACH domain, e.g., a fragment containing a 5' end); a 3' fragment (e.g., derived from a cleavage in the TH domain, e.g., a fragment containing a 3' end); or an internal fragment (e.g., derived from a cleavage in any one of the ACH domain, DH domain or TH domain). Examples of TREM fragments include TREM halves (e.g., derived from a cleavage in ACHD, e.g., a 5' TREM half or a 3' TREM half), 5' fragments (e.g., derived from a cleavage in DHD or ACHD, e.g., a fragment containing the 5' end), 3' fragments (e.g., derived from a cleavage in THD, e.g., a fragment containing the 3' end of a TREM), or internal fragments (e.g., derived from a cleavage in one or more of ACHD, DHD, or THD).

[0131] In some embodiments, the TREM, TREM core fragment, or TREM fragment may be loaded with an amino acid (e.g., a homogeneous amino acid); may be loaded with a non-homogeneous amino acid (e.g., a misloaded TREM (mTREM)); or may not be loaded with an amino acid (e.g., an unloaded TREM (uTREM)). In some embodiments, the TREM, TREM core fragment, or TREM fragment may be loaded with an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, methionine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0132] In some embodiments, the unextended anticodon is an anticodon consisting of three or fewer nucleotides. In some embodiments, the unextended anticodon pairs with three or fewer codon nucleotides on the nucleic acid being translated.

[0133] In some embodiments, the TREM, TREM core fragment, or TREM fragment is a cognate TREM. In some embodiments, the TREM, TREM core fragment, or TREM fragment is a non-cognate TREM. In some embodiments, the TREM, TREM core fragment, or TREM fragment recognizes codons provided in Table 2 or Table 3.

[0134] [Table 2]

[0135] [Table 3]

[0136] In one embodiment, the TREM includes a ribonucleic acid (RNA) sequence encoded by a deoxyribonucleic acid (DNA) sequence disclosed in Table 1, for example, one of the SEQ ID NOs. 1 to 451 disclosed in Table 1. In another embodiment, the TREM includes an RNA sequence that is at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the DNA sequence provided in Table 1, for example, one of the RNA sequences encoded by one of the SEQ ID NOs. 1 to 451 disclosed in Table 1. In one embodiment, the TREM includes an RNA sequence encoded by a DNA sequence that is at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of the DNA sequences provided in Table 1, e.g., SEQ ID NOs 1 to 451 disclosed in Table 1.

[0137] In one embodiment, a TREM, TREM core fragment, or TREM fragment contains at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence encoded by a DNA sequence disclosed in Table 1, for example, at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence encoded by any one of SEQ ID NOs. 1 to 451 disclosed in Table 1. In one embodiment, a TREM, TREM core fragment, or TREM fragment contains at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence that is at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the DNA sequence provided in Table 1, for example, an RNA sequence encoded by any one of SEQ ID NOs. 1 to 451 disclosed in Table 1. In one embodiment, the TREM, TREM core fragment, or TREM fragment comprises at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence encoded by a DNA sequence that is at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of the SEQ ID NOs. 1 to 451 disclosed in Table 1.

[0138] In one embodiment, the TREM core fragment or TREM fragment comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the DNA sequence provided in Table 1, for example, the RNA sequence encoded by any one of SEQ ID NOs. In one embodiment, the TREM core fragment or TREM fragment comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of an RNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the DNA sequence provided in Table 1, for example, the RNA sequence encoded by any one of SEQ ID NOs. In one embodiment, the TREM core fragment or TREM fragment includes at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 98%, or 99% of an RNA sequence encoded by a DNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the DNA sequences provided in Table 1, e.g., SEQ ID NOs 1 to 451 disclosed in Table 1.

[0139] In one embodiment, the TREM core fragment or TREM fragment comprises at least 5 ribonucleotides (nt), 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, or 60 nt (but less than the full length) of an RNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the RNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the DNA sequence provided in Table 1, for example, an RNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the RNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the RNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the RNA sequence that is at least 60% identical to the DNA sequence provided in Table 1, for example, an60% identical to the DNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the RNA sequence that is at In one embodiment, the TREM core fragment or TREM fragment includes at least 5 ribonucleotides (nt), 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, or 60 nt (but less than the full length) of an RNA sequence encoded by a DNA sequence having at least 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs. 1 to 451 disclosed in Table 1.

[0140] In one embodiment, the TREM core fragment or TREM fragment includes a sequence of lengths of 10-90 ribonucleotides (rnt), 10-80 rnt, 10-70 rnt, 10-60 rnt, 10-50 rnt, 10-40 rnt, 10-30 rnt, 10-20 rnt, 20-90 rnt, 20-80 rnt, 20-70 rnt, 20-60 rnt, 20-50 rnt, 20-40 rnt, 30-90 rnt, 30-80 rnt, 30-70 rnt, 30-60 rnt, or 30-50 rnt.

[0141] [Table 1-1]

[0142] Table 1-2

[0143] Table 1-3

[0144] Table 1-4

[0145] Table 1-5

[0146] Table 1-6

[0147] Table 1-7

[0148] Table 1-8

[0149] Table 1-9

[0150] Table 1-10

[0151] Table 1-11

[0152] Table 1-12

[0153] Table 1-13

[0154] Table 1-14

[0155] Table 1-15

[0156] Table 1-16

[0157] Table 1-17

[0158] Table 1-18

[0159] Table 1-19

[0160] Table 1-20

[0161] Table 1-21

[0162] Table 1-22

[0163] Table 1-23

[0164] Table 1-24

[0165] Table 1-25

[0166] Table 1-26

[0167] Table 1-27

[0168] Table 1-28

[0169] Table 1-29

[0170] Table 1-30

[0171] Table 1-31

[0172] Table 1-32

[0173] [Table 1-33]

[0174] [Table 1-34]

[0175] [Table 1-35]

[0176] [Table 1-36]

[0177] [Table 1-37]

[0178] Nucleotide sequence modification This disclosure describes, for example, TREMs comprising nucleotide sequence modifications that can modulate the functional parameters of a TREM with respect to a first TREM nucleotide sequence. These TREMs may or may not further include non-natural chemical modifications. For example, TREMs comprising nucleotide sequence modifications may exhibit improved activity or stability, for example, in vitro or intracellularly. Nucleotide sequence modifications may be nucleotide substitutions, for example, changes from one nucleotide at a given position in the TREM sequence to a different nucleotide. In some embodiments, the nucleotide substitution is an A to U substitution. In some embodiments, the nucleotide substitution is an A to G substitution. In some embodiments, the nucleotide substitution is an A to C substitution. In some embodiments, the nucleotide substitution is an A to A substitution. In some embodiments, the nucleotide substitution is an A to G substitution. In some embodiments, the nucleotide substitution is an A to C substitution. In some embodiments, the nucleotide substitution is an A to A substitution. In some embodiments, the nucleotide substitution is an A to U substitution. In some embodiments, the nucleotide substitution is an A to C substitution. In one embodiment, the nucleotide substitution is a substitution from C to A. In another embodiment, the nucleotide substitution is a substitution from C to U. In yet another embodiment, the nucleotide substitution is a substitution from C to G.

[0179] In some embodiments, the present disclosure features a TREM comprising a nucleotide substitution in a nucleotide within the TREM sequence. In some embodiments, the nucleotide substitution is located within a domain of the TREM, for example, [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], or [ASt domain 2]. In some embodiments, the nucleotide substitution is located within [ASt domain 1]. In some embodiments, the nucleotide substitution is located within the [DH domain]. In some embodiments, the nucleotide substitution is located within the [ACH domain]. In some embodiments, the nucleotide substitution is located within the [VL domain]. In some embodiments, the nucleotide substitution is located within the [TH domain]. In some embodiments, the nucleotide substitution is located within [ASt domain 2]. In some embodiments, the nucleotide substitution is located at any of positions 1, 2, 3, 4, 5, 6, 7, 8, or 9 within [ASt domain 1]. In some embodiments, the nucleotide substitution is located at any of the positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 within the [DH domain]. In some embodiments, the nucleotide substitution is located at any of the positions 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 within the [ACH domain]. In some embodiments, the nucleotide substitution is located at any of the positions 44, 45, 46, 47, 48, or 49 within the [VL domain]. In some embodiments, the nucleotide substitution is located at any of the positions 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 within the [TH domain]. In one embodiment, the nucleotide substitution is located at any of the positions 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 within the [ASt domain 2].In one embodiment, the nucleotide substitution is at a specific position within the TREM sequence, for example, nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In some embodiments, the nucleotide substitution is at position 1 in the TREM sequence. In some embodiments, the nucleotide substitution is at position 2 in the TREM sequence. In some embodiments, the nucleotide substitution is at position 3 in the TREM sequence. In some embodiments, the nucleotide substitution is at position 4 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 5 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 6 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 7 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 8 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 9 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 10 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 11 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 12 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 13 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 14 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 15 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 16 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 17 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 18 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 19 in the TREM sequence.In one embodiment, the nucleotide substitution is located at position 20 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 21 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 22 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 23 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 24 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 25 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 26 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 27 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 28 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 29 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 30 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 31 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 32 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 33 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 34 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 35 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 36 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 37 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 38 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 39 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 40 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 41 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 42 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 43 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 44 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 45 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 46 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 47 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 48 in the TREM sequence.In one embodiment, the nucleotide substitution is located at position 49 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 50 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 51 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 52 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 53 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 54 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 55 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 56 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 57 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 58 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 59 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 60 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 61 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 62 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 63 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 64 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 65 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 66 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 67 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 68 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 69 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 70 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 71 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 72 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 73 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 74 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 75 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 76 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 77 in the TREM sequence.In one embodiment, the nucleotide substitution is located at position 78 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 79 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 80 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 81 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 82 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 83 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 84 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 85 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 86 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 87 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 88 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 89 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 90 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 91 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 92 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 93 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 94 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 95 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 96 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 97 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 98 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 99 in the TREM sequence. In one embodiment, the nucleotide substitution is located at position 100 in the TREM sequence. In one embodiment, the TREM contains multiple nucleotide substitutions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide substitutions. In one embodiment, the TREM contains one nucleotide substitution. In one embodiment, the TREM contains two nucleotide substitutions. In one embodiment, the TREM contains three nucleotide substitutions. In one embodiment, TREM includes four nucleotide substitutions.In one embodiment, TREM includes 5 nucleotide substitutions. In another embodiment, TREM includes 6 nucleotide substitutions. In another embodiment, TREM includes 7 nucleotide substitutions. In another embodiment, TREM includes 8 nucleotide substitutions. In another embodiment, TREM includes 9 nucleotide substitutions. In another embodiment, TREM includes 10 nucleotide substitutions. In another embodiment, TREM includes 11 nucleotide substitutions. In another embodiment, TREM includes 12 nucleotide substitutions. In another embodiment, TREM includes 13 nucleotide substitutions. In another embodiment, TREM includes 14 nucleotide substitutions. In another embodiment, TREM includes 15 nucleotide substitutions. In yet another embodiment, TREM includes... In one embodiment, the TREM contains 16 nucleotide substitutions. In one embodiment, the TREM contains 17 nucleotide substitutions. In one embodiment, the TREM contains 18 nucleotide substitutions. In one embodiment, the TREM contains 19 nucleotide substitutions. In one embodiment, the TREM contains 20 nucleotide substitutions. In one embodiment, the TREM contains more than 20 nucleotide substitutions. In one embodiment, the TREM contains nucleotide substitutions in two or more TREM domains, for example, two or more of [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], or [ASt domain 2]. In one embodiment, the TREM contains nucleotide substitutions in one TREM domain. In one embodiment, the TREM contains nucleotide substitutions in two TREM domains. In one embodiment, the TREM contains nucleotide substitutions in three TREM domains. In one embodiment, the TREM contains nucleotide substitutions in four TREM domains. In one embodiment, the TREM contains nucleotide substitutions in five TREM domains. In one embodiment, the TREM contains nucleotide substitutions in six TREM domains. In some embodiments, TREM includes nucleotide substitutions in [ASt domain 1] and [DH domain]. In some embodiments, TREM includes nucleotide substitutions in [ASt domain 1] and [ACH domain]. In some embodiments, TREM includes nucleotide substitutions in [ASt domain 1] and [VL domain]. In some embodiments, TREM includes nucleotide substitutions in [ASt domain 1] and [TH domain]. In some embodiments, TREM includes nucleotide substitutions in [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM includes nucleotide substitutions in [DH domain] and [ACH domain]. In some embodiments, TREM includes nucleotide substitutions in [DH domain] and [ACH domain]. In some embodiments, TREM includes nucleotide substitutions in [DH domain] and [VL domain]. In some embodiments, TREM includes nucleotide substitutions in [DH domain] and [TH domain]. In some embodiments, TREM includes nucleotide substitutions in [DH domain] and [ASt domain 2].In some embodiments, TREM includes nucleotide substitutions in the [ACH domain] and [VL domain]. In some embodiments, TREM includes nucleotide substitutions in the [ACH domain] and [TH domain]. In some embodiments, TREM includes nucleotide substitutions in the [ACH domain] and [ASt domain 2]. In some embodiments, TREM includes nucleotide substitutions in the [VL domain] and [TH domain]. In some embodiments, TREM includes nucleotide substitutions in the [VL domain] and [ASt domain 2]. In some embodiments, TREM includes nucleotide substitutions in the [TH domain] and [ASt domain 2]. In some embodiments, TREM includes nucleotide substitutions in the [ASt domain 1] and [ASt domain 1]. In some embodiments, TREM includes nucleotide substitutions in the [DH domain] and [DH domain]. In some embodiments, TREM includes nucleotide substitutions in the [ACH domain] and [ACH domain]. In some embodiments, TREM includes nucleotide substitutions in the [VL domain] and [VL domain]. In some embodiments, TREM includes nucleotide substitutions in the [TH domain] and [TH domain]. In one embodiment, the TREM includes [ASt domain 2] and a nucleotide substitution in [ASt domain 2]. In one embodiment, the nucleotide substitution is located within a loop of the TREM secondary structure, for example, within an unpaired region of the TREM. In one embodiment, the nucleotide substitution is located at any of the unpaired positions 8, 9, 14, 15, 16, 17, 17A, 18, 19, 20, 20A, 20B, 21, 32, 33, 34, 35, 36, 37, 38, 45, e11, e12, e13, e14, e15, e16, e17, e1, e2, e3, e4, e5, e27, e26, e25, e24, e23, e22, e21, 46, 47, 48, 54, 55, 56, 57, 58, 59, 60, 73, 74, 75, or 76, according to a universal tRNA numbering scheme, for example, a nucleotide position numbering scheme as provided in Figure 4.In one embodiment, the nucleotide substitution is located at any of the base-pairing positions 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 22, 23, 24, 25, 27, 28, 29, 30, 31, 39, 40, 41, 42, 43, 49, 50, 51, 52, 53, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72, according to a universal tRNA numbering scheme, for example, a nucleotide position numbering scheme as provided in Figure 4. In one embodiment, the compensatory nucleotide substitution for the first A nucleotide substitution is U. In another embodiment, the compensatory nucleotide substitution for the first U nucleotide substitution is A. In another embodiment, the compensatory nucleotide substitution for the first G nucleotide substitution is C. In another embodiment, the compensatory nucleotide substitution for the first C nucleotide substitution is G. In some embodiments, both nucleotide positions of a base pair include a nucleotide substitution, for example, both nucleotide positions of base pairs 1:72, 2:71, 3:70, 4:69, 5:68, 6:67, 7:66, 10:25, 11:24, 12:23, 13:22, 27:43, 28:42, 29:41, 30:40, 31:39, 49:65, 50:64, 51:63, 52:62, or 53:61, where the number before the colon represents the first nucleotide position of the base pair, and the number after the colon represents the second nucleotide position of the base pair. In some embodiments, both nucleotides of a base pair at positions 1 and 72 include a nucleotide substitution. In some embodiments, both nucleotides of a base pair at positions 2 and 71 include a nucleotide substitution. In some embodiments, both nucleotides of a base pair at positions 3 and 70 include a nucleotide substitution. In one embodiment, both nucleotides of the base pair at positions 4 and 69 include a nucleotide substitution.In some embodiments, both nucleotides in the base pairs at positions 5 and 68 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 6 and 67 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 7 and 68 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 10 and 25 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 11 and 24 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 12 and 23 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 13 and 22 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 27 and 43 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 28 and 42 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 29 and 41 include nucleotide substitutions. In some embodiments, both nucleotides in the base pairs at positions 30 and 40 include nucleotide substitutions. In some embodiments, both nucleotides of the base pair at positions 31 and 39 include a nucleotide substitution. In some embodiments, both nucleotides of the base pair at positions 49 and 65 include a nucleotide substitution. In some embodiments, both nucleotides of the base pair at positions 50 and 64 include a nucleotide substitution. In some embodiments, both nucleotides of the base pair at positions 51 and 63 include a nucleotide substitution. In some embodiments, both nucleotides of the base pair at positions 52 and 63 include a nucleotide substitution. In some embodiments, both nucleotides of the base pair at positions 53 and 61 include a nucleotide substitution. In some embodiments, TREM includes multiple pairs of nucleotide substitutions and compensatory nucleotide substitutions, for example, nucleotide substitutions that maintain multiple pairs of base pair formation, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, TREM includes one pair of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, TREM includes two pairs of nucleotide substitutions and compensatory nucleotide substitutions.In some embodiments, the TREM includes three pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes four pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes five pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes six pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes seven pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes eight pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes nine pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes ten pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the TREM includes eleven or more pairs of nucleotide substitutions and compensatory nucleotide substitutions. In some embodiments, the nucleotide substitutions at the first and second compensatory positions result in a change from lower energy base pairs, e.g., G:C or C:G base pairs to A:U or U:A base pairs. In some embodiments, nucleotide substitutions at the first and second compensatory positions result in a change from a higher-energy base pair, e.g., A:U or U:A base pair to a G:C or C:G base pair. In some embodiments, nucleotide substitutions at the first and second compensatory positions result in a change from a neutral-energy base pair, e.g., from an A:U base pair to a U:A base pair, or from a C:G base pair to a G:C base pair. In some embodiments, nucleotide substitutions result in a wobble base pair, e.g., a G:U base pair. In some embodiments, nucleotide substitutions at the first and second compensatory positions result in a wobble base pair, e.g., a G:U base pair. In some embodiments, nucleotide substitutions disrupt base pairing.

[0180] In some embodiments, nucleotide substitutions result in modulation of TREM activity, for example, an increase or decrease in TREM activity. For example, nucleotide substitutions can modulate TREM's ability to: (i) assist in protein synthesis, (ii) be loaded by tRNA synthetase, (iii) be bound by elongation factors, (iv) introduce amino acids into peptide chains, (v) assist in protein elongation, (vi) assist in the initiation of protein synthesis, or (vii) read through immature stop codons (PTCs), for example, within a cell. In some embodiments, nucleotide substitutions modulate TREM's ability to assist in protein synthesis. In some embodiments, nucleotide substitutions modulate TREM's ability to be loaded by tRNA synthetase. In some embodiments, nucleotide substitutions modulate TREM's ability to be bound by elongation factors. In some embodiments, nucleotide substitutions modulate TREM's ability to introduce amino acids into peptide chains. In some embodiments, nucleotide substitutions modulate TREM's ability to assist in protein elongation. In some embodiments, nucleotide substitutions modulate the ability of TREM to assist in the initiation of protein synthesis. In some embodiments, nucleotide substitutions modulate the ability of TREM to read through PTCs. In some embodiments, nucleotide substitutions at specific positions within TREM increase TREM's ability to assist in protein synthesis, for example, in a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM increase the TREM's ability to be loaded by RNA synthetase, for example, in a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard.In one embodiment, nucleotide substitutions at specific positions in TREM increase the TREM's ability to be bound by elongation factors, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in the TREM increase the TREM's ability to introduce amino acids into peptide chains, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM increase the TREM's ability to assist protein elongation, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM increase TREM's ability to assist in initiating protein synthesis, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard. In one embodiment, a nucleotide substitution at a specific position in the TREM increases the TREM's ability to read through immature stop codons, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard.In one embodiment, nucleotide substitutions at specific positions in TREM reduce the TREM's ability to assist in protein synthesis, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM reduce the TREM's ability to be loaded by RNA synthetase, for example, intracellularly, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM reduce the TREM's ability to be bound by elongation factors, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in the TREM reduce the TREM's ability to introduce amino acids into peptide chains, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM reduce the TREM's ability to assist protein elongation, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard.In one embodiment, nucleotide substitutions at specific positions in TREM reduce TREM's ability to assist in initiating protein synthesis, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard. In one embodiment, a nucleotide substitution at a specific position in the TREM reduces the TREM's ability to read through immature stop codons, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to, for example, a reference standard.

[0181] In some embodiments, nucleotide substitutions result in the regulation of TREM stability, for example, by increasing or decreasing TREM stability. For example, nucleotide substitutions may regulate, for example, within a cell, (i) TREM localization, (ii) the amount of time before TREM degradation, (iii) the amount of time TREM is loaded with amino acids, (iv) the amount of time TREM interacts with ribosomes, (v) the amount of time TREM interacts with elongation factors, (vi) TREM modification, or (vii) the interaction between TREM and tRNA degradation proteins. In some embodiments, nucleotide substitutions regulate TREM localization. In some embodiments, nucleotide substitutions regulate the amount of time before TREM degradation. In some embodiments, nucleotide substitutions regulate the amount of time TREM is loaded with amino acids. In some embodiments, nucleotide substitutions regulate the amount of time TREM interacts with ribosomes. In some embodiments, nucleotide substitutions regulate the amount of time TREM interacts with elongation factors. In some embodiments, nucleotide substitutions regulate TREM modification. In some embodiments, nucleotide substitutions modulate the interaction between TREM and tRNA degradation proteins. In some embodiments, nucleotide substitutions at specific locations in TREM increase the proper localization of TREM, for example, intracellularly, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM increase the amount of time it takes for TREM to be degraded, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard.In one embodiment, nucleotide substitution at a specific position of TREM increases the amount of time TREM is loaded with amino acids, for example, in a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to, for example, a reference standard. In one embodiment, a nucleotide substitution at a specific position in the TREM increases the amount of time the TREM interacts with ribosomes, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to, for example, a reference standard. In one embodiment, a nucleotide substitution at a specific position of TREM increases the amount of time that TREM interacts with elongation factors, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to, for example, a reference standard. In one embodiment, a nucleotide substitution at a specific position of TREM increases the modification of TREM, for example, intracellularly, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to, for example, a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM increase the interaction between TREM and tRNA degradation proteins, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more compared to, for example, a reference standard.In one embodiment, nucleotide substitutions at specific positions in TREM reduce the amount of time it takes for TREM to degrade, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard. In one embodiment, nucleotide substitution at a specific position in TREM reduces the amount of time TREM is loaded with amino acids, for example, in a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM reduce the amount of time TREM interacts with ribosomes, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to, for example, a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM reduce the amount of time TREM interacts with elongation factors, for example, within a cell, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to, for example, a reference standard. In one embodiment, nucleotide substitutions at specific positions of TREM reduce the modification of TREM, for example, intracellularly, by approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to a reference standard.In one embodiment, nucleotide substitutions at specific positions of TREM reduce the modification of TREM, for example, intracellularly, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to, for example, a reference standard. In one embodiment, nucleotide substitutions at specific positions in TREM reduce the interaction between TREM and tRNA degradation proteins, for example, intracellularly, by, for example, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or more, compared to, for example, a reference standard.

[0182] Typical nucleotide sequence modifications are shown below.

[0183] A TREM may contain a nucleotide mutation in the nucleotide sequence of SEQ ID NO: 734. In some embodiments, the nucleotide mutation in the nucleotide sequence of SEQ ID NO: 734 is a nucleotide substitution. In some embodiments, a TREM contains a nucleotide substitution in the nucleotide sequence of SEQ ID NO: 734 in the [VL domain]. In some embodiments, a TREM contains one nucleotide substitution. In some embodiments, the nucleotide substitution is located at a nucleotide position between positions 40 and 50. In some embodiments, the nucleotide substitution is located at nucleotide positions 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the nucleotide substitution is located at position 44. In some embodiments, the nucleotide substitution is an A to G substitution. In some embodiments, the nucleotide substitution is A44G. In some embodiments, a TREM has the nucleotide sequence of SEQ ID NO: 743. In some embodiments, a TREM having SEQ ID NO: 743 contains a non-natural modification. In one embodiment, TREM having sequence number 743 has non-natural modifications listed in Table 6, for example, one of pattern numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0184] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734 are present in each of [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM includes eight nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 1 and 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 60 and 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 70 and 80. In some embodiments, the nucleotide substitutions are located at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In some embodiments, the nucleotide substitution is located at position 2. In one embodiment, the nucleotide substitution is at position 4. In one embodiment, the nucleotide substitution is at position 5. In one embodiment, the nucleotide substitution is at position 6. In one embodiment, the nucleotide substitution is at position 67. In one embodiment, the nucleotide substitution is at position 68. In one embodiment, the nucleotide substitution is at position 69. In one embodiment, the nucleotide substitution is at position 71. In one embodiment, the nucleotide substitution is a G to C substitution. In one embodiment, the nucleotide substitution is a U to C substitution. In one embodiment, the nucleotide substitution is a C to G substitution. In one embodiment, the nucleotide substitution is a C to A substitution. In one embodiment, the nucleotide substitution is a G to U substitution. In one embodiment, the nucleotide substitution is an A to G substitution. In one embodiment, the nucleotide substitution is a U to G substitution. In one embodiment, the nucleotide substitution is G2C. In one embodiment, the nucleotide substitution is U4C. In one embodiment, the nucleotide substitution is C5G. In one embodiment, the nucleotide substitution is C6A. In one embodiment, the nucleotide substitution is G67U.In one embodiment, the nucleotide substitution is G68C. In one embodiment, the nucleotide substitution is A69G. In one embodiment, the nucleotide substitution is U71G. In one embodiment, the TREM has the nucleotide sequence of SEQ ID NO: 639. In one embodiment, the TREM having SEQ ID NO: 639 includes non-natural modifications. In one embodiment, the TREM having SEQ ID NO: 639 has non-natural modifications listed in Table 6, for example, one of pattern numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0185] TREM may contain nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are located in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], and [ASt domain 2]. In some embodiments, TREM contains 17 nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between 1 and 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions between 10 and 20. In some embodiments, the nucleotide substitutions are located at nucleotide positions between 20 and 30. In some embodiments, the nucleotide substitutions are located at nucleotide positions between 30 and 40. In some embodiments, the nucleotide substitutions are located at nucleotide positions between 40 and 50. In some embodiments, the nucleotide substitutions are located at nucleotide positions between 60 and 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions between 70 and 80. In some embodiments, the nucleotide substitutions are located at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleotide substitution is located at nucleotide positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the nucleotide substitution is located at nucleotide positions 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the nucleotide substitution is located at nucleotide positions 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In some embodiments, the nucleotide substitution is located at nucleotide positions 40, 41, 41, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the nucleotide substitution is located at nucleotide positions 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. In some embodiments, the nucleotide substitution is located at nucleotide positions 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In some embodiments, the nucleotide substitution is located at position 2. In some embodiments, the nucleotide substitution is located at position 12. In some embodiments, the nucleotide substitution is located at position 13. In some embodiments, the nucleotide substitution is located at position 22. In some embodiments, the nucleotide substitution is located at position 23.In one embodiment, the nucleotide substitution is at position 28. In one embodiment, the nucleotide substitution is at position 31. In one embodiment, the nucleotide substitution is at position 39. In one embodiment, the nucleotide substitution is at position 40. In one embodiment, the nucleotide substitution is at position 42. In one embodiment, the nucleotide substitution is at position 43. In one embodiment, the nucleotide substitution is at position 44. In one embodiment, the nucleotide substitution is at position 46. In one embodiment, the nucleotide substitution is at position 49. In one embodiment, the nucleotide substitution is at position 65. In one embodiment, the nucleotide substitution is at position 71. In one embodiment, the nucleotide substitution is at position 72. In one embodiment, the nucleotide substitution is a G to C substitution. In one embodiment, the nucleotide substitution is a C to U substitution. In one embodiment, the nucleotide substitution is a G to A substitution. In one embodiment, the nucleotide substitution is a C to G substitution. In one embodiment, the nucleotide substitution is a U to C substitution. In one embodiment, the nucleotide substitution is an A to C substitution. In one embodiment, the nucleotide substitution is a U to G substitution. In one embodiment, the nucleotide substitution is from A to G. In one embodiment, the nucleotide substitution is from C to G. In one embodiment, the nucleotide substitution is from G to U. In one embodiment, the nucleotide substitution is G2C. In one embodiment, the nucleotide substitution is G12C. In one embodiment, the nucleotide substitution is C13U. In one embodiment, the nucleotide substitution is G22A. In one embodiment, the nucleotide substitution is C23G. In one embodiment, the nucleotide substitution is U28C. In one embodiment, the nucleotide substitution is A31C. In one embodiment, the nucleotide substitution is U39G. In one embodiment, the nucleotide substitution is U40C. In one embodiment, the nucleotide substitution is A42G. In one embodiment, the nucleotide substitution is A43G. In one embodiment, the nucleotide substitution is A44G. In one embodiment, the nucleotide substitution is G46A. In one embodiment, the nucleotide substitution is C49G.In one embodiment, the nucleotide substitution is G65U. In one embodiment, the nucleotide substitution is U71G. In one embodiment, the nucleotide substitution is C72U. In one embodiment, the TREM has the nucleotide sequence of SEQ ID NO: 1149. In one embodiment, the TREM having SEQ ID NO: 1149 includes non-natural modifications. In one embodiment, the TREM having SEQ ID NO: 1149 has non-natural modifications listed in Table 6, for example, one of pattern numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0186] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are located in each of [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM includes four nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 1 and 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 60 and 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions 60, 61, 62, 63, 64, 64, 65, 66, 67, 68, 69, or 70. In some embodiments, the nucleotide substitution is at position 4. In some embodiments, the nucleotide substitution is at position 7. In some embodiments, the nucleotide substitution is at position 66. In some embodiments, the nucleotide substitution is at position 69. In some embodiments, the nucleotide substitution is a U to C substitution. In some embodiments, the nucleotide substitution is a G to A substitution. In one embodiment, the nucleotide substitution is a C to U substitution. In one embodiment, the nucleotide substitution is an A to G substitution. In one embodiment, the nucleotide substitution is U4C. In one embodiment, the nucleotide substitution is G7A. In one embodiment, the nucleotide substitution is C66U. In one embodiment, the nucleotide substitution is A69G. In one embodiment, the TREM has the nucleotide sequence of SEQ ID NO: 652. In one embodiment, the TREM having SEQ ID NO: 652 includes non-natural modifications. In one embodiment, the TREM having SEQ ID NO: 652 has one of the non-natural modifications listed in Table 6, e.g., pattern numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0187] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are located within the [ACH domain]. In some embodiments, TREM includes nine nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 20 and 30. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 30 and 40. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 40 and 50. In some embodiments, the nucleotide substitutions are located at nucleotide positions 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the nucleotide substitutions are located at nucleotide positions 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In some embodiments, the nucleotide substitutions are located at nucleotide positions 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the nucleotide substitution is located at position 27. In one embodiment, the nucleotide substitution is at position 28. In one embodiment, the nucleotide substitution is at position 29. In one embodiment, the nucleotide substitution is at position 31. In one embodiment, the nucleotide substitution is at position 39. In one embodiment, the nucleotide substitution is at position 40. In one embodiment, the nucleotide substitution is at position 41. In one embodiment, the nucleotide substitution is at position 42. In one embodiment, the nucleotide substitution is at position 43. In one embodiment, the nucleotide substitution is a substitution from U to A. In one embodiment, the nucleotide substitution is a substitution from U to C. In one embodiment, the nucleotide substitution is a substitution from G to C. In one embodiment, the nucleotide substitution is a substitution from A to U. In one embodiment, the nucleotide substitution is a substitution from C to G. In one embodiment, the nucleotide substitution is a substitution from A to G. In one embodiment, the nucleotide substitution is U27A. In one embodiment, the nucleotide substitution is U28C. In one embodiment, the nucleotide substitution is G29C. In one embodiment, the nucleotide substitution is A31U. In one embodiment, the nucleotide substitution is U39A. In one embodiment, the nucleotide substitution is U40C.In one embodiment, the nucleotide substitution is C41G. In one embodiment, the nucleotide substitution is A42G. In one embodiment, the nucleotide substitution is A43U. In one embodiment, the TREM has the nucleotide sequence of SEQ ID NO: 702. In one embodiment, the TREM having SEQ ID NO: 702 includes non-natural modifications. In one embodiment, the TREM having SEQ ID NO: 702 has one of the non-natural modifications listed in Table 6, for example, pattern numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0188] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are located in each of [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM includes eight nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 1 and 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 60 and 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 70 and 80. In some embodiments, the nucleotide substitutions are located at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In some embodiments, the nucleotide substitution is located at position 1. In one embodiment, the nucleotide substitution is at position 2. In one embodiment, the nucleotide substitution is at position 4. In one embodiment, the nucleotide substitution is at position 5. In one embodiment, the nucleotide substitution is at position 68. In one embodiment, the nucleotide substitution is at position 69. In one embodiment, the nucleotide substitution is at position 71. In one embodiment, the nucleotide substitution is at position 72. In one embodiment, the nucleotide substitution is a substitution from G to A. In one embodiment, the nucleotide substitution is a substitution from G to C. In one embodiment, the nucleotide substitution is a substitution from U to A. In one embodiment, the nucleotide substitution is a substitution from C to A. In one embodiment, the nucleotide substitution is a substitution from G to U. In one embodiment, the nucleotide substitution is a substitution from A to U. In one embodiment, the nucleotide substitution is a substitution from U to G. In one embodiment, the nucleotide substitution is a substitution from C to U. In one embodiment, the nucleotide substitution is G1A. In one embodiment, the nucleotide substitution is G2C. In one embodiment, the nucleotide substitution is U4A. In one embodiment, the nucleotide substitution is C5A.In one embodiment, the nucleotide substitution is G68U. In one embodiment, the nucleotide substitution is A69U. In one embodiment, the nucleotide substitution is U71G. In one embodiment, the nucleotide substitution is C72U. In one embodiment, the TREM has the nucleotide sequence of SEQ ID NO: 627. In one embodiment, the TREM having SEQ ID NO: 627 includes non-natural modifications. In one embodiment, the TREM having SEQ ID NO: 627 has non-natural modifications listed in Table 6, for example, one of pattern numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0189] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are located in each of the [ASt domain 1], [DH domain], [VL domain], and [ASt domain 2]. In some embodiments, TREM includes five nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 1 and 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 20 and 30. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 40 and 50. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 60 and 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the nucleotide substitution is located at nucleotide positions 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the nucleotide substitution is located at nucleotide positions 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. In some embodiments, the nucleotide substitution is located at position 4. In some embodiments, the nucleotide substitution is located at position 26. In some embodiments, the nucleotide substitution is located at position 49. In some embodiments, the nucleotide substitution is located at position 65. In some embodiments, the nucleotide substitution is located at position 69. In some embodiments, the nucleotide substitution is a U to C substitution. In some embodiments, the nucleotide substitution is an A to G substitution. In some embodiments, the nucleotide substitution is a C to G substitution. In some embodiments, the nucleotide substitution is a G to C substitution. In some embodiments, the nucleotide substitution is U4C. In some embodiments, the nucleotide substitution is A26G. In some embodiments, the nucleotide substitution is C49G. In one embodiment, the nucleotide substitution is G65C. In another embodiment, the nucleotide substitution is A69G. In another embodiment, TREM has the nucleotide sequence of SEQ ID NO: 1150.In one embodiment, the TREM having sequence number 1150 includes a non-natural modification. In one embodiment, the TREM having sequence number 1150 has one of the non-natural modifications listed in Table 6, for example, pattern numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0190] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are present in both the [VL domain] and the [ASt domain 2]. In some embodiments, TREM includes two nucleotide substitutions. In some embodiments, the nucleotide substitution is located at a nucleotide position between positions 40 and 50. In some embodiments, the nucleotide substitution is located at a nucleotide position between positions 60 and 70. In some embodiments, the nucleotide substitution is located at nucleotide positions 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the nucleotide substitution is located at nucleotide positions 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. In some embodiments, the nucleotide substitution is located at position 49. In some embodiments, the nucleotide substitution is located at position 65. In some embodiments, the nucleotide substitution is a C to G substitution. In some embodiments, the nucleotide substitution is a G to C substitution. In some embodiments, the nucleotide substitution is C49G. In one embodiment, the nucleotide substitution is G65C. In one embodiment, the TREM has the nucleotide sequence of SEQ ID NO: 1151. In one embodiment, the TREM having SEQ ID NO: 1151 includes non-natural modifications. In one embodiment, the TREM having SEQ ID NO: 1151 has one of the non-natural modifications listed in Table 6, e.g., pattern numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0191] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are located in each of [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM includes five nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 1 and 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 60 and 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 70 and 80. In some embodiments, the nucleotide substitutions are located at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. In some embodiments, the nucleotide substitutions are located at nucleotide positions 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In some embodiments, the nucleotide substitution is located at position 1. In some embodiments, the nucleotide substitution is at position 2. In some embodiments, the nucleotide substitution is at position 4. In some embodiments, the nucleotide substitution is at position 69. In some embodiments, the nucleotide substitution is at position 72. In some embodiments, the nucleotide substitution is a G to U substitution. In some embodiments, the nucleotide substitution is a G to A substitution. In some embodiments, the nucleotide substitution is a U to A substitution. In some embodiments, the nucleotide substitution is an A to U substitution. In some embodiments, the nucleotide substitution is a C to A substitution. In some embodiments, the nucleotide substitution is G1U. In some embodiments, the nucleotide substitution is G2A. In some embodiments, the nucleotide substitution is U4A. In some embodiments, the nucleotide substitution is A69U. In some embodiments, the nucleotide substitution is C72A. In some embodiments, TREM has the nucleotide sequence of SEQ ID NO: 794. In some embodiments, TREM having SEQ ID NO: 794 includes unnatural modifications.In one embodiment, TREM having sequence number 794 has one of the non-natural modifications listed in Table 6, for example, pattern numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0192] TREM may include nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitutions are located in each of the [ASt domain 1], [ACH domain], and [ASt domain 2]. In some embodiments, TREM includes four nucleotide substitutions. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 1 and 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 20 and 30. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 40 and 50. In some embodiments, the nucleotide substitutions are located at nucleotide positions between positions 70 and 80. In some embodiments, the nucleotide substitutions are located at nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleotide substitutions are located at nucleotide positions 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the nucleotide substitution is located at nucleotide positions 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the nucleotide substitution is located at nucleotide positions 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In some embodiments, the nucleotide substitution is located at position 2. In some embodiments, the nucleotide substitution is located at position 27. In some embodiments, the nucleotide substitution is located at position 43. In some embodiments, the nucleotide substitution is located at position 71. In some embodiments, the nucleotide substitution is a G to C substitution. In some embodiments, the nucleotide substitution is a U to G substitution. In some embodiments, the nucleotide substitution is an A to C substitution. In some embodiments, the nucleotide substitution is G2C. In some embodiments, the nucleotide substitution is U27G. In some embodiments, the nucleotide substitution is A43C. In some embodiments, the nucleotide substitution is U71G. In some embodiments, TREM has the nucleotide sequence of SEQ ID NO: 641. In one embodiment, TREM having sequence number 641 includes unnatural modifications.In one embodiment, TREM having sequence number 641 has one of the non-natural modifications listed in Table 6, for example, pattern numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0193] TREM may include a nucleotide substitution in the nucleotide sequence of SEQ ID NO: 734, where the nucleotide substitution is located in the [ACH domain]. In some embodiments, TREM includes four nucleotide substitutions. In some embodiments, the nucleotide substitution is located at a nucleotide position between positions 20 and 30. In some embodiments, the nucleotide substitution is located at a nucleotide position between positions 30 and 40. In some embodiments, the nucleotide substitution is located at a nucleotide position between positions 40 and 50. In some embodiments, the nucleotide substitution is located at nucleotide positions 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the nucleotide substitution is located at nucleotide positions 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In some embodiments, the nucleotide substitution is located at nucleotide positions 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the nucleotide substitution is located at position 28. In one embodiment, the nucleotide substitution is at position 31. In one embodiment, the nucleotide substitution is at position 39. In one embodiment, the nucleotide substitution is at position 42. In one embodiment, the nucleotide substitution is a U to C substitution. In one embodiment, the nucleotide substitution is an A to U substitution. In one embodiment, the nucleotide substitution is a U to A substitution. In one embodiment, the nucleotide substitution is an A to G substitution. In one embodiment, the nucleotide substitution is U28C. In one embodiment, the nucleotide substitution is A31U. In one embodiment, the nucleotide substitution is U39A. In one embodiment, the nucleotide substitution is A42G. In one embodiment, TREM has the nucleotide sequence of SEQ ID NO: 710. In one embodiment, TREM having SEQ ID NO: 710 includes unnatural modifications. In one embodiment, TREM having sequence number 710 has non-natural modifications listed in Table 6, for example, one of pattern numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32.

[0194] Table 7

[0195] In one embodiment, the TREM may include a nucleotide sequence scaffold, for example, one of scaffold numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In one embodiment, the TREM includes a chemical modification pattern, for example, one of pattern numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32. In some embodiments, a TREM may include one of a nucleotide sequence scaffold, e.g., scaffold numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and one of a chemical modification pattern, e.g., pattern numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32. In some embodiments, the functional parameters of a TREM may be regulated by the sequence scaffold. In some embodiments, the functional parameters of a TREM may be regulated by the chemical modification pattern. In some embodiments, the functional parameters of a TREM may be regulated by both the sequence scaffold and the modification pattern. In some embodiments, the functional parameter of TREM, modulated by a nucleotide sequence scaffold, a chemical modification pattern, or both, is the log2 activity of TREM relative to a mock, e.g., the log2 activity of TREM shown in Figure 1. In some embodiments, the log2 activity of TREM may be tuned to a range of 1 to 10²⁴. In some embodiments, the log2 activity of TREM may be tuned to a range of 1 to 4, 4 to 16, 16 to 64, 64 to 256, or 256 to 10²⁴. In some embodiments, the log2 activity of TREM may be tuned to a range of about 1 to about 40. In some embodiments, the log2 activity of TREM may be tuned to a range of about 2 to about 60. In some embodiments, the log2 activity of TREM may be tuned to a range of about 3 to about 60. In some embodiments, the log2 activity of TREM may be tuned to a range of about 3 to about 50.In some embodiments, the log2 activity of TREM can be adjusted to a range of about 2 to about 128. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 3 to about 30. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 3 to about 5¹². In some embodiments, the log2 activity of TREM can be adjusted to a range of about 4 to about 15. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 8 to about 100. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 3 to about 70. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 3 to about 5¹². In some embodiments, the log2 activity of TREM can be adjusted to a range of about 3 to about 128. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 3 to about 70. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 4 to about 128. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 32 to about 256. In some embodiments, the log2 activity of TREM is adjustable to a range of about 30 to about 512. In some embodiments, the log2 activity of TREM is adjustable to a range of about 60 to about 512. In some embodiments, the log2 activity of TREM is adjustable to a range of about 32 to about 512. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 64 to about 512. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 8 to about 256. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 10 to about 256. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 10 to about 400. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 64 to about 500. In one embodiment, the log2 activity of TREM can be adjusted to a range of about 128 to about 800. In another embodiment, the log2 activity of TREM can be adjusted to a range of about 14 to about 400. In yet another embodiment, the log2 activity of TREM can be adjusted to a range of about 128 to about 700.In some embodiments, the log2 activity of TREM can be adjusted to a range of about 8 to about 300. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 40 to about 512. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 100 to about 512. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 128 to about 400. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 64 to about 512. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 200 to about 512. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 1 to about 40 by pattern number 1. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 2 to about 60 by pattern number 2. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 3 to about 60 by pattern number 3. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 50 by pattern number 4. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 2 to approximately 128 by pattern number 5. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 30 by pattern number 6. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 5¹² by pattern number 7. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 4 to approximately 15 by pattern number 8. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 8 to approximately 100 by pattern number 9. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 70 by pattern number 10. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 5¹² by pattern number 11. In one embodiment, the log2 activity of TREM can be adjusted to a range of about 3 to about 128 by pattern number 12. In another embodiment, the log2 activity of TREM can be adjusted to a range of about 3 to about 70 by pattern number 13.In some embodiments, the log2 activity of TREM can be adjusted to a range of about 4 to about 128 by pattern number 14. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 32 to about 256 by pattern number 15. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 30 to about 512 by pattern number 16. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 60 to about 512 by pattern number 17. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 32 to about 512 by pattern number 18. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 64 to about 512 by pattern number 19. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 8 to about 256 by pattern number 20. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 10 to about 256 by pattern number 21. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 10 to about 400 by pattern number 22. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 64 to about 500 by pattern number 23. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 128 to about 800 by pattern number 24. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 14 to about 400 by pattern number 25. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 128 to about 700 by pattern number 26. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 8 to about 300 by pattern number 27. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 40 to about 512 by pattern number 28. In some embodiments, the log2 activity of TREM can be adjusted to a range of about 100 to about 512 by pattern number 29. In one embodiment, the log2 activity of TREM can be adjusted to a range of approximately 128 to approximately 400 by pattern number 30.In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 64 to approximately 512 by pattern number 31. In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 200 to approximately 512 by pattern number 32. In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 1 to approximately 800 by scaffold number 1. In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 4 to approximately 400 by scaffold number 2. In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 512 by scaffold number 3. In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 400 by scaffold number 4. In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 3 to approximately 512 by scaffold number 5. In some embodiments, the log2 activity of TREM can be adjusted to a range of approximately 2 to approximately 256 by scaffold number 6. In one embodiment, the log2 activity of TREM can be adjusted by scaffold number 7 to a range of about 3 to about 700. In another embodiment, the log2 activity of TREM can be adjusted by scaffold number 8 to a range of about 2 to about 600. In yet another embodiment, the log2 activity of TREM can be adjusted by scaffold number 9 to a range of about 3 to about 200. In yet another embodiment, the log2 activity of TREM can be adjusted by scaffold number 10 to a range of about 3 to about 800. In yet another embodiment, the log2 activity of TREM can be adjusted by scaffold number 11 to a range of about 3 to about 300. In yet another embodiment, the log2 activity of TREM can be adjusted by scaffold number 12 to a range of about 1 to about 400.

[0196] In one embodiment, TREM may include any one of nucleotide sequence scaffolds, for example, scaffold numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In one embodiment, TREM may include unnatural modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or more unnatural modifications. In one embodiment, TREM includes, for example, one of the scaffold numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and non-natural modifications, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or more. In one embodiment, the functional parameters of TREM may be adjusted by the sequence scaffold. In one embodiment, the functional parameters of TREM may be adjusted by the number of non-natural modifications. In one embodiment, the functional parameters of TREM may be adjusted by the number of sequence scaffolds and non-natural modifications. In some embodiments, a functional parameter of TREM, modulated by the number of nucleotide sequence scaffolds, the number of unnatural modifications, or both, is the log2 activity of TREM relative to a mock, e.g., the log2 activity of TREM shown in Figure 2. In some embodiments, the log2 activity of TREM can be modulated to a range of about 60 to about 256 by 0 unnatural modifications. In some embodiments, the log2 activity of TREM can be modulated to a range of about 128 to about 800 by 4 unnatural modifications. In some embodiments, the log2 activity of TREM can be modulated to a range of about 256 to about 600 by 5 unnatural modifications. In some embodiments, the log2 activity of TREM can be modulated to a range of about 200 to about 512 by 6 unnatural modifications.In one embodiment, the log2 activity of TREM can be regulated to a range of about 150 to about 700 by eight unnatural modifications. In one embodiment, the log2 activity of TREM can be regulated to a range of about 3 to about 100 by ten unnatural modifications. In one embodiment, the log2 activity of TREM can be regulated to a range of about 3 to about 100 by thirteen unnatural modifications. In one embodiment, the log2 activity of TREM can be regulated to a range of about 8 to about 400 by fourteen unnatural modifications. In one embodiment, the log2 activity of TREM can be regulated to a range of about 32 to about 250 by fifteen unnatural modifications. In one embodiment, the log2 activity of TREM can be regulated to a range of about 8 to about 400 by sixteen unnatural modifications. In one embodiment, the log2 activity of TREM can be regulated to a range of about 60 to about 400 by nineteen unnatural modifications. In some embodiments, the log2 activity of TREM can be regulated by unnatural modification 21 to a range of about 3 to about 128. In some embodiments, the log2 activity of TREM can be regulated by unnatural modification 22 to a range of about 8 to about 256. In some embodiments, the log2 activity of TREM can be regulated by unnatural modification 24 to a range of about 12 to about 500. In some embodiments, the log2 activity of TREM can be regulated by unnatural modification 25 to a range of about 2 to about 60. In some embodiments, the log2 activity of TREM can be regulated by unnatural modification 26 to a range of about 3 to about 55. In some embodiments, the log2 activity of TREM can be regulated by unnatural modification 27 to a range of about 60 to about 500. In some embodiments, the log2 activity of TREM can be regulated by unnatural modification 28 to a range of about 2 to about 100. In one embodiment, the log2 activity of TREM can be regulated by 29 unnatural modifications to range from about 55 to about 600. In another embodiment, the log2 activity of TREM can be regulated by 30 unnatural modifications to range from about 4 to about 128. In yet another embodiment, the log2 activity of TREM can be regulated by 31 unnatural modifications to range from about 1 to about 32.In some embodiments, the log2 activity of TREM can be regulated by 32 unnatural modifications to range from about 3 to about 128. In some embodiments, the log2 activity of TREM can be regulated by 33 unnatural modifications to range from about 64 to about 5¹². In some embodiments, the log2 activity of TREM can be regulated by 34 unnatural modifications to range from about 32 to about 5¹². In some embodiments, the log2 activity of TREM can be regulated by 35 unnatural modifications to range from about 20 to about 5¹². In some embodiments, the log2 activity of TREM can be regulated by 36 unnatural modifications to range from about 2 to about 5¹². In some embodiments, the log2 activity of TREM can be regulated by 37 unnatural modifications to range from about 3 to about 5¹². In some embodiments, the log2 activity of TREM can be regulated by 40 unnatural modifications to range from about 100 to about 5¹². In some embodiments, the log2 activity of TREM can be regulated to a range of about 6 to about 256 by the non-native modification 41. In some embodiments, the log2 activity of TREM can be regulated to a range of about 1 to about 400 by scaffold number 1. In some embodiments, the log2 activity of TREM can be regulated to a range of about 4 to about 512 by scaffold number 2. In some embodiments, the log2 activity of TREM can be regulated to a range of about 4 to about 512 by scaffold number 3. In some embodiments, the log2 activity of TREM can be regulated to a range of about 3 to about 400 by scaffold number 4. In some embodiments, the log2 activity of TREM can be regulated to a range of about 3 to about 700 by scaffold number 5. In some embodiments, the log2 activity of TREM can be regulated to a range of about 3 to about 256 by scaffold number 6. In some embodiments, the log2 activity of TREM can be regulated to a range of about 3 to about 800 by scaffold number 7. In one embodiment, the log2 activity of TREM can be regulated by scaffold number 8 to a range of about 2 to about 600. In another embodiment, the log2 activity of TREM can be regulated by scaffold number 9 to a range of about 3 to about 256.In one embodiment, the log2 activity of TREM can be adjusted by scaffold number 10 to a range of about 3 to about 800. In another embodiment, the log2 activity of TREM can be adjusted by scaffold number 11 to a range of about 3 to about 500. In yet another embodiment, the log2 activity of TREM can be adjusted by scaffold number 12 to a range of about 1 to about 256.

[0197] In some embodiments, TREM is not the TREM shown in Figure 3. In some embodiments, TREM does not include the nucleotide sequence of TREM shown in Figure 3. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 743. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 652. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 702. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 627. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 1150. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 1151. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 794. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 641. In some embodiments, TREM does not include the nucleotide sequence of SEQ ID NO: 710.

[0198] In some embodiments, TREM does not include a pattern of unnatural modification according to the patterns shown in Table 6 (for example, a pattern selected from pattern numbers 1 to 32). In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 1 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 2 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 3 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 4 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 5 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 6 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 7 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 8 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern 9 shown in Table 6. In some embodiments, TREM does not include a pattern of unnatural modification according to pattern number 10 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 11 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 12 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 13 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 14 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 15 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 16 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 17 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 18 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 19 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 20 shown in Table 6.In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 21 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 22 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 23 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 24 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 25 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 26 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 27 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 28 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 29 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern number 30 shown in Table 6. In some embodiments, TREM does not include the unnatural modification pattern indicated by pattern 31 shown in Table 6. In one embodiment, the TREM does not include the unnatural modification pattern shown in Table 6, pattern 32.

[0199] In some embodiments, the TREM comprises a nucleotide sequence, for example, one of the sequences shown in Figure 3 or Figure 23. In some embodiments, the functional parameters of the TREM are modulated by nucleotide substitutions, for example, nucleotide substitutions relative to a reference TREM sequence. In some embodiments, the functional parameter of the TREM modulated by nucleotide substitutions is the immature stop codon (PTC) read-through activity of the TREM, for example, the TREM's ability to decode an immature stop codon and continue translation within the protein coding sequence. For example, nucleotide substitutions can modulate the relative abundance of the TREM in a pool of hits obtained by screening a TREM library containing nucleotide substitutions related to PTC read-through activity. In some embodiments, a nucleotide substitution at a predetermined position within the TREM sequence increases the hit abundance of the TREM containing the nucleotide substitution compared to the composition of the TREM library.

[0200] In some embodiments, a TREM includes nucleotide substitutions in a specific region of the TREM. In some embodiments, nucleotide substitutions in a specific region of the TREM modulate functional parameters of the TREM, such as activity or stability. In some embodiments, nucleotide substitutions in a specific region of the TREM modulate the activity of the TREM (e.g., PTC readthrough activity). For example, nucleotide substitutions in a specific region of the TREM can increase the activity of the TREM, while nucleotide substitutions in a second region of the TREM can decrease the activity of the TREM. In some embodiments, the activity modulated by nucleotide substitution is the PTC readthrough activity of the TREM, for example, the TREM's ability to assist in the translation of immature stop codons (PTCs) in protein-coding sequences. In some embodiments, the PTC readthrough activity of the TREM modulates the abundance of the TREM in a pool of hits obtained by pooled screening of multiple TREMs including nucleotide substitutions. In some embodiments, a TREM with increased PTC readthrough activity has a higher abundance in a pool of hits obtained from pooled screening of TREMs including nucleotide substitutions, for example, compared to a reference standard. In one embodiment, a TREM with reduced PTC readthrough activity has a lower abundance in the pool of hits obtained from pooled screening of TREMs containing nucleotide substitutions, for example, compared to a reference standard. In one embodiment, a nucleotide substitution in a specific region of the TREM modulates the abundance of TREM in the pool of hits obtained from pooled screening of PTC readthrough activity. In one embodiment, a nucleotide substitution in a specific region of the TREM increases the abundance of TREM in the pool of hits obtained from pooled screening. In one embodiment, a nucleotide substitution in a stem formed between [ASt domain 1] and [ASt domain 2] (e.g., a base-pairing region), for example, in a stem formed between positions 1-7 and 66-72, increases the abundance of TREM in the pool of hits obtained from pooled screening.In some embodiments, nucleotide substitutions in stems formed between the base pairs at positions 1 and 72, 2 and 71, 3 and 70, 4 and 69, 5 and 68, 6 and 67, and 7 and 66 increase the abundance of TREM in the pool of hits obtained from pooled screening, for example, as shown in Figure 4A. In some embodiments, nucleotide substitutions in stems of the [ACH domain], for example, in stems formed between positions 27-31 and 39-43, increase the abundance of TREM in the pool of hits obtained from pooled screening. In some embodiments, nucleotide substitutions in stems formed between the base pairs at positions 27 and 43, 28 and 42, 29 and 41, 30 and 40, and 31 and 39, for example, as shown in Figure 4A, increase the abundance of TREM in the pool of hits obtained from pooled screening. In some embodiments, nucleotide substitutions in specific regions of TREM decrease the abundance of TREM in the pool of hits obtained from pooled screening. In some embodiments, nucleotide substitutions in the loop region of the [ACH domain], for example, at positions 32-38, reduce the abundance of TREM in the pool of hits obtained from pooled screening. In some embodiments, for example, as shown in Figure 4A, nucleotide substitutions in the loop formed at positions 32, 33, 34, 35, 36, 37, and 38 reduce the abundance of TREM in the pool of hits obtained from pooled screening. In some embodiments, nucleotide substitutions in a specific region of TREM regulate the abundance of various TREM scaffolds (e.g., TREMs specific to various amino acids, e.g., TREMs with the ability to be loaded with various amino acids) in the pool of hits obtained from pooled screening. In some embodiments, nucleotide substitutions at a specific position of TREM (e.g., nucleotide substitutions in the base pair formed between positions 51 and 63) increase the abundance of a particular TREM scaffold in the pool of hits obtained from pooled screening.In one embodiment, as shown in Figure 4A, a nucleotide substitution in the base pair formed between positions 51 and 63 increases the abundance of a particular TREM scaffold in the pool of hits obtained from pooled screening.

[0201] In some embodiments, the abundance of nucleotide substitutions at specific positions of TREM in the hit pool obtained from pooled screening is regulated by the activity of TREM, e.g., the PTC readthrough activity of TREM. In some embodiments, the normalized abundance of nucleotide substitutions in the hit pool is about 0 to about 4.5, as shown in Figure 4B, for example. In some embodiments, the normalized abundance of nucleotide substitutions in the hit pool is about 0 to about 3.5. In some embodiments, the normalized abundance of nucleotide substitutions in the hit pool is about 0 to about 2.5. In some embodiments, the normalized abundance of nucleotide substitutions in the hit pool is about 0 to about 2.

[0202] In one embodiment, the enrichment (e.g., abundance) of nucleotide substitutions at specific positions in the TREM within the pool of hits obtained from pooled screening is regulated by the activity of the TREM (e.g., the PTC read-through activity of the TREM). In one embodiment, nucleotide substitutions at specific positions within the TREM are concentrated within the pool of hits obtained from pooled screening, for example, being more abundant in the hit pool than the initial pool composition. In another embodiment, nucleotide substitutions at specific positions within the TREM are drastically reduced within the pool of hits obtained from pooled screening, for example, being more abundant in the hit pool than the initial pool composition. In yet another embodiment, nucleotide substitutions at specific positions within the TREM are concentrated to a range of approximately 0 to approximately 4, as shown in Figure 5. In one embodiment, a nucleotide substitution at any of the positions 1, 2, 3, 4, 5, 6, 7, 16, 17, 20a, 27, 28, 29, 30, 31, 39, 40, 41, 42, 43, 51, 59, 60, 63, 66, 67, 68, 69, 70, 71, 72, or 73 increases the abundance of TREM in the pool of hits obtained from pooled screening, for example, as shown in Figure 5. In one embodiment, nucleotide substitutions at any of the positions 1, 2, 3, 4, 5, 6, 7, 16, 17, 20a, 27, 28, 29, 30, 31, 39, 40, 41, 42, 43, 51, 59, 60, 63, 66, 67, 68, 69, 70, 71, 72, or 73 increase the immature stop codon (PTC) readthrough activity of TREM, as shown in Figure 5, for example. In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by a nucleotide substitution at any of positions 1, 2, 3, 4, 5, 6, or 7 within the [ASt domain 1], e.g., as shown in Figure 5.In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by a nucleotide substitution at either position 16 or 17 in the [DH domain], e.g., as shown in Figure 5. In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by, for example, a nucleotide substitution at position 20a in the [DH domain], as shown in Figure 5. In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by a nucleotide substitution at any of the positions 27, 28, 29, 30, or 31 in the [ACH domain], e.g., as shown in Figure 5. In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by a nucleotide substitution at any of the positions 39, 40, 41, 42, or 43 within the [ACH domain], e.g., as shown in Figure 5.In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by, for example, a nucleotide substitution at position 51 in the [TH domain], as shown in Figure 5. In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by a nucleotide substitution at either position 59 or 60 in the [TH domain], e.g., as shown in Figure 5. In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by, for example, the nucleotide position at position 63 in the [TH domain], as shown in Figure 5. In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is increased by a nucleotide substitution at any of the positions 66, 67, 68, 69, 70, 71, 72, or 73 in the [ASt domain 2], e.g., as shown in Figure 5.In one embodiment, the activity of a TREM corresponding to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1835, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, 2019, 1654, or 1660, e.g., the PTC readthrough activity of the TREM, is reduced by a nucleotide substitution at any of the positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], e.g., as shown in Figure 5.

[0203] In some embodiments, the abundance of nucleotide substitutions at specific positions in the TREM within the pool of hits obtained from pooled screening is regulated by the activity of the TREM, for example, the PTC read-through activity of the TREM. In some embodiments, the normalized abundance of nucleotide substitutions in the pool of hits is about 0 to about 3.6, as shown in Figure 6. In some embodiments, the normalized abundance of nucleotide substitutions in the pool of hits is about 0 to about 8, as shown in Figure 7. In some embodiments, the normalized abundance of nucleotide substitutions in the pool of hits is about 0 to about 3.5, as shown, for example, in Figure 8. In some embodiments, the normalized abundance of nucleotide substitutions in the pool of hits is about 0 to about 5, as shown, for example, in Figure 9. In some embodiments, the normalized abundance of nucleotide substitutions in the pool of hits is about 0 to about 5, as shown, for example, in Figure 10. In some embodiments, the normalized abundance of nucleotide substitutions in the pool of hits is about 0 to about 12, as shown, for example, in Figure 11. In one embodiment, the normalized frequency of nucleotide substitutions in the hit pool is approximately 0 to approximately 2.1, as shown, for example, in Figure 12. In another embodiment, the normalized frequency of nucleotide substitutions in the hit pool is approximately 0 to approximately 2.4, as shown, for example, in Figure 13. In yet another embodiment, the normalized abundance of nucleotide substitutions in the hit group is approximately 0 to approximately 4, as shown, for example, in Figure 14. In yet another embodiment, the normalized abundance of nucleotide substitutions in the hit group is approximately 0 to approximately 4.5, as shown, for example, in Figure 15.

[0204] In one embodiment, the enrichment (e.g., abundance) of nucleotide substitutions at specific positions in the TREM within the pool of hits obtained from pooled screening is regulated by the activity of the TREM, e.g., the PTC readthrough activity of the TREM. In one embodiment, the nucleotide substitution disrupts base pairing at specific positions within the TREM. In one embodiment, the nucleotide substitutions that disrupt base pairing are concentrated within the pool of hits obtained from pooled screening. In one embodiment, the nucleotide substitutions that disrupt base pairing are drastically reduced within the pool of hits obtained from pooled screening. In one embodiment, the nucleotide substitutions that disrupt base pairing are concentrated to approximately 0 to approximately 4 times, as shown in Figure 16.

[0205] In one embodiment, enrichment of TREMs, including nucleotide substitutions within a pool of hits, e.g., TREMs having increased activity, e.g., increased PTC readthrough activity, is reproducible between repeated screenings, as shown, for example, in Figures 17 and 19A-B.

[0206] In some embodiments, the log2 PTC readthrough activity of a TREM containing nucleotide substitutions is regulated by nucleotide substitutions with respect to, for example, the parent TREM sequence, for example, the start TREM sequence. In some embodiments, the log2 PTC readthrough activity of a TREM is regulated by nucleotide substitutions to be approximately -0.5 to approximately 6.8, for example, as shown in Figure 18. In some embodiments, the log2 PTC readthrough activity of a TREM is regulated by nucleotide substitutions to be approximately -0.5 to approximately 4 at low TREM doses. In some embodiments, the log2 PTC readthrough activity of a TREM is regulated by nucleotide substitutions to be approximately 0.5 to approximately 6.8 at high TREM doses.

[0207] In some embodiments, delivery of TREM containing nucleotide substitutions does not affect the activity of TREM, for example, the PTC readthrough activity of TREM. In some embodiments, the PTC readthrough activity of TREM delivered as synthetic oligonucleotides or by lentiviruses is substantially equivalent, as shown, for example, in Figures 22 and 25. In some embodiments, the normalized log2 activity of TREM containing nucleotide substitutions delivered by lentiviruses is about -2 to about 4. In some embodiments, the normalized log2 activity of TREM containing nucleotide substitutions delivered as synthetic oligonucleotides, for example, oligos, is about -2.1 to about 4.

[0208] In some embodiments, the TREM includes nucleotide substitutions at nucleotide positions relative to a reference sequence, e.g., a nucleotide substitution within the reference TREM. In some embodiments, the TREM including nucleotide substitutions modulates (e.g., increases or decreases) a functional parameter of the TREM relative to a reference TREM, e.g., the activity or stability of the TREM. In some embodiments, the functional parameter of the TREM modulated by the nucleotide substitution relative to the reference TREM is the immature stop codon (PTC) readthrough activity of the TREM. In some embodiments, the PTC readthrough activity of the TREM is modulated by nucleotide substitutions relative to a reference TREM, e.g., one of the reference TREMs SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023. In some embodiments, the PTC readthrough activity of the TREM is modulated by a nucleotide substitution relative to SEQ ID NO: 1638. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 1654. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 1660. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 1835. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 1867. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 2000. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 2001. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 2016. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 2017. In some embodiments, the PTC read-through activity of TREM is regulated by nucleotide substitution for SEQ ID NO: 2018.In some embodiments, the PTC read-through activity of TREM is modified by nucleotide substitution for SEQ ID NO: 2019. In some embodiments, the PTC read-through activity of TREM is modified by nucleotide substitution for SEQ ID NO: 2020. In some embodiments, the PTC read-through activity of TREM is modified by nucleotide substitution for SEQ ID NO: 2021. In some embodiments, the PTC read-through activity of TREM is modified by nucleotide substitution for SEQ ID NO: 2022. In some embodiments, the PTC read-through activity of TREM is modified by nucleotide substitution for SEQ ID NO: 2023. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for any of the parent TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 1638. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 1654. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 1660. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 1835. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 1867. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2000. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2001. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2016. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2017.In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2018. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2019. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2020. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2021. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2022. In some embodiments, the PTC read-through activity of TREM is increased by nucleotide substitution for SEQ ID NO: 2023.

[0209] In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 1% to over 500%, e.g., approximately 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more than 500%. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 1%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 5%. In one embodiment, a nucleotide substitution on any of the parent TREMs of a reference TREM (e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023) increases the PTC readthrough of the TREM by approximately 10%. In another embodiment, a nucleotide substitution on any of the reference TREMs (e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023) increases the PTC readthrough of the TREM by approximately 20%.In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 30%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 40%. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 50%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 60%. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 70%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 80%.In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 90%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 100%. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 150%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 200%. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 250%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 300%.In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 350%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 400%. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 450%. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 500%. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 500 or more.

[0210] In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 2 to 500 times or more, e.g., approximately 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more than 500. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately twofold. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately fivefold. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 10 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 20 times.In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 30 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 40 times. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 50 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 60 times. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 70 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 80 times.In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 90 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 100 times. In one embodiment, a nucleotide substitution on a reference TREM (e.g., any of the reference TREMs SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023) increases the PTC readthrough of the TREM by approximately 150 times. In another embodiment, a nucleotide substitution on a reference TREM (e.g., any of the reference TREMs SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023) increases the PTC readthrough of the TREM by approximately 200 times. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 250 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 300 times.In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 350 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 400 times. In one embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 450 times. In another embodiment, a nucleotide substitution on any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increases the PTC readthrough of the TREM by approximately 500 times. In some embodiments, nucleotide substitutions to any of the reference TREMs, e.g., SEQ ID NOs: 1638, 1654, 1660, 1835, 1867, 2000, 2001, 2016, 2017, 2018, 2019, 2020, 2021, 2022, or 2023, increase the PTC readthrough of the TREM by more than 500 times. In some embodiments, nucleotide substitutions, e.g., those listed in Figure 24, modulate the activity of the TREM relative to the reference TREM. In some embodiments, the nucleotide substitutions listed in Figure 24 increase the activity of the TREM relative to the reference TREM. In some embodiments, the nucleotide substitutions listed in Figure 24 decrease the activity of the TREM relative to the reference TREM.

[0211] In one embodiment, the TREM having a nucleotide substitution is one of the TREMs listed in Figure 24, for example, SEQ ID NOs: 1457-2001. In one embodiment, the nucleotide substitution is one of the nucleotide substitutions listed in Figure 24. In one embodiment, the nucleotide substitution increases the immature stop codon (PTC) readthrough activity of the TREM, for example, as shown in Figure 24. In one embodiment, the PTC read-through activity-increasing nucleotide substitutions of TREM are SEQ ID NOs: 1469, 1473, 1480, 1639, 1640, 1641, 1642, 1645, 1646, 1649, 1651, 1652, 1654, 1655, 1658, 1659, 1660, 1661, 1662, 1665, 1666, 1668, 1669, 1671, 1672, 1673, 1677, 1679, 1680, 1682, 1685, 1686, 1687, 1688, 1689, 1694, 1695, 1696, 1697, 1699, This is a nucleotide substitution that is included in any of the following: 1704, 1705, 1707, 1709, 1711, 1725, 1726, 1727, 1729, 1730, 1731, 1732, 1736, 1738, 1739, 1740, 1746, 1750, 1751, 1752, 1758, 1764, 1767, 1768, 1769, 1770, 1775, 1777, 1780, 1783, 1792, 1793, 1795, 1797, 1799, 1803, 1808, 1814, 1818, 1820, 1823, 1824, 1830, 1832, or 1833.

[0212] In one embodiment, a TREM containing an Arg-TGA scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1638, contains a nucleotide substitution at any one of the positions shown in Figure 6. In one embodiment, a TREM containing an Arg-TGA scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1638, contains a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, or 7 in the [ASt domain 1], for example, as shown in Figure 6. In one embodiment, a TREM containing an Arg-TGA scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1638, contains a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 6. In one embodiment, a TREM containing an Arg-TGA scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1638, includes a nucleotide substitution at any of nucleotide positions 65, 66, 67, 68, 69, 70, 71, 72, or 73 in the [ASt domain 2], for example, as shown in Figure 6. In one embodiment, the activity of a TREM containing an Arg-TGA scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1638, which includes a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, or 7, is increased by, for example, about 1.5 times, about 2 times, about 3 times, or about 3.6 times compared to the reference sequence, for example, as shown in Figure 6. In one embodiment, the activity of a TREM containing an Arg-TGA scaffold with a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1638, is reduced by, for example, about 2 times compared to, for example, the reference sequence, as shown in Figure 6. In another embodiment, the activity of a TREM containing an Arg-TGA scaffold with a nucleotide substitution at any of nucleotide positions 65, 66, 67, 68, 69, 70, 71, 72, or 73, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1638, is increased by, for example, about 1.5 times, about 2 times, or about 3.6 times compared to, for example, the reference sequence, as shown in Figure 6.

[0213] In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1835, contains a nucleotide substitution at any one of the positions shown in Figure 7. In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1835, has a nucleotide substitution at any of nucleotide positions 26, 27, 28, 29, 30, or 31 in the [ACH domain], for example, as shown in Figure 7. In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1835, has a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 7. In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1835, includes a nucleotide substitution at any of nucleotide positions 39, 40, 41, 42, 43, or 44, for example, within the [ACH domain], as shown in Figure 7. In one embodiment, the activity of a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1835, which includes a nucleotide substitution at any of nucleotide positions 26, 27, 28, 29, 30, or 31, is increased compared to the reference sequence, for example, as shown in Figure 7, for example, by about 1.5 times, about 5 times, about 6 times, about 7 times, or about 8 times. In one embodiment, the activity of a TREM containing a Gln-TAG scaffold with a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1835, is decreased compared to the reference sequence, for example, as shown in Figure 7, for example, for a decrease of, for example, about twofold. In another embodiment, the activity of a TREM containing a Gln-TAG scaffold with a nucleotide substitution at any of nucleotide positions 39, 40, 41, 42, 43, or 44, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1835, is increased compared to the reference sequence, for example, as shown in Figure 7, for example, for an increase of, for example, about twofold, about fivefold, about sixfold, about sevenfold, or about eightfold.

[0214] In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, includes a nucleotide substitution at any one of the positions shown in Figure 8. In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, has a nucleotide substitution at any of nucleotide positions 3, 4, 5, 6, or 7 in, for example, the [ASt domain 1], as shown in Figure 8. In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, includes a creotide substitution at nucleotide position 20a in, for example, the [DH domain], as shown in Figure 8. In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, has a nucleotide substitution at any of nucleotide positions 26, 27, 28, 29, or 30 in the [ACH domain], for example, as shown in Figure 8. In another embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, includes a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 8. In yet another embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, includes a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, 44, or 45 in the [ACH domain], for example, as shown in Figure 8. In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, includes a nucleotide substitution at nucleotide position 49, for example, within the [TH domain], as shown in Figure 8.In one embodiment, a TREM containing a Gln-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, includes a nucleotide substitution at any of nucleotide positions 65, 66, 67, 68, 69, 70, 71, 72, or 73, for example, within the [ASt domain 2], as shown in Figure 8. In one embodiment, the activity of a TREM containing a Gln-TAG scaffold having a nucleotide substitution at any of nucleotide positions 3, 4, 5, 6, or 7, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, is increased compared to the reference sequence, for example, as shown in Figure 8, for example, by about 2.5 times, about 3 times, or about 3.5 times. In one embodiment, the activity of a TREM containing a Gln-TAG scaffold having a nucleotide substitution at nucleotide position 20a, for example, the activity of a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, is increased compared to the reference sequence, for example, as shown in Figure 8, for example, by about 2.5 times or about 3 times. In another embodiment, the activity of a TREM containing a Gln-TAG scaffold having a nucleotide substitution at any of nucleotide positions 26, 27, 28, 29, or 30, for example, the activity of a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, is increased compared to the reference sequence, for example, as shown in Figure 8, for example, by about 1.5 times, about 2 times, or about 2.5 times. In one embodiment, the activity of a TREM containing a Gln-TAG scaffold having a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, the activity of a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, is decreased compared to the reference sequence, for example, as shown in Figure 8, for example, for example, for a decrease of, for example, about 2 times. In another embodiment, the activity of a TREM containing a Gln-TAG scaffold having a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, 44, or 45, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, is increased compared to the reference sequence, for example, as shown in Figure 8, for example, for an increase of, for example, about 1.5 times, about 2 times, or about 2.5 times.In one embodiment, the activity of a TREM containing a Gln-TAG scaffold having a substitution at nucleotide position 49, for example, the activity of a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, is increased compared to the reference sequence, for example, as shown in Figure 8, for example, by about 2 times. In another embodiment, the activity of a TREM containing a Gln-TAG scaffold having a nucleotide substitution at any of nucleotide positions 65, 66, 67, 68, 69, 70, 71, 72, or 73, for example, the activity of a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1660 or 1654, is increased compared to the reference sequence, for example, as shown in Figure 8, for example, by about 1.5 times, about 2 times, about 2.5 times, about 3 times, or about 3.5 times.

[0215] In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, e.g., SEQ ID NO: 1867, 2000, or 2001, contains a nucleotide substitution at any one of the positions shown in Figure 9. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs: 1867, 2000, or 2001, includes a nucleotide substitution at any of nucleotide positions 20a, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 in the [DH domain] or [ACH domain], for example, as shown in Figure 9. In another embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs: 1867, 2000, or 2001, includes a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 9. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs: 1867, 2000, or 2001, includes a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, 44, or 45 in the [ACH domain], for example, as shown in Figure 9. In another embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs: 1867, 2000, or 2001, includes a nucleotide substitution at any of nucleotide positions 46 or 48 in the [VL domain], for example, as shown in Figure 9.In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs. 1867, 2000, or 2001, includes a nucleotide substitution at either nucleotide position 59 or 60, for example, in the [TH domain], as shown in Figure 9. In another embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs. 1867, 2000, or 2001, includes a nucleotide substitution at either nucleotide position 66, 67, 68, 69, 70, 71, 72, or 73, for example, in the [ASt domain 2], as shown in Figure 9. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs. 1867, 2000, or 2001, is increased compared to the reference sequence, for example, as shown in Figure 9, for example, by about 1.5 times, about 2 times, or about 2.5 times. In another embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 20a, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs. 1867, 2000, or 2001, is increased compared to the reference sequence, for example, as shown in Figure 9, for example, by about 1.5 times, about 2 times, about 2.5 times, or about 3 times. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NOs. 1867, 2000, or 2001, is reduced, for example, by about twofold, compared to the reference sequence, as shown in Figure 9.In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, 44, or 45, for example, the activity of a TREM corresponding to a reference sequence, for example, SEQ ID NOs. 1867, 2000, or 2001, is increased compared to the reference sequence, for example, as shown in Figure 9, for example, by about 1.5 times or about 2 times. In another embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 46 or 48, for example, the activity of a TREM corresponding to a reference sequence, for example, SEQ ID NOs. 1867, 2000, or 2001, is increased compared to the reference sequence, for example, as shown in Figure 9, for example, by about 1.5 times or about 2 times. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at either nucleotide position 59 or 60, for example, the activity of a TREM corresponding to a reference sequence, e.g., SEQ ID NOs. 1867, 2000, or 2001, is increased compared to the reference sequence, e.g., by about 1.5 times, as shown in Figure 9. In another embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at either nucleotide position 66, 67, 68, 69, 70, 71, 72, or 73, for example, the activity of a TREM corresponding to a reference sequence, e.g., SEQ ID NOs. 1867, 2000, or 2001, is increased compared to the reference sequence, e.g., by about 1.5 times, about 2 times, or about 2.5 times, as shown in Figure 9.

[0216] In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at any one of the positions shown in Figure 10. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 in, for example, the [ASt domain 1] or [DH domain], as shown in Figure 10. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at any of nucleotide positions 20a, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 in, for example, the [DH domain] or [ACH domain], as shown in Figure 10. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 10. In another embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, 44, or 45 in the [ACH domain], for example, as shown in Figure 10. In yet another embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at any of nucleotide positions 46, 48, or 49 in the [VL domain], for example, as shown in Figure 10. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at either nucleotide position 59 or 60, for example, within the [TH domain], as shown in Figure 10.In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, includes a nucleotide substitution at any of nucleotide positions 66, 67, 68, 69, 70, 71, 72, or 73, for example, in the [ASt domain 2], as shown in Figure 10. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, is increased compared to the reference sequence, for example, as shown in Figure 10, for example, by about 1.5 times, about 2 times, about 2.5 times, or about 3 times. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 20a, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, is increased compared to the reference sequence, for example, as shown in Figure 10, for example, by about 1.5 times, about 2 times, about 2.5 times, or about 3 times. In another embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, is decreased compared to the reference sequence, for example, as shown in Figure 10, for example, by about 2 times. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, 44, or 45, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, is increased compared to the reference sequence, for example, as shown in Figure 10, for example, by about 1.5 times or about 2 times. In another embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 46 or 48, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, is increased compared to the reference sequence, for example, as shown in Figure 10, for example, by about 1.5 times or about 2 times.In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at either nucleotide position 59 or 60, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, is increased compared to the reference sequence, for example, as shown in Figure 10, for example, by about 1.5 times. In another embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 66, 67, 68, 69, 70, 71, 72, or 73, for example, the TREM corresponding to a reference sequence, for example, SEQ ID NO: 1867, is increased compared to the reference sequence, for example, as shown in Figure 10, for example, by about 1.5 times or about 2 times.

[0217] In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2000, includes a nucleotide substitution at one of the positions shown in Figure 11. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2000, includes a nucleotide substitution at either nucleotide position 31 or 32, for example, within the [ACH domain], as shown in Figure 11. In one embodiment, a TREM containing a Glu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2000, includes a nucleotide substitution at either nucleotide position 32, 33, 34, 35, 36, 37, or 38, for example, within the [ACH domain], as shown in Figure 11. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at either nucleotide position 31 or 32, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2000, is increased compared to the reference sequence, for example, as shown in Figure 11, for example, by about 5 times or about 11 times. In one embodiment, the activity of a TREM containing a Glu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2000, is reduced, for example, by about twofold, compared to the reference sequence, as shown in Figure 11, for example.

[0218] In one embodiment, a TREM containing a Leu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, sequence number 2016 or 2017, includes a nucleotide substitution at any one of the positions shown in Figure 12. In one embodiment, a TREM containing a Leu-TAG scaffold, for example, a TREM corresponding to a reference sequence (e.g., SEQ ID NO: 2016 or 2017), includes a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 12. In one embodiment, a TREM containing a Leu-TAG scaffold, for example, a TREM corresponding to a reference sequence (e.g., SEQ ID NO: 2016 or 2017), includes a nucleotide substitution at any of nucleotide positions 39, 40, 41, 42, 43, 44, 45, V11, V12, V13, V14, V15, V16, V17, V1, V2, V3, V4, V5, V27, V2 The nucleotide position is included at any of 6, V25, V24, V23, V22, V21, 46, 47, 48, 49, or 50. In one embodiment, a TREM containing a Leu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2016 or 2017, includes a nucleotide substitution at nucleotide position 59, for example, in the [TH domain], as shown in Figure 12. In one embodiment, a TREM containing a Leu-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2016 or 2017, includes a nucleotide substitution at any of nucleotide positions 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73, for example, in the [TH domain] or [ASt domain 2], as shown in Figure 12.In one embodiment, the activity of a TREM containing a Leu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2016 or 2017, is increased compared to the reference sequence, for example, as shown in Figure 12, for example, by about 1.5 times or about 2 times. In another embodiment, the activity of a TREM containing a Leu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2016 or 2017, is decreased compared to the reference sequence, for example, as shown in Figure 12, for example, by about 2 times compared to the reference sequence. In one embodiment, the activity of a TREM containing a Leu-TAG scaffold having a nucleotide position at any of nucleotide positions 39, 40, 41, 42, 43, 44, 45, V11, V12, V13, V14, V15, V16, V17, V1, V2, V3, V4, V5, V27, V26, V25, V24, V23, V22, V21, 46, 47, 48, 49, or 50, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2016 or 2017, is increased compared to the reference sequence, for example, as shown in Figure 12, for example, by about 1.5 times or about 2 times. In one embodiment, the activity of a TREM containing a Leu-TAG scaffold having a substitution at nucleotide position 59, for example, the activity of a TREM corresponding to a reference sequence, for example, sequence number 2016 or 2017, is increased compared to the reference sequence, for example, as shown in Figure 12, for example, by about 1.5 times or about 2 times. In another embodiment, the activity of a TREM containing a Leu-TAG scaffold having a nucleotide substitution at any of nucleotide positions 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73, for example, the activity of a TREM corresponding to a reference sequence, for example, sequence number 2016 or 2017, is increased compared to the reference sequence, for example, as shown in Figure 12, for example, by about 1.5 times or about 2 times.

[0219] In one embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, includes a nucleotide substitution at any one of the positions shown in Figure 13. In one embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, includes a nucleotide substitution at any of nucleotide positions 4, 5, 6, or 7 in, for example, the [ASt domain 1], as shown in Figure 13. In one embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, includes a nucleotide substitution at any of nucleotide positions 16, 17, or 20 in, for example, the [DH domain], as shown in Figure 13. In one embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, includes a nucleotide substitution at any of nucleotide positions 28, 29, or 30 in the [ACH domain], for example, as shown in Figure 13. In another embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, has a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 13. In yet another embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, includes a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, or 44 in the [ACH domain], for example, as shown in Figure 13. In one embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, includes a nucleotide substitution at any of nucleotide positions 47, 48, 49, 50, or 51, for example, in the [VL domain] or [TH domain], as shown in Figure 13.In one embodiment, a TREM containing a Tyr-TAG scaffold, for example, a TREM corresponding to reference sequence 2022 or 2023, includes a nucleotide substitution at any of nucleotide positions 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 in, for example, the [TH domain] or [ASt domain 2], as shown in Figure 13. In one embodiment, the activity of a TREM containing a Tyr-TAG scaffold having a nucleotide substitution at any of nucleotide positions 4, 5, 6, or 7, for example, a TREM corresponding to a reference sequence, for example, sequence number 2022 or 2023, is increased compared to, for example, the reference sequence, for example, as shown in Figure 13, for example, by about 1.5 times, about 2 times, or about 2.5 times. In one embodiment, the activity of a TREM containing a Tyr-TAG scaffold having a nucleotide substitution at any of nucleotide positions 16, 17, or 20, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, is increased compared to the reference sequence, for example, as shown in Figure 13, for example, by about 1.5 times or about 2 times. In another embodiment, the activity of a TREM containing a Tyr-TAG scaffold having a nucleotide substitution at any of nucleotide positions 28, 29, or 30, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, is increased compared to the reference sequence, for example, as shown in Figure 13, for example, by about 1.5 times, about 2 times, or about 2.5 times. In one embodiment, the activity of a TREM containing a Tyr-TAG scaffold having a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, is decreased compared to the reference sequence, for example, as shown in Figure 13, for example, for example, for about 2 times. In another embodiment, the activity of a TREM containing a Tyr-TAG scaffold having a nucleotide substitution at any of nucleotide positions 40, 41, 42, 43, or 44, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, is increased compared to the reference sequence, for example, as shown in Figure 13, for example, for about 1.5 times, about 2 times, or about 2.5 times.In one embodiment, the activity of a TREM containing a Tyr-TAG scaffold having a nucleotide substitution at any of nucleotide positions 47, 48, 49, 50, or 51, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, is increased compared to the reference sequence, for example, as shown in Figure 13, for example, by about 1.5 times, about 2 times, or about 2.5 times. In one embodiment, the activity of a TREM containing a Tyr-TAG scaffold having a nucleotide substitution at any of nucleotide positions 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2022 or 2023, is increased compared to the reference sequence, for example, as shown in Figure 13, for example, by about 1.5 times, about 2 times, or about 2.5 times.

[0220] In one embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a creotide substitution at any one of the positions shown in Figure 14. In one embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a nucleotide substitution at any of nucleotide positions 3, 4, 5, or 6 in, for example, the [ASt domain 1], as shown in Figure 14. In one embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a nucleotide substitution at any of nucleotide positions 29, 30, 31, 32, or 33 in, for example, the [ACH domain], as shown in Figure 14. In one embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a nucleotide substitution at any of nucleotide positions 34, 35, or 36 in the [ACH domain], for example, as shown in Figure 14. In another embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a nucleotide substitution at any of nucleotide positions 37, 38, 39, 40, or 41 in the [ACH domain], for example, as shown in Figure 14. In yet another embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a nucleotide substitution at nucleotide position 51 in the [TH domain], for example, as shown in Figure 14. In one embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a nucleotide substitution at nucleotide position 63, for example, in the [TH domain], as shown in Figure 14. In another embodiment, a TREM containing a Ser-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, includes a nucleotide substitution at nucleotide position 67, for example, in the [ASt domain 2], as shown in Figure 14.In one embodiment, the activity of a TREM containing a Ser-TAG scaffold having a nucleotide substitution at any of nucleotide positions 3, 4, 5, or 6, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, is increased compared to the reference sequence, for example, as shown in Figure 14, for example, by about 2 or 3 times. In another embodiment, the activity of a TREM containing a Ser-TAG scaffold having a nucleotide substitution at any of nucleotide positions 29, 30, 31, 32, or 33, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, is increased compared to the reference sequence, for example, as shown in Figure 14, for example, by about 1.5 or 2 times. In yet another embodiment, the activity of a TREM containing a Ser-TAG scaffold having a nucleotide substitution at any of nucleotide positions 34, 35, or 36, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, is decreased compared to the reference sequence, for example, by about 2 times, as shown in Figure 14. In one embodiment, the activity of a TREM containing a Ser-TAG scaffold having a nucleotide substitution at any of nucleotide positions 37, 38, 39, 40, or 41, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, is increased compared to the reference sequence, for example, as shown in Figure 14, for example, by about 1.5 times, about 2 times, about 2.5 times, or about 3 times. In one embodiment, the activity of a TREM containing a Ser-TAG scaffold having a nucleotide substitution at nucleotide position 51, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, is increased compared to the reference sequence, for example, as shown in Figure 14, for example, by about 4 times. In one embodiment, the activity of a TREM containing a Ser-TAG scaffold having a nucleotide substitution at nucleotide position 63, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, is increased compared to the reference sequence, for example, as shown in Figure 14, for example, by about 4 times.In one embodiment, the activity of a TREM containing a Ser-TAG scaffold having a nucleotide substitution at nucleotide position 67, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2020 or 2021, is increased, for example, by about three times, compared to the reference sequence, as shown in Figure 14.

[0221] In one embodiment, a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, includes a nucleotide substitution at any one of the positions shown in Figure 15. In one embodiment, a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, includes a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, or 7 in, for example, the [ASt domain 1], as shown in Figure 15. In one embodiment, a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, includes a nucleotide substitution at any of nucleotide positions 17 or 18 in, for example, the [DH domain], as shown in Figure 15. In one embodiment, a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, sequence number 2018 or 2019, includes a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38 in the [ACH domain], for example, as shown in Figure 15. In one embodiment, a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, sequence number 2018 or 2019, includes a nucleotide substitution at nucleotide position 37 in the [ACH domain], for example, as shown in Figure 15. In one embodiment, a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, sequence number 2018 or 2019, includes a nucleotide substitution at nucleotide position 50 in the [TH domain], for example, as shown in Figure 15. In one embodiment, a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, includes a nucleotide substitution at any of the nucleotide positions 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73 within, for example, the [DH domain] or [ASt domain 2], as shown in Figure 15.In one embodiment, the activity of a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, having a nucleotide substitution at any of nucleotide positions 1, 2, 3, 4, 5, 6, or 7, is increased compared to the reference sequence, for example, as shown in Figure 15, for example, by about 1.5 times, about 2 times, or about 4 times. In another embodiment, the activity of a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, having a nucleotide substitution at either nucleotide position 17 or 18, is increased compared to the reference sequence, for example, as shown in Figure 15, for example, by about 2 times or about 3 times. In one embodiment, the activity of a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, having a nucleotide substitution at any of nucleotide positions 32, 33, 34, 35, 36, 37, or 38, increases compared to the reference sequence, for example, as shown in Figure 15, and decreases by, for example, about twofold. In another embodiment, the activity of a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, having a nucleotide substitution at nucleotide position 37, increases compared to the reference sequence, for example, as shown in Figure 15, and increases by, for example, about threefold. In yet another embodiment, the activity of a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, for example, SEQ ID NO: 2018 or 2019, having a nucleotide substitution at nucleotide position 50, increases compared to the reference sequence, for example, as shown in Figure 15, and increases by, for example, about threefold. In one embodiment, the activity of a TREM containing a Lys-TAG scaffold, for example, a TREM corresponding to a reference sequence, e.g., SEQ ID NO. 2018 or 2019, having a nucleotide substitution at any of nucleotide positions 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73, is increased compared to the reference sequence, for example, as shown in Figure 15, by, for example, about 1.5 times, about 2 times, about 2.5 times, about 3.5 times, or about 4 times.

[0222] non-natural modification The TREMs, TREM core fragments, or TREM fragments described herein may include unnatural modifications, such as those listed in Table 5. Unnatural modifications can be prepared according to methods known in the art. In some embodiments, the unnatural modification is a modification that a cell, e.g., a human cell, does not prepare on endogenous tRNA. In some embodiments, the unnatural modification is a modification that a cell, e.g., a human cell, may prepare on endogenous tRNA, but such a modification is located at a position where it does not occur on natural tRNA. In some embodiments, the unnatural modification is located in a domain, linker, or arm where such a modification does not occur naturally. In some embodiments, the unnatural modification is located at a position within a domain, linker, or arm where such a modification does not occur naturally. In some embodiments, the unnatural modification is located on a nucleotide where such a modification does not occur naturally. In some embodiments, the unnatural modification is located on a nucleotide at a position within a domain, linker, or arm where such a modification does not occur naturally.

[0223] In some embodiments, the TREMs, TREM core fragments, or TREM fragments described herein include the modifications shown in Table 5, or combinations thereof. The modifications shown in Table 5 are either not naturally present in RNA or are naturally present, and are used herein for synthetic TREMs, TREM core fragments, or TREM fragments at positions not found in nature.

[0224] [Table 5-1]

[0225] [Table 5-2]

[0226] [Table 5-3]

[0227] Table 5-4

[0228] Table 5-5

[0229] Table 5-6

[0230] Table 5-7

[0231] Table 5-8

[0232] Table 5-9

[0233] Table 5-10

[0234] Table 5-11

[0235] Table 5-12

[0236] Table 5-13

[0237] Table 5-14

[0238] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [DH domain] and [ACH domain]. In some embodiments, TREM contains 21 unnatural modifications. In some embodiments, TREM contains 15 2'-O-methyl modifications. In some embodiments, TREM contains one 2'-fluoro modification. In some embodiments, TREM contains five phosphorothioate modifications. In some embodiments, TREM includes 15 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], one 2'-fluoro modification in [ACH domain], and five phosphorothioate modifications in [DH domain] and [ACH domain]. In some embodiments, TREM includes the unnatural modification pattern of pattern number 1 shown in Table 6: 1-m, 14-m, 15-m, 16-m, 17-m, 18-m, 19-*, 20-m, 21-*, 33-f, 34-m, 35-*, 37-*, 38-*, 41-m, 42-m, 43-m, 44-m, 50-m, 52-m, and 73-m.

[0239] TREM may contain unnatural modifications in each of [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications and internucleotide modifications in each of [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM contains six unnatural modifications. In some embodiments, TREM contains three 2'-O-methyl modifications. In some embodiments, TREM contains three phosphorothioate modifications. In some embodiments, TREM contains three 2'-O-methyl modifications in [ASt domain 1] and [AST domain 2], and three phosphorothioate modifications in [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM contains the unnatural modification pattern of pattern number 2 shown in Table 6: 1-m*, 2-m*, 74-*, 75-m.

[0240] TREM may contain unnatural modifications in each of the [ASt domain 1], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ASt domain 1], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains seven unnatural modifications. In some embodiments, TREM contains three 2'-O-methyl modifications. In some embodiments, TREM contains four phosphorothioate modifications. In some embodiments, TREM contains three 2'-O-methyl modifications in the [ASt domain 1] and [ASt domain 2], and four phosphorothioate modifications in the [ASt domain 1], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains the unnatural modification pattern of pattern number 3 shown in Table 6: 1-m*, 2-m*, 52-*, 74-*, 75-m.

[0241] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of its [DH domain], [ACH domain], and [TH domain]. In some embodiments, TREM contains 31 unnatural modifications. In some embodiments, TREM contains 23 2'-O-methyl modifications. In some embodiments, TREM contains 8 phosphorothioate modifications. In some embodiments, TREM includes 23 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], and 8 phosphorothioate modifications in [DH domain], [ACH domain], and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 4 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 13-m, 14-m, 15-m, 16-m, 17-m, 18-m, 19-*, 20-m, 21-*, 25-m, 33-*, 34-m, 35-*, 37-*, 38-*, 49-m, 50-m, 51-m, 52-m, 53-m, 54-*, 55-*, 61-m, 65-m, and 73-m.

[0242] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of its [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM contains 10 unnatural modifications. In some embodiments, TREM contains 4 2'-O-methyl modifications. In some embodiments, TREM contains 3 2'-fluoro modifications. In some embodiments, TREM contains 3 phosphorothioate modifications. In some embodiments, TREM contains 4 2'-O-methyl modifications in [ASt domain 1], [TH domain], and [ASt domain 2], 3 2'-fluoro modifications in [DH domain] and [TH domain], and 3 phosphorothioate modifications in [ASt domain 1] and [ASt domain 2]. In some embodiments, TREM includes non-natural modification patterns of pattern number 5 shown in Table 6: 1-m*, 2-m*, 13-f, 14-f, 54-f, 59-m, 75-*, and 76-m.

[0243] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ACH domain] and [TH domain]. In some embodiments, TREM contains 26 unnatural modifications. In some embodiments, TREM contains 16 2'-O-methyl modifications. In some embodiments, TREM contains 2 2'-fluoro modifications. In some embodiments, TREM contains 8 phosphorothioate modifications. In some embodiments, TREM includes 16 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 2 2'-fluoro modifications in [ACH domain] and [VL domain], and 8 phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the unnatural modification pattern of pattern number 6 shown in Table 6: 1-m, 13-m, 17-m, 18-m, 25-m, 33-*, 34-m, 35-*, 37-*, 38-*, 41-f, 42-m, 43-m, 44-f, 49-m, 50-m, 52-m, 54-*, 55-*, 56-m, 57-*, 58-*, 59-m, 61-m, 65-m, and 73-m.

[0244] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of its [DH domain] and [TH domain]. In some embodiments, TREM contains 25 unnatural modifications. In some embodiments, TREM contains 19 2'-O-methyl modifications. In some embodiments, TREM contains two 2'-fluoro modifications. In some embodiments, TREM contains four phosphorothioate modifications. In some embodiments, TREM includes 19 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], two 2'-fluoro modifications in [ACH domain] and [VL domain], and four phosphorothioate modifications in [DH domain] and [TH domain]. In some embodiments, TREM includes the unnatural modification pattern of pattern number 7 shown in Table 6: 1-m, 13-m, 14-m, 15-m, 16-m, 17-m, 18-m, 19-*, 20-m, 21-*, 25-m, 41-f, 42-m, 43-m, 44-f, 49-m, 50-m, 51-m, 52-m, 53-m, 54-*, 55-*, 61-m, 65-m, and 73-m.

[0245] TREM may include unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], and [ASt domain 2]. In some embodiments, TREM includes nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], and [ASt domain 2]. In some embodiments, TREM includes 10 unnatural modifications. In some embodiments, TREM includes 8 2'-O-methyl modifications. In some embodiments, TREM includes 2 2'-fluoro modifications. In some embodiments, TREM includes 8 2'-O-methyl modifications in the [ASt domain 1], [DH domain], [ACH domain], and [ASt domain 2], and 2 2'-fluoro modifications in the [DH domain] and [ACH domain]. In some embodiments, TREM includes non-natural modification patterns of pattern number 8 shown in Table 6: 1-m, 19-m, 20-m, 21-f, 22-m, 40-f, 41-m, 42-m, 43-m, and 73-m.

[0246] TREM may include unnatural modifications in each of [ASt domain 1], [ACH domain], and [ASt domain 2]. In some embodiments, TREM includes nucleotide sugar modifications in each of [ASt domain 1], [ACH domain], and [ASt domain 2]. In some embodiments, TREM includes internucleotide modifications in [ASt domain 1]. In some embodiments, TREM includes 16 unnatural modifications. In some embodiments, TREM includes 13 2'-O-methyl modifications. In some embodiments, TREM includes two 2'-fluoro modifications. In some embodiments, TREM includes one phosphorothioate modification. In some embodiments, TREM includes 13 2'-O-methyl modifications in [ASt domain 1], [ACH domain], and [ASt domain 2], two 2'-fluoro modifications in [ACH domain], and one phosphorothioate modification in [ASt domain 1]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 9 shown in Table 6: 1-m*, 2-m, 3-m, 4-m, 5-m, 6-m, 27-m, 28-m, 29-f, 30-m, 40-f, 41-m, 42-m, 43-m, and 73-m.

[0247] TREM may include unnatural modifications in each of the [ASt domain 1], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM includes nucleotide sugar modifications in each of the [ASt domain 1], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM includes internucleotide modifications in [ASt domain 1]. In some embodiments, TREM includes 23 unnatural modifications. In some embodiments, TREM includes 21 2'-O-methyl modifications. In some embodiments, TREM includes one 2'-fluoro modification. In some embodiments, TREM includes one phosphorothioate modification. In some embodiments, TREM includes 21 2'-O-methyl modifications in [ASt domain 1], [VL domain], [TH domain], and [ASt domain 2], one 2'-fluoro modification in the [TH domain], and one phosphorothioate modification in [ASt domain 1]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 10 shown in Table 6: 1-m*, 2-m, 3-m, 4-m, 5-m, 6-m, 49-m, 50-m, 51-m, 52-m, 53-m, 61-m, 62-m, 63-f, 64-m, 65-m, 66-m, 67-m, 68-m, 69-m, 71-m, and 72-m.

[0248] TREM may include unnatural modifications in each of its [ASt domain 1], [DH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM includes nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM includes internucleotide modifications in [ASt domain 1]. In some embodiments, TREM includes 31 unnatural modifications. In some embodiments, TREM includes 28 2'-O-methyl modifications. In some embodiments, TREM includes one 2'-fluoro modification. In some embodiments, TREM includes two phosphorothioate modifications. In some embodiments, TREM includes 28 2'-O-methyl modifications in [ASt domain 1], [DH domain], [VL domain], [TH domain], and [ASt domain 2], one 2'-fluoro modification in [TH domain], and two phosphorothioate modifications in [ASt domain 1]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 11 shown in Table 6: 1-m*, 2-m*, 3-m, 4-m, 5-m, 6-m, 10-m, 11-m, 12-m, 13-m, 22-m, 23-m, 24-m, 25-m, 49-m, 50-m, 51-m, 52-m, 53-m, 54-m, 61-m, 62-m, 63-f, 64-m, 65-m, 66-m, 67-m, 68-m, and 73-m.

[0249] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of its [ASt domain 1] and [ACH domain]. In some embodiments, TREM contains 32 unnatural modifications. In some embodiments, TREM contains 23 2'-O-methyl modifications. In some embodiments, TREM contains 4 2'-fluoro modifications. In some embodiments, TREM contains 5 phosphorothioate modifications. In some embodiments, TREM includes 23 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 4 2'-fluoro modifications in [ACH domain] and [VL domain], and 5 phosphorothioate modifications in [ASt domain 1] and [ACH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 12 shown in Table 6: 1-m*, 2-m*, 3-m, 4-m, 5-m, 6-m, 16-m, 17-m, 18-m, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-m, 44-m, 45-m, 46-f, 47-f, 48-f, 49-m, 50-m, 51-m, 52-m, 53-m, 54-m, 56-m, 59-m, and 73-m.

[0250] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in the [ACH domain]. In some embodiments, TREM contains 29 unnatural modifications. In some embodiments, TREM contains 21 2'-O-methyl modifications. In some embodiments, TREM contains 5 2'-fluoro modifications. In some embodiments, TREM contains 3 phosphorothioate modifications. In some embodiments, TREM includes 21 2'-O-methyl modifications in [ASt domain 1], [DH domain], [VL domain], [TH domain], and [ASt domain 2], 5 2'-fluoro modifications in [ACH domain], [VL domain], and [TH domain], and 3 phosphorothioate modifications in [ACH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 13 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 16-m, 17-m, 18-m, 33-f, 35-*, 37-*, 38-*, 44-m, 45-m, 46-f, 47-f, 48-f, 56-m, 59-m, 61-m, 62-m, 63-f, 64-m, 65-m, 66-m, 67-m, 68-m, and 73-m.

[0251] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of its [ASt domain 1] and [ACH domain]. In some embodiments, TREM contains 43 unnatural modifications. In some embodiments, TREM contains 31 2'-O-methyl modifications. In some embodiments, TREM contains 7 2'-fluoro modifications. In some embodiments, TREM contains 5 phosphorothioate modifications. In some embodiments, TREM includes 43 2'-O-methyl modifications in [ASt domain 1], [DH domain], [VL domain], [TH domain], and [ASt domain 2], 7 2'-fluoro modifications in [DH domain], [ACH domain], [VL domain], and [TH domain], and 5 phosphorothioate modifications in [ASt domain 1] and [ACH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 14 shown in Table 6: 1-m*, 2-m*, 3-m, 4-m, 5-m, 6-m, 10-m, 11-m, 12-m, 13-m, 14-m, 15-f, 16-m, 17-m, 18-m, 19-f, 20-m, 22-m, 23-m, 24-m, 25-m, 33-f, 35-*, 37-*, 38-*, 44-m, 45-m, 46-f, 47-f, 48-f, 56-m, 59-m, 61-m, 62-m, 63-f, 64-m, 65-m, 66-m, 67-m, 68-m, and 73-m.

[0252] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ASt domain 1] and [ACH domain]. In some embodiments, TREM contains 26 unnatural modifications. In some embodiments, TREM contains 17 2'-O-methyl modifications. In some embodiments, TREM contains 4 2'-fluoro modifications. In some embodiments, TREM contains 5 phosphorothioate modifications. In some embodiments, TREM includes 17 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 4 2'-fluoro modifications in [ACH domain] and [VL domain], and 5 phosphorothioate modifications in [ASt domain 1] and [ACH domain]. In some embodiments, TREM includes the unnatural modification pattern number 15 shown in Table 6: 1-m*, 2-m*, 3-m, 4-m, 5-m, 6-m, 16-m, 17-m, 18-m, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-m, 44-m, 45-m, 46-f, 47-f, 48-f, 56-m, 59-m, and 73-m.

[0253] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains six unnatural modifications. In some embodiments, TREM contains six 2'-O-methyl modifications. In some embodiments, TREM contains six 2'-O-methyl modifications in its [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains the unnatural modification pattern number 16 shown in Table 6: 1-m, 17-m, 18-m, 50-m, 52-m, and 73-m.

[0254] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications within the [TH domain]. In some embodiments, TREM contains 15 unnatural modifications. In some embodiments, TREM contains 11 2'-O-methyl modifications. In some embodiments, TREM contains two 2'-fluoro modifications. In some embodiments, TREM contains two phosphorothioate modifications. In some embodiments, TREM contains 11 2'-O-methyl modifications in [ASt domain 1], [DH domain], [TH domain], and [ASt domain 2], two 2'-fluoro modifications in the [DH domain], and two phosphorothioate modifications in the [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 17 shown in Table 6: 1-m, 14-m, 15-f, 16-m, 17-m, 18-m, 19-f, 20-m, 54-m*, 55-*, 56-m, 57-m, 59-m, and 73-m.

[0255] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [DH domain], [ACH domain], [VL domain], and [TH domain] regions. In some embodiments, TREM contains 29 unnatural modifications. In some embodiments, TREM contains 15 2'-O-methyl modifications. In some embodiments, TREM contains 4 2'-fluoro modifications. In some embodiments, TREM contains 10 phosphorothioate modifications. In some embodiments, TREM comprises 15 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [TH domain], and [ASt domain 2], 4 2'-fluoro modifications in [ACH domain], [VL domain], and [TH domain], and 10 phosphorothioate modifications in [DH domain], [ACH domain], [VL domain], and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 18 shown in Table 6: 1-m, 10-m, 13-m, 17-*, 18-m, 19-*, 20-m, 25-m, 29-m, 33-f, 35-*, 37-*, 38-*, 41-f*, 43-m, 44-f, 46-*, 50-m, 52-m, 54-*, 55-*, 56-m, 57-*, 62-m, 63-f, 65-m, 71-m, and 73-m.

[0256] TREM may include unnatural modifications in each of the [ASt domain 1], [TH domain], and [ASt domain 2]. In some embodiments, TREM includes nucleotide sugar modifications in each of the [ASt domain 1], [TH domain], and [ASt domain 2]. In some embodiments, TREM includes 13 unnatural modifications. In some embodiments, TREM includes 12 2'-O-methyl modifications. In some embodiments, TREM includes one 2'-fluoro modification. In some embodiments, TREM includes 12 2'-O-methyl modifications in the [ASt domain 1], [TH domain], and [ASt domain 2], and one 2'-fluoro modification in the [ASt domain 2]. In some embodiments, TREM includes non-natural modification patterns of pattern number 19 shown in Table 6: 1-m, 61-m, 62-m, 64-m, 65-m, 66-m, 67-m, 68-m, 69-m, 70-f, 71-m, 72-m, and 73-m.

[0257] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in the [ACH domain]. In some embodiments, TREM contains 19 unnatural modifications. In some embodiments, TREM contains 13 2'-O-methyl modifications. In some embodiments, TREM contains 3 2'-fluoro modifications. In some embodiments, TREM contains 3 phosphorothioate modifications. In some embodiments, TREM includes 13 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 3 2'-fluoro modifications in [ACH domain], and 3 phosphorothioate modifications in [ACH domain]. In some embodiments, TREM includes the unnatural modification pattern number 20 shown in Table 6: 1-m, 14-m, 15-f, 16-m, 17-m, 18-m, 19-f, 20-m, 33-f, 35-*, 37-*, 38-*, 41-m, 42-m, 43-m, 44-m, 50-m, 52-m, and 73-m.

[0258] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications within the [TH domain]. In some embodiments, TREM contains 28 unnatural modifications. In some embodiments, TREM contains 26 2'-O-methyl modifications. In some embodiments, TREM contains one 2'-fluoro modification. In some embodiments, TREM contains one phosphorothioate modification. In some embodiments, TREM includes [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and 26 2'-O-methyl modifications in [ASt domain 2], one 2'-fluoro modification in [TH domain], and one phosphorothioate modification in [TH domain]. In some embodiments, TREM includes the non-natural modification pattern number 21 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 14-m, 15-m, 16-m, 17-m, 18-m, 20-m, 25-m, 41-m, 42-m, 43-m, 44-m, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 65-m, and 73-m.

[0259] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ACH domain] and [TH domain]. In some embodiments, TREM contains 32 unnatural modifications. In some embodiments, TREM contains 25 2'-O-methyl modifications. In some embodiments, TREM contains 3 2'-fluoro modifications. In some embodiments, TREM contains 4 phosphorothioate modifications. In some embodiments, TREM includes 25 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 3 2'-fluoro modifications in [ACH domain], [VL domain], and [TH domain], and 4 phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 22 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 14-m, 15-m, 16-m, 17-m, 18-m, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-m, 42-m, 43-m, 44-f, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 65-m, and 73-m.

[0260] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ACH domain] and [TH domain]. In some embodiments, TREM contains 27 unnatural modifications. In some embodiments, TREM contains 21 2'-O-methyl modifications. In some embodiments, TREM contains one 2'-fluoro modification. In some embodiments, TREM contains five phosphorothioate modifications. In some embodiments, TREM includes 21 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], one 2'-fluoro modification in [ACH domain], and five phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the unnatural modification pattern of pattern number 23 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 16-m, 17-m, 18-m, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-m, 42-m, 43-m, 44-m, 49-m, 50-m, 52-m, 54-*, 55-*, 61-m, 65-m, and 73-m.

[0261] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ACH domain] and [TH domain]. In some embodiments, TREM contains 34 unnatural modifications. In some embodiments, TREM contains 26 2'-O-methyl modifications. In some embodiments, TREM contains 4 2'-fluoro modifications. In some embodiments, TREM contains 4 phosphorothioate modifications. In some embodiments, TREM includes 26 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 4 phosphorothioate modifications in [ACH domain], [VL domain], and [TH domain], and 4 phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 24 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 10-m, 11-m, 12-m, 13-m, 16-m, 17-m, 18-m, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-f, 42-m, 43-m, 44-f, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 65-m, and 73-m.

[0262] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ACH domain] and [TH domain]. In some embodiments, TREM contains 37 unnatural modifications. In some embodiments, TREM contains 29 2'-O-methyl modifications. In some embodiments, TREM contains 4 2'-fluoro modifications. In some embodiments, TREM contains 4 phosphorothioate modifications. In some embodiments, TREM includes 29 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 4 2'-fluoro modifications in [DH domain], [ACH domain], and [TH domain], and 4 phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 25 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 10-m, 11-m, 12-m, 13-m, 14-m, 15-f, 16-m, 17-m, 18-m, 19-f, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-m, 42-m, 43-m, 44-m, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 65-m, and 73-m.

[0263] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ACH domain] and [TH domain]. In some embodiments, TREM contains 36 unnatural modifications. In some embodiments, TREM contains 28 2'-O-methyl modifications. In some embodiments, TREM contains 4 2'-fluoro modifications. In some embodiments, TREM contains 4 phosphorothioate modifications. In some embodiments, TREM includes 28 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 4 2'-fluoro modifications in [ACH domain], [VL domain], and [TH domain], and 4 phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 26 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 16-m, 17-m, 18-m, 20-m, 22-m, 23-m, 24-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-m, 42-m, 43-m, 44-m, 45-m, 46-f, 48-f, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 65-m, and 73-m.

[0264] TREM may contain unnatural modifications in the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2] regions. In some embodiments, TREM contains nucleotide sugar modifications in each of [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in the [TH domain]. In some embodiments, TREM contains 31 unnatural modifications. In some embodiments, TREM contains 28 2'-O-methyl modifications. In some embodiments, TREM contains two 2'-fluoro modifications. In some embodiments, TREM contains one phosphorothioate modification. In some embodiments, TREM includes [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and 28 2'-O-methyl modifications in [ASt domain 2], two 2'-fluoro modifications in [TH domain], and one phosphorothioate modification in [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 27 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 14-m, 15-m, 16-m, 17-m, 18-m, 20-m, 25-m, 41-m, 42-m, 43-m, 44-m, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 62-m, 63-f, 64-m, 65-m, and 73-m.

[0265] TREM may contain unnatural modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of its [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of its [ACH domain] and [TH domain]. In some embodiments, TREM contains 33 unnatural modifications. In some embodiments, TREM contains 24 2'-O-methyl modifications. In some embodiments, TREM contains 5 2'-fluoro modifications. In some embodiments, TREM contains 4 phosphorothioate modifications. In some embodiments, TREM includes 24 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 5 2'-fluoro modifications in [ACH domain], [VL domain], and [TH domain], and 4 phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 28 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 16-m, 17-m, 18-m, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-f, 42-m, 43-m, 44-f, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 62-m, 63-f, 64-m, 65-m, and 73-m.

[0266] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains internucleotide modifications in each of the [ACH domain] and [TH domain]. In some embodiments, TREM contains 35 unnatural modifications. In some embodiments, TREM contains 26 2'-O-methyl modifications. In some embodiments, TREM contains 5 2'-fluoro modifications. In some embodiments, TREM contains 4 phosphorothioate modifications. In some embodiments, TREM includes 26 2'-O-methyl modifications in [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2], 5 2'-fluoro modifications in [ACH domain], [VL domain], and [TH domain], and 4 phosphorothioate modifications in [ACH domain] and [TH domain]. In some embodiments, TREM includes the non-natural modification patterns of pattern number 29 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 14-m, 15-m, 16-m, 17-m, 18-m, 20-m, 25-m, 33-f, 35-*, 37-*, 38-*, 41-f, 42-m, 43-m, 44-f, 49-m, 50-m, 52-m, 54-m, 55-*, 56-m, 57-f, 59-m, 61-m, 62-m, 63-f, 64-m, 65-m, and 73-m.

[0267] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], and [ASt domain 2]. In some embodiments, TREM contains 16 unnatural modifications. In some embodiments, TREM contains 15 2'-O-methyl modifications. In some embodiments, TREM contains one 2'-fluoro modification. In some embodiments, TREM contains 15 2'-O-methyl modifications in the [ASt domain 1], [DH domain], [ACH domain], and [ASt domain 2], and one 2'-fluoro modification in the [ACH domain]. In some embodiments, TREM includes non-natural modification patterns of pattern number 30 shown in Table 6: 1-m, 2-m, 3-m, 4-m, 5-m, 6-m, 14-m, 17-m, 18-m, 20-m, 22-m, 25-m, 27-m, 33-f, 71-m, and 73-m.

[0268] TREM may include unnatural modifications in each of the [ASt domain 1], [VL domain], and [ASt domain 2]. In some embodiments, TREM includes nucleotide sugar modifications in each of the [ASt domain 1], [VL domain], and [ASt domain 2]. In some embodiments, TREM includes internucleotide modifications in [ASt domain 1]. In some embodiments, TREM includes 14 unnatural modifications. In some embodiments, TREM includes 11 2'-O-methyl modifications. In some embodiments, TREM includes two 2'-fluoro modifications. In some embodiments, TREM includes one phosphorothioate modification. In some embodiments, TREM includes 11 2'-O-methyl modifications in [ASt domain 1], [VL domain], and [ASt domain 2], two 2'-fluoro modifications in the [VL domain], and one phosphorothioate modification in [ASt domain 1]. In some embodiments, TREM includes non-natural modification patterns of pattern number 31 shown in Table 6: 1-m*, 2-m, 3-m, 4-m, 5-m, 6-m, 44-m, 45-m, 46-f, 47-f, 48-m, 49-m, and 73-m.

[0269] TREM may contain unnatural modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain], and [ASt domain 2]. In some embodiments, TREM contains nucleotide sugar modifications in each of the [ASt domain 1], [DH domain], [ACH domain], [VL domain], [TH domain...

Claims

1. The array of formula (I): [L1]x - [AST Domain 1] - [L2]x - [DH Domain] - [L3]x - [ACH Domain] - [VL Domain] - [TH Domain] - [L4]x - [AST Domain 2] - [L5]x (I) A tRNA effector molecule (TREM) containing, During the ceremony: Independently, the [L1] and [VL domains] are optional; x is 0 or 1; A tRNA effector molecule (TRM) comprising a nucleotide substitution (e.g., a nucleotide mutation) within the TREM that can modulate the functional parameters of the TREM.

2. The TREM according to claim 1, wherein the functional parameter includes the activity of the TREM or the stability of the TREM.

3. The TREM according to claim 1 or 2, wherein the functional parameter includes, for example, the immature stop codon (PTC) readthrough activity of the TREM in a sample (e.g., cells) or subject (e.g., an increase in the level of PTC readthrough activity or a decrease in PTC readthrough activity).

4. The TREM according to any one of claims 1 to 3, wherein the functional parameter includes, for example, the stability of the TREM in a sample (e.g., cells) or subject (e.g., increased stability of the TREM or decreased stability of the TREM).

5. The aforementioned functional parameters are as follows: (a) Protein translation; (b) Expression level (e.g., of polypeptides, proteins, or mRNA); (c) Post-translational modification of polypeptides or proteins; (d) Folding (e.g., of polypeptides or proteins, or mRNA); (e) Structure (e.g., of a polypeptide or protein, or of mRNA); (f) Transduction (e.g., of polypeptides or proteins); (g) Computation (e.g., of polypeptides or proteins, or mRNA), (h) Incorporation into supramolecular structures (e.g., polypeptides or proteins or mRNA), e.g., incorporation into membranes, proteasomes or ribosomes, (i) Incorporation into a multimer polypeptide, e.g., a homodimer or heterodimer, and / or (j) Stability A TREM according to any one of claims 1 to 4, selected from the above.

6. The aforementioned functional parameters are further as follows: (1) Regulation of signaling pathways, such as cellular signaling pathways, downstream or upstream of proteins encoded by endogenous ORFs having a first sequence or PTC; (2) Regulation of cell fate; (3) Regulation of ribosome occupation; (4) Regulation of protein translation; (5) mRNA stability regulation; (6) Protein folding and structural regulation; (7) Protein transduction or compartmentalization control; and / or (8) Regulation of protein stability TREM according to claim 5, including the above.

7. The TREM according to any one of claims 1 to 6, wherein the TREM includes a TREM having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the TREM shown in Figure 3 or Figure 23.

8. The TREM according to any one of claims 1 to 7, wherein the TREM includes a nucleotide sequence that differs by only 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23.

9. The TREM according to any one of claims 1 to 8, wherein the TREM includes a nucleotide sequence that differs by only 10, 15, 20, 25, 30, 35, or 40 nucleotides from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23.

10. The TREM according to any one of claims 1 to 9, wherein the TREM includes a nucleotide sequence that is more than 5 nucleotides different from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23.

11. The TREM according to any one of claims 1 to 10, wherein the TREM includes a nucleotide sequence that is more than 10 nucleotides different from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23.

12. The TREM according to any one of claims 1 to 11, wherein the TREM includes a sequence that differs from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

13. The TREM according to any one of claims 1 to 12, wherein the TREM includes a nucleotide sequence that differs from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23 by only 10, 15, 20, 25, 30, 35, or 40 nucleotides.

14. The TREM according to any one of claims 1 to 13, wherein the TREM includes a nucleotide sequence that differs by only one nucleotide from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23.

15. The TREM according to any one of claims 1 to 14, wherein the TREM includes a nucleotide sequence that differs by only 5 nucleotides from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23.

16. The TREM according to any one of claims 1 to 15, wherein the TREM includes a nucleotide sequence that differs by only 10 nucleotides from the nucleotide sequence of the TREM shown in Figure 3 or Figure 23.

17. The TREM according to any one of claims 1 to 16, wherein the TREM includes a nucleotide substitution within the [ASt domain 1], for example, the nucleotide substitution is located at any of positions 1, 2, 3, 4, 5, 6, 7, 8, or 9 within the [ASt domain 1].

18. The TREM according to any one of claims 1 to 17, wherein the TREM includes a nucleotide substitution within the [DH domain], for example, the nucleotide substitution is located at any of positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 within the [DH domain].

19. The TREM according to any one of claims 1 to 18, wherein the TREM includes a nucleotide substitution within the [ACH domain], for example, the nucleotide substitution is located at any of positions 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 within the [ACH domain].

20. The TREM according to any one of claims 1 to 19, wherein the TREM includes a nucleotide substitution within the [VL domain], for example, the nucleotide substitution is located at any of positions 44, 45, 46, 47, 48, or 49 within the [VL domain].

21. The TREM according to any one of claims 1 to 20, wherein the TREM includes a nucleotide substitution within the [TH domain], for example, the nucleotide substitution is located at any of positions 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 within the [TH domain].

22. The TREM according to any one of claims 1 to 21, wherein the TREM includes a nucleotide substitution within the [ASt domain 2], for example, the nucleotide substitution is located at any of positions 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 within the [ASt domain 2].

23. The TREM according to any one of claims 1 to 22, wherein the TREM has an array selected from the arrays shown in Figure 3 or Figure 23.

24. The TREM according to any one of claims 1 to 23, wherein the TREM comprises a plurality of nucleotide substitutions.

25. The TREM according to any one of claims 1 to 24, wherein the nucleotide substitution comprises substituting the first native nucleotide with adenosine, guanosine, cytosine, or uracil nucleotide.

26. The TREM according to any one of claims 1 to 25, wherein the TREM is selected from sequence numbers 625 to 700, 701 to 800, 801 to 900, 901 to 1000, 1001 to 1100, 1101 to 1151, 1290 to 1300, 1301 to 1400, or 1401 to 1456 in Figure 3.

27. The TREM according to any one of claims 1 to 26, wherein the TREM containing nucleotide substitutions does not show any difference in functional parameters compared to the TREM of Figure 3 or Figure 23.

28. The TREM according to any one of claims 1 to 26, wherein the TREM containing nucleotide substitutions exhibits differences in functional parameters compared to the TREM of Figure 3 or Figure 23.

29. The TREM according to any one of claims 1 to 28, wherein the TREM containing nucleotide substitutions shows improvement in functional parameters compared to the TREM of Figure 3 or Figure 23.

30. The TREM according to any one of claims 1 to 29, wherein the improvements include improvements of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, compared to, for example, the functional parameters of the TREM shown in Figure 3 or Figure 23.

31. The TREM according to any one of claims 1 to 30, wherein the TREM containing nucleotide substitutions exhibits a reduction in functional parameters compared to the TREM of Figure 3 or Figure 23.

32. The TREM according to any one of claims 1 to 31, wherein the reduction includes a reduction of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to, for example, the functional parameters of the TREM shown in Figure 3 or Figure 23.

33. The TREM according to any one of claims 1 to 32, wherein the TREM includes, for example, a nucleotide substitution at any of positions 1, 2, 3, 4, 5, 6, or 7 within the [ASt domain 1].

34. The TREM according to any one of claims 1 to 33, wherein the TREM includes, for example, a nucleotide substitution at any of the positions 16, 17, or 20a within the [DH domain].

35. The TREM according to any one of claims 1 to 34, wherein the TREM includes, for example, a nucleotide substitution at any of positions 27, 28, 29, 30, 31, 39, 40, 41, 42, or 43 within the [ACH domain].

36. The TREM according to any one of claims 1 to 35, wherein the TREM includes, for example, a nucleotide substitution at any of positions 51, 59, 60, or 63 within the [TH domain].

37. The TREM according to any one of claims 1 to 36, wherein the TREM includes, for example, a nucleotide substitution at any of positions 66, 67, 68, 69, 70, 71, 72, or 73 within the [ASt domain 2].

38. The TREM according to any one of claims 1 to 37, wherein the TREM includes, for example, a nucleotide substitution at any of positions 32, 33, 34, 35, 36, 37, or 38 within the [ACH domain].

39. For example, the TREM according to any one of claims 1 to 38, wherein the TREM includes a nucleotide substitution at any of positions 1, 2, 3, 4, 5, 6, or 7 within the [ASt domain 1], and exhibits an improvement in the functional parameters of the TREM.

40. For example, the TREM according to any one of claims 1 to 39, wherein the TREM includes a nucleotide substitution at any of positions 16, 17, or 20a within the [DH domain], thereby demonstrating an improvement in the functional parameters of the TREM.

41. For example, the TREM according to any one of claims 1 to 40, wherein the TREM includes a nucleotide substitution at any of positions 27, 28, 29, 30, 31, 39, 40, 41, 42, or 43 within the [ACH domain], which shows an improvement in the functional parameters of the TREM.

42. For example, the TREM according to any one of claims 1 to 41, wherein the TREM includes a nucleotide substitution at any of positions 51, 59, 60, or 63 within the [TH domain], thereby demonstrating an improvement in the functional parameters of the TREM.

43. For example, the TREM according to any one of claims 1 to 42, wherein the TREM includes a nucleotide substitution at any of positions 66, 67, 68, 69, 70, 71, 72, or 73 within the [ASt domain 2], thereby demonstrating an improvement in the functional parameters of the TREM.

44. For example, the TREM according to any one of claims 1 to 43, wherein the TREM having a nucleotide substitution at any of positions 32, 33, 34, 35, 36, 37, or 38 within the [ACH domain] exhibits a decrease in the functional parameters of the TREM.

45. The TREM according to any one of claims 1 to 44, wherein the TREM comprising nucleotide substitutions comprises a TREM corresponding to a reference sequence of a scaffold, for example, Arg-TGA, Gln-TAG, Glu-TAG, Leu-TAG, Tyr-TAG, Ser-TAG, or Lys-TAG scaffold, for example, SEQ ID NOs: 1638, 1835, 1660, 1654, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, or 2019.

46. The TREM according to any one of claims 1 to 45, wherein the TREM containing the nucleotide substitution corresponds to the reference sequence of SEQ ID NO: 1638, 1835, 1660, 1654, 1867, 2000, 2001, 2016, 2017, 2022, 2023, 2020, 2021, 2018, or 2019.

47. The TREM according to any one of claims 1 to 46, wherein the TREM includes non-natural modifications.

48. The TREM according to claim 47, wherein the unnatural modification is located at the 2' position of the nucleotide sugar or within the internucleotide region (e.g., a skeletal modification).

49. The TREM according to claim 47 or 48, wherein the non-natural modification is selected from 2'-O-methyl (2'-OMe), 2'-halo (e.g., 2'-F or 2'-Cl), 2'-O-methoxyethyl (2'-MOE), or 2'-deoxy modification.

50. The TREM according to any one of claims 47 to 49, wherein the non-natural modification is a phosphorothioate modification.

51. The TREM according to any one of claims 47 to 50, wherein the TREM comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional non-natural modifications (e.g., 2'-ribose modifications or internucleotide modifications, e.g., 2'OMe, 2'-halo, 2'-MOE, 2'-deoxy, or phosphorothioate modifications) compared to the TREM shown in Figure 3.

52. The TREM according to any one of claims 47 to 51, wherein the TREM includes a pattern of non-natural modifications selected from the patterns listed in Table 6.

53. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 1 to 9 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 52, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

54. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 10-19 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 53, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

55. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 20-29 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 54, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

56. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 30-39 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 55, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

57. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 40-49 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 56, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

58. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 50-59 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 57, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

59. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 60-69 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 58, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

60. (i) The non-natural modification is located at a nucleotide position corresponding to one or more nucleotides 70-76 of SEQ ID NO: 734; and / or (ii) The TREM according to any one of claims 1 to 59, wherein the TREM differs from the nucleotide sequence of SEQ ID NO: 734 by only about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

61. The TREM according to any one of claims 1 to 60, wherein the non-natural modification is located at a nucleotide position corresponding to one or more of nucleotide positions 1 to 76, according to CtNS.

62. A pharmaceutical composition comprising TREM as described in any one of claims 1 to 61.

63. The pharmaceutical composition according to claim 90, further comprising pharmaceutically acceptable components, for example, excipients.

64. A lipid nanoparticle formulation comprising TREM according to any one of claims 1 to 91.

65. A lipid nanoparticle formulation comprising the pharmaceutical composition according to claim 92.

66. A composition used for treating a subject having a disease or disorder related to PTC, comprising administering to the subject a TREM described herein (for example, a TREM described in any one of claims 1 to 61).

67. The composition for use according to claim 66, wherein the disease or disorder associated with PTC includes hemophilia B, Fabry disease, Usher syndrome, or CLN2 disease.

68. A method for treating a subject having a disease or disorder related to PTC, comprising administering to the subject a TREM described herein (for example, a TREM described in any one of claims 1 to 61), thereby treating the subject having the disease or disorder.

69. The method according to claim 68, wherein the disease or disorder related to PTC includes hemophilia B, Fabry disease, Usher syndrome, or CLN2 disease.

70. A method for regulating the functional parameters of a tRNA effector molecule (TRM), wherein the TRM is defined by formula (A): [L1]x - [AST Domain 1] - [L2]x - [DH Domain] - [L3]x - [ACH Domain] - [VL Domain] - [TH Domain] - [L4]x - [AST Domain 2] - [L5]x (A), (In the formula: Independently, the [L1] and [VL domains] are optional; x is either 0 or 1. Includes an array of; and The TREM includes nucleotide substitutions (e.g., nucleotide mutations) within the TREM that can modulate the functional parameters of the TREM; A method for adjusting the functional parameters of the TREM.

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