Methods and compositions for oligonucleotide bioconjugation

An oligonucleotide bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

An oligonucleotide bioconjugate of formula (III)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1—C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

An oligonucleotide bioconjugate of formula (IV)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

An oligonucleotide bioconjugate of formula (V)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of the L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

A method of making an oligonucleotide bioconjugate of formula (I)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a carbohydrate, a lipid, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage, the method comprising a step of reacting the thiol of formula (VI) with the maleimide of formula (VII) to form the oligonucleotide bioconjugate of formula (I)
• wherein A and Y of formula (VI) are defined as above for formula (I), and wherein Z and B of formula (VII) are defined as above for formula (I).

A method of making an oligonucleotide bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2—groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage,
• the method comprising the steps of
• (i) reacting the thiol of formula (VI) with one of the two maleimides of formula (VIII) to form a compound of formula (IX)
• wherein A and Y of formula (VI) are defined as above for formula (II), and wherein L of formula (VIII) is defined as above for formula (II), and
• (ii) reacting the maleimide of formula (IX) with the thiol of formula (X) to form the oligonucleotide bioconjugate of formula (II)
• wherein Z and B of formula (X) are as defined above for formula (II).

A method of making an oligonucleotide bioconjugate of formula (III)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2—groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1—C alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage,
• the method comprising the steps of
• (i) reacting the thiol of formula (VI) with one of the two maleimides of formula (VIII) to form a compound of formula (IX)
• wherein A and Y of formula (VI) are defined as above for formula (III), and wherein L of formula (VIII) is defined as above for formula (III),
• (ii) reacting the maleimide of formula (IX) with the thiol of formula (X) to form a compound of formula (II)
• wherein Z and B of formula (X) are as defined above for formula (III), and
• (iii) hydrolyzing one of the succinimides of formula (II) to form the oligonucleotide bioconjugate of formula (III)

A method of making an oligonucleotide bioconjugate of formula (IV)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage,
• the method comprising the steps of
• (i) reacting the thiol of formula (VI) with the maleimide of formula (VIII) to form a compound of formula (IX)
• wherein A and Y of formula (VI) are defined as above for formula (IV), and wherein
• L of formula (VIII) is defined as above for formula (IV),
• (ii) reacting the maleimide of formula (IX) with the thiol of formula (X) to form a compound of formula (II)
• wherein Z and B of formula (X) are as defined above for formula (IV), and
• (iii) hydrolyzing one of the succinimides of formula (II) to form the oligonucleotide bioconjugate of formula (IV)

A method of making an oligonucleotide bioconjugate of formula (V)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is an oligonucleotide having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a small molecule, a lipid, a carbohydrate, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage,
• the method comprising the steps of
• (i) reacting the thiol of formula (VI) with one of the two maleimides of formula (VIII) to form a compound of formula (IX)
• wherein A and Y of formula (VI) are defined as above for formula (V), and wherein
• L is defined as above for formula (V),
• (ii) reacting the maleimide of formula (IX) with the thiol of formula (X) to form a compound of formula (II)
• wherein Z and B of formula (X) are as defined above for formula (V), and
• (iii) hydrolyzing both of the succinimides of formula (II) to form the oligonucleotide bioconjugate of formula (V)

A method of co-expressing a first polypeptide and a second polypeptide in a cell, the method comprising a step of contacting the cell with an mRNA bioconjugate of formula (I)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes the first polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second mRNA molecule having a 3′-end and a 5′-end, wherein B encodes the second polypeptide,
• wherein Y is covalently attached to the 3′-end of A through a first linkage, and wherein Z is covalently attached to the 3′-end of B through a second linkage.

A method of co-expressing a first polypeptide and a second polypeptide in a cell, the method comprising a step of contacting the cell with an mRNA bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes the first polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second mRNA molecule having a 3′-end and a 5′-end, wherein B encodes the second polypeptide;
• wherein Y is covalently attached to the 3′-end of A through a first linkage, and wherein Z is covalently attached to the 3′-end of B through a second linkage.

A method of delivering equimolar amounts of a first mRNA molecule and a second mRNA molecule to a cell, the method comprising a step of contacting the cell with an mRNA bioconjugate of formula (I)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second mRNA molecule having a 3′-end and a 5′-end,
• wherein Y is covalently attached to the 3′-end of A through a first linkage, and wherein Z is covalently attached to the 3′-end of B through a second linkage.

A method of delivering equimolar amounts of a first mRNA molecule and a second mRNA molecule to a cell, the method comprising a step of contacting the cell with an mRNA bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second mRNA molecule having a 3′-end and a 5′-end;
• wherein Y is covalently attached to the 3′-end of A through a first linkage, and wherein Z is covalently attached to the 3′-end of B through a second linkage.

A method of targeted therapy, the method comprising a step of administering to a patient in need thereof a pharmaceutical formulation comprising an mRNA bioconjugate of formula (I)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first therapeutic polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second therapeutic polypeptide, a therapeutic small molecule, or a second mRNA molecule having a 3′-end and a 5′-end encoding a second therapeutic polypeptide,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

A method of targeted therapy, the method comprising a step of administering to a patient in need thereof a pharmaceutical formulation comprising an mRNA bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first therapeutic polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of L is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second therapeutic polypeptide, a therapeutic small molecule, or a second mRNA molecule having a 3′-end and a 5′-end encoding a second therapeutic polypeptide,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

A method of treating, preventing, slowing the progression, or reducing the severity of cancer in a patient in need thereof, the method comprising a step of administering to the patient in need thereof a pharmaceutical formulation comprising an mRNA bioconjugate of formula (I)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first therapeutic polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second therapeutic polypeptide, a therapeutic small molecule, or a second mRNA molecule having a 3′-end and a 5′-end encoding a second therapeutic polypeptide,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

A method of treating, preventing, slowing the progression, or reducing the severity of cancer in a patient in need thereof, the method comprising a step of administering to the patient in need thereof a pharmaceutical formulation comprising an mRNA bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first therapeutic polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of the C2-C50 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second therapeutic polypeptide, a therapeutic small molecule, or a second mRNA molecule having a 3′-end and a 5′-end encoding a second therapeutic polypeptide,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

A method of treating, preventing, slowing the progression, or reducing the severity of obesity in a patient in need thereof, the method comprising a step of administering to the patient in need thereof a pharmaceutical formulation comprising an mRNA bioconjugate of formula (I)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first therapeutic polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second therapeutic polypeptide, a therapeutic small molecule, or a second mRNA molecule having a 3′-end and a 5′-end encoding a second therapeutic polypeptide,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

A method of treating, preventing, slowing the progression, or reducing the severity of obesity in a patient in need thereof, the method comprising a step of administering to the patient in need thereof a pharmaceutical formulation comprising an mRNA bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first therapeutic polypeptide;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of the C2-C50 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is a second therapeutic polypeptide, a therapeutic small molecule, or a second mRNA molecule having a 3′-end and a 5′-end encoding a second therapeutic polypeptide,
• wherein Y is covalently attached to the 3′-end of A through a first linkage.

A method of enzyme replacement therapy, the method comprising a step of administering to a patient in need of enzyme replacement therapy a pharmaceutical formulation comprising an mRNA bioconjugate of formula (I)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first enzyme;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a second enzyme, a small molecule, a carbohydrate, a lipid, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage, and wherein the patient in need of enzyme replacement therapy is deficient of the first enzyme.

A method of enzyme replacement therapy, the method comprising a step of administering to a patient in need of enzyme replacement therapy a pharmaceutical formulation comprising an mRNA bioconjugate of formula (II)

• or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent,
• wherein
• A is a first mRNA molecule having a 3′-end and a 5′-end, wherein A encodes a first enzyme;
• Y is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• L is a C2-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C8 cycloalkyl, aryl, heteroaryl, C4-C50 alkyl-cycloalkyl, C7-C50 alkyl-aryl, or C6-C50 alkyl-heteroaryl, wherein any one or more —CH2— groups of the C2-C50 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of L is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• Z is a C1-C20 alkyl, wherein any one or more —CH2— groups of the C1-C20 alkyl is each optionally replaced independently with —C(═O)—, —CF2—, or a heteroatomic moiety selected from the group consisting of —O—, —S—, —NH—, and —N(C1-C6 alkyl)-, wherein any one or more —CH3 groups of the C1-C20 alkyl is each optionally replaced independently with —CF3, —CF2H, —CH2F, or a heteroatomic moiety selected from the group consisting of —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —O(C1-C6 alkyl), and —S(C1-C6 alkyl), and wherein any two heteroatomic moieties are separated from one another by at least two carbon atoms;
• and
• B is an oligonucleotide, a polypeptide, a protein, a second enzyme, a small molecule, a carbohydrate, a lipid, a polyethylene glycol (PEG) molecule, or a biopolymer,
• wherein Y is covalently attached to the 3′-end of A through a first linkage, and wherein the patient in need of enzyme replacement therapy is deficient of the first enzyme.