3′ protected nucleotides

A method for sequencing a nucleic acid molecule, the method comprising:

• (a) obtaining a chip comprising a plurality of individually addressable nanopores, wherein each individually addressable nanopore of the plurality of individually addressable nanopores comprises a nanopore in a membrane that is disposed adjacent to an electrode, wherein the nanopore is linked to an enzyme and wherein each individually addressable nanopore is adapted to detect a tag that is released from a 3′ protected nucleotide, wherein the 3′ protected nucleotide is embodied by Formula (IIIA):
• wherein
• X is a tagged nucleobase;
• Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH) [O—P(O)(OH)]z—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5;
• R1 is a bond, —CH2—, —C(O)—O—, —C(O)—NRa—, or —C(O)—Rx—;
• R2 and R3 are each independently H, a saturated or unsaturated C1-C6 alkyl group, a C5-C6 aryl or heteroaryl group, a halogen, —[(C(Ra)(Rb))p—O]q—(Ra), —C(O)—ORa, —C(O)—N(Ra)(Rb), —(C(Ra)(Rb))n—N+C−, —CN, or —NO2;
• R4 is a bond, a substituted or unsubstituted 5- to 7-membered aryl group, —CH═CH—, a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group, or —O—C(O)-aryl-;
• R5 is —(C(Ra)(Rb))n—N3, —(C(Ra)(Rb))n—N+C−, —(C(Ra)(Rb))n—CN, a 5- to 8-membered cycloalkyl group comprising two sulfur atoms positioned 1, 3 relative to each other, or a group having the structure:
• Z1 and Z2 are independently H, a C1-C4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups;
• Q1 and Q3 are each independently a bond, C(Re)(Rf)—, or —C(O)—;
• Q2 is a bond, o-phenylene, or —[C(Re)(Rf)]w—, where w is 1 or 2;
• Ra and Rb are each independently H or a saturated C1-C6 alkyl group;
• Re and Rf are independently H, methyl, ethyl, isopropyl, or a substituted or unsubstituted 5- or 6-membered aryl group;
• Rx is a substituted or unsubstituted 5- or 6-membered aromatic group or heteroaromatic group;
• n is 0 or an integer ranging from 1 to 3;
• and
• p and q are each independently zero or an integer ranging from 1 to 3;
• provided that when R1 and R4 are both bonds and when R2 and R3 are both H, then R5 is not an azide group;
• (b) directing the nucleic acid molecule adjacent to or in proximity to the nanopore;
• (c) with the aid of the enzyme, polymerizing the 3′ protected nucleotide along the nucleic acid molecule to generate a polynucleotide strand that is complementary to at least a portion of the nucleic acid molecule;
• (d) deprotecting the 3′ protected nucleotide after incorporation of the 3′protected nucleotide into the polynucleotide strand;
• and
• (e) detecting the tag released from the 3′ protected nucleotide with the aid of the electrode, wherein the released tag flows through or in proximity to the nanopore,
• where steps (d) and (e) may be performed in any order.

A method of sequencing a nucleic acid sample with the aid of a nanopore, comprising:

• (a) performing a polymerization reaction with the aid of a single polymerase coupled to the nanopore, the polymerization reaction incorporating at least one protected nucleotide into a growing polynucleotide strand complementary to a single stranded nucleic acid molecule derived from the nucleic acid sample, detecting, with the aid of the nanopore, the incorporation of the at least one protected nucleotide or a byproduct thereof into the growing polynucleotide strand;
• and either simultaneously with or subsequent to the step of detecting the incorporation of the protected nucleotide or the byproduct thereof, deprotecting the protected nucleotide to provide a 3′ deprotected nucleotide, wherein the at least one protected nucleotide has Formula (IIIA):
• wherein
• X is a nucleobase or a tagged nucleobase;
• Y is —O—P(O)(OH)—[O—P(O)(OH)]z—OH or —O—P(O)(OH)—[O—P(O)(OH)]z—O-oligonucleotide, where z is 0 or an integer ranging from 1 to 5;
• R1 is a bond, —CH2—, —C(O)—O—, —C(O)—NRa—, or —C(O)—Rx—;
• R2 and R3 are each independently H, a saturated or unsaturated C1-C6 alkyl group, a C5-C6 aryl or heteroaryl group, a halogen, —[(C(Ra)(Rb))p—O]q—(Ra), —C(O)—ORa, —C(O)—N(Ra)(Rb), —(C(Ra)(Rb))n—N+C−, —CN, or —NO2;
• R4 is a bond, a substituted or unsubstituted 5- to 7-membered aryl group, —CH═CH—, a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group, or —O—C(O)-aryl-;
• R5 is —(C(Ra)(Rb))n—N3, —(C(Ra)(Rb))n—N+C−, a 5- to 8-membered cycloalkyl group comprising two sulfur atoms positioned 1, 3 relative to each other, or a group having the structure:
• Z1 and Z2 are independently H, a C1-C4 alkyl group, or a 5- to 6-membered aryl group optionally substituted with one or more hydroxyl groups;
• Q1 and Q3 are each independently a bond, —C(Re)(Rf)—, or —C(O)—;
• Q2 is a bond, o-phenylene, or —[C(Re)(Rf)]w—, where w is 1 or 2;
• Ra and Rb are each independently H or a saturated C1-C6 alkyl group;
• Re and Rf are independently H, methyl, ethyl, isopropyl, or a substituted or unsubstituted 5- or 6-membered aryl group;
• Rx is a substituted or unsubstituted 5- or 6-membered aromatic group or heteroaromatic group;
• n is 0 or an integer ranging from 1 to 3;
• and
• p and q are each independently zero or an integer ranging from 1 to 3;
• provided that when R1 and R4 are both bonds and when R2 and R3 are both H, then R5 is not an azide.