IMMOBILIZED ENZYME COMPOSITIONS AND METHODS
The present disclosure relates, according to some embodiments, to immobilized enzyme compositions and methods for cleaving polynucleotide molecules including, for example, double-stranded DNA. Immobilized enzymes may comprise, for example, an enzyme (e.g., a type IIS restriction endonuclease, an RNAP, a capping enzyme), a support (e.g., a magnetic bead), and optionally, a linker disposed between the enzyme and the support. In some embodiments, methods may include contacting an immobilized enzyme with a polynucleotide substrate to form reaction products, separating the immobilized enzyme from the reaction products, and optionally reusing the immobilized enzymes in one or more subsequent reactions. preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase.
1 . An immobilized enzyme comprising:
an enzyme selected from a type IIS restriction endonuclease, an RNA polymerase, and a capping enzyme,
a peptide linker attached to the enzyme by a peptide bond,
a SNAP-tag attached to the linker by a peptide bond,
O 6 -benzyleguanine bound to the SNAP-tag; and
magnetic beads having a surface modification comprising the O 6 -benzyleguanine.
2 . An immobilized enzyme according to claim 1 , wherein the type IIS restriction endonuclease has a recognition sequence and cleave site of 5′ GCTCTTC N1 3′ or 5′ GCTCTTC N1/N4 3′ or the RNA polymerase is T7 RNA polymerase or the capping enzyme is Faustovirus capping enzyme or 2′O-methyltransferase.
3 . An immobilized BspQI comprising:
BspQI,
a glycine-serine linker attached to the BspQI by a peptide bond,
a SNAP-tag attached to the linker by a peptide bond,
O 6 -benzyleguanine bound to the SNAP-tag; and
magnetic beads having a surface modification comprising the O 6 -benzyleguanine.
4 . An immobilized T7 RNA polymerase comprising:
T7 RNA polymerase,
a glycine-serine linker attached to the T7 RNA polymerase by a peptide bond,
a SNAP-tag attached to the linker by a peptide bond,
O 6 -benzyleguanine bound to the SNAP-tag; and
magnetic beads having a surface modification comprising the O 6 -benzyleguanine.
5 . An immobilized Faustovirus capping enzyme comprising:
Faustovirus capping enzyme,
a glycine-serine linker attached to the Faustovirus capping enzyme by a peptide bond,
a SNAP-tag attached to the linker by a peptide bond,
O 6 -benzyleguanine bound to the SNAP-tag; and
magnetic beads having a surface modification comprising the O 6 -benzyleguanine.
6 . A method of cleaving a double stranded DNA substrate, the method comprising:
contacting a first portion of the double stranded DNA substrate with an immobilized enzyme comprising a type IIS restriction endonuclease to produce double stranded DNA cleavage products;
separating the immobilized enzyme from the double stranded DNA cleavage products to form separated immobilized enzyme and separated double stranded DNA cleavage products; and
contacting a second portion of the double stranded DNA substrate with the separated immobilized enzyme comprising a type IIS restriction endonuclease to produce more double stranded DNA cleavage products.
7 . A method according to claim 6 , further comprising repeating the separating and subsequent contacting steps from 2 to 50 times.
8 . A method according to claim 6 , further comprising combining the separated double stranded DNA cleavage products with the more double stranded DNA cleavage products to produce pooled products.
9 . A method according to claim 6 , wherein the enzyme is BspQI or Nt.BspQI.
10 . A method of cleaving a double stranded DNA substrate, the method comprising:
contacting the double stranded DNA substrate with an immobilized enzyme comprising a type IIS restriction endonuclease to produce double stranded DNA cleavage products, wherein the double stranded DNA cleavage products comprise at least one nick;
separating the immobilized enzyme from the double stranded DNA cleavage products to form separated immobilized enzyme and separated double stranded DNA cleavage products; and
contacting the separated double stranded DNA cleavage products with a second enzyme.
11 . A method according to claim 10 , wherein the double stranded DNA substrate comprises, in a 5′-3′ direction, a coding sequence, a poly(U) sequence, and a type IIS restriction endonuclease recognition site.
12 . A method according to claim 11 , wherein the type IIS restriction endonuclease is BspQI and the type IIS restriction endonuclease recognition site is a BspQI recognition site.
13 . A method of producing RNA comprising:
(a) contacting a polynucleotide template with an immobilized enzyme to form a cleaved polynucleotide template; and
(b) contacting the cleaved polynucleotide template with an RNA polymerase to produce transcription products comprising the RNA,
wherein the polynucleotide template comprises, in a 5′-3′ direction, a coding sequence, a poly(U) sequence, and a type IIS restriction endonuclease recognition site and the immobilized enzyme comprises a type IIS restriction endonuclease, a support, and a linker disposed between the type IIS restriction endonuclease and the support.
14 . A method according to claim 13 , wherein the (a) contacting and the (b) contacting are performed as a coupled reaction.
15 . A method according to claim 13 , wherein the RNA comprises, in a 5′-3′ direction, a sequence complementary to the coding sequence and a poly(A) sequence.
16 . A method according to claim 13 , wherein the type IIS restriction endonuclease is BspQI and the type IIS restriction endonuclease recognition site is a BspQI recognition site.
17 . A method according to claim 13 , wherein the RNA polymerase is a type II RNA polymerase.
18 . A method according to claim 13 , further comprising contacting the RNA with a capping enzyme to form a capped RNA product.
19 . A method according to claim 13 , further comprising chemically capping the RNA to form a capped RNA product.
20 . An in vitro transcription method, the method comprising:
(a) contacting a double stranded DNA template with a first enzyme to form a nicked template, the first enzyme comprising a type IIS restriction endonuclease;
(b) contacting the nicked template with a second enzyme to form a transcription product, the second enzyme comprising an RNA polymerase; and
(c) optionally contacting the transcription product with a third enzyme to form a capped transcription product, the third enzyme comprising a capping enzyme, wherein at least one of the first enzyme, the second enzyme, and the third enzyme are immobilized enzymes.
21 . A method according to claim 20 , wherein the type IIS restriction endonuclease is BspQI or Nt.BspQI.
22 . A method according to claim 20 , wherein the RNA polymerase is T7 RNA polymerase.
23 . A method according to claim 20 , wherein the capping enzyme is Faustovirus capping enzyme.
24 . A method according to claim 20 , wherein the contacting the transcription product with the capping enzyme further comprises contacting the transcription product with a Cap-0 capping enzyme to form a Cap-0 transcription product and optionally contacting the Cap-0 transcription product with a Cap-1 capping enzyme to form a Cap-1 transcription product.
25 . A method according to claim 22 , wherein the Cap-0 enzyme is Faustovirus capping enzyme.
26 . A method according to claim 22 , wherein the Cap-1 enzyme is 2′O-methyltransferase.
27 . A method according to claim 22 , wherein the capping efficiency is at least 40%.
28 . An in vitro transcription system, the system comprising a first reactor having an inlet, an outlet, and a reaction chamber disposed between the inlet and the outlet, wherein the first reaction chamber holds one or more enzymes each independently selected from a soluble enzyme, a soluble fusion protein comprising one or more enzymes, an immobilized enzyme, and an immobilized fusion protein comprising one or more enzymes, and wherein the first reactor comprises:
a soluble type IIS restriction endonuclease,
a soluble RNA polymerase,
a soluble capping enzyme,
a soluble fusion protein comprising an RNA polymerase and a capping enzyme,
an immobilized type IIS restriction endonuclease
an immobilized RNA polymerase,
an immobilized capping enzyme,
an immobilized fusion protein comprising an RNA polymerase and a capping enzyme, or
combinations thereof,
optionally wherein the immobilized type IIS restriction endonuclease, the immobilized RNA polymerase, or the immobilized capping enzyme is co-immobilized on a support with one or both of the other two enzymes.