Compositions and Methods For High Fidelity Assembly of Nucleic Acids

Abstract

Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.

1 . A method of producing a target nucleic acid having a predefined sequence, the method comprising:
 providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends of each of the plurality of blunt-end double-stranded nucleic acid fragments;   producing a plurality of cohesive-end double-stranded nucleic acid fragments that together comprises the target nucleic acid sequence via enzymatic digestion of the plurality of blunt-end double-stranded nucleic acid fragments, wherein the plurality of cohesive-end double-stranded nucleic acid fragments each have two different and non-complementary overhangs;   ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase, wherein a first overhang of a first cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a second overhang of a second cohesive-end double-stranded nucleic acid fragment; and   forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid having a predefined sequence.   
     
     
         2 . The method of  claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments are generated from a plurality of single-stranded oligonucleotides immobilized on a solid support. 
     
     
         3 . The method of  claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments comprises:
 releasing a plurality of oligonucleotides synthesized on a solid support; and   synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.   
     
     
         4 . The method of  claim 3 , wherein the plurality of oligonucleotides each comprise a universal primer binding site, and wherein a universal primer complementary to said universal primer binding site is used in said polymerase based reaction. 
     
     
         5 . The method of  claim 4 , wherein the plurality of oligonucleotides each comprise the restriction enzyme recognition sequence. 
     
     
         6 . The method of  claim 5 , wherein said the restriction enzyme recognition sequence is part of the universal primer binding site and is located at the 5′ or 3′ end of the universal primer binding site or the restriction enzyme recognition sequence is located upstream or downstream to the universal primer binding site. 
     
     
         7 . The method of  claim 4 , wherein the universal primer has an affinity tag to facilitate affinity removal of undesirable enzymatic digestion products. 
     
     
         8 . The method of  claim 7 , wherein the affinity tag is biotin. 
     
     
         9 . The method of  claim 1 , wherein the plurality of blunt-end double-stranded nucleic acids comprises at least 3, 4, 5, 6, 7, 8, 10, 15 or 20 different blunt-end double-stranded nucleic acid fragments. 
     
     
         10 . The method of  claim 1 , wherein each of the plurality of blunt-end double-stranded nucleic acid fragments is at least 50, 100, 200, or 300 bases long. 
     
     
         11 . The method of  claim 1 , wherein the restriction enzyme recognition sequence is the same for all blunt-end double-stranded nucleic acid fragments. 
     
     
         12 . The method of  claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments comprise at least two different restriction enzyme recognition sequences recognizable by two different restriction enzymes that are selected to produce overhangs having the same number of bases. 
     
     
         13 . The method of  claim 1 , wherein the restriction enzyme recognition sequence is capable of being recognized by a type IIs restriction enzyme. 
     
     
         14 . The method of  claim 13 , wherein the type IIs restriction enzyme is BsaI, BsmBI, BspQI, BtgZI, BsmFI, FokI, BbvI, any variant thereof, or any combination thereof. 
     
     
         15 . The method of  claim 1 , wherein the plurality of cohesive-end double-stranded nucleic acid fragments are designed such that the a cohesive end in a cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a next cohesive end in an adjacent cohesive-end double-stranded nucleic acid fragment. 
     
     
         16 . The method of  claim 1 , wherein the overhangs are at least 3, 4, 5, 6, 7, or 8 bases long. 
     
     
         17 . The method of  claim 1 , wherein the overhangs differ from one another by at least 1, 2, 3 or 4 bases. 
     
     
         18 . The method of  claim 1 , wherein the overhangs are 5′ or 3′ overhangs. 
     
     
         19 . The method of  claim 1 , further comprising, before the ligating step, purifying the plurality of cohesive-end double-stranded nucleic acid fragments to remove undesirable enzymatic digestion products. 
     
     
         20 . The method of  claim 19 , wherein the undesirable enzymatic digestion products include fragments less than about 40, about 35, about 30, about 25, about 20, or about 15 bases long. 
     
     
         21 . The method of  claim 19 , wherein said purifying includes differential affinity to silica, size filtration, differential precipitation with polyethylene glycol or cetyltrimethlyammonium bromide, or any combination thereof. 
     
     
         22 . The method of  claim 1 , wherein the ligase is T3 DNA ligase, T4 DNA ligase, T7 DNA ligase,  E. coli  DNA ligase, any variant thereof, or any combination thereof. 
     
     
         23 . The method of  claim 1 , wherein the target nucleic acid is a non-naturally occurring nucleic acid. 
     
     
         24 . The method of  claim 1 , wherein the target nucleic acid is at least 500, 800, 1000, 1500, 2000, or 3000 bases long. 
     
     
         25 . The method of  claim 1 , further comprising amplifying the target nucleic acid using a pair of primers specific to the target nucleic acid and a polymerase. 
     
     
         26 . The method of  claim 1 , further comprising confirming the sequence of the target nucleic acid. 
     
     
         27 . The method of  claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments are hierarchically assembled from synthetic oligonucleotides. 
     
     
         28 . The method of  claim 1  wherein the plurality of nucleic acid fragments are ligated in a single pool. 
     
     
         29 . The method of  claim 1  wherein the plurality of nucleic acid fragments are in at least two pools, each nucleic acid fragment of the first pool having a terminal end complementary to a nucleic acid fragment of the second pool. 
     
     
         30 . The method of  claim 29  wherein the plurality of nucleic acid fragments are oligonucleotide dimers. 
     
     
         31 . A method for designing a plurality of starting nucleic acids to be assembled into a target nucleic acid, the method comprising:
 (a) obtaining an input target sequence of a target nucleic acid;   (b) selecting a plurality of subsequences therein such that every two adjacent subsequences overlap with each other by N bases;   (c) storing the resulting overlapping N-base sequences in a memory;   (d) comparing the overlapping N-base sequences to one another to ensure that they differ from one another by at least one base; and   (e) repeating steps (b) to (d) until a plurality of satisfactory nucleic acid fragments are obtained wherein any two adjacent starting nucleic acid fragments uniquely overlap with each other by N bases.   
     
     
         32 . The method of  claim 31  further comprising designing flanking sequences at its 5′ end and 3′ end, the flanking sequences comprising a restriction enzyme recognition site, capable of being recognized by a type IIS restriction enzyme. 
     
     
         32 . The method of  claim 32 , wherein the restriction enzyme recognition site is a type IIS recognition site. 
     
     
         33 . The method of  claim 32  wherein the flanking sequences further comprise a stretch of nucleotides such that any two adjacent starting nucleic acid fragments have uniquely complementary cohesive ends after cleavage with the restriction enzyme. 
     
     
         34 . The method of  claim 32  wherein the flanking sequences further comprise a primer binding site. 
     
     
         35 . The method of  claim 31 , wherein the target nucleic acid is a non-naturally occurring nucleic acid. 
     
     
         36 . The method of  claim 31 , wherein the target nucleic acid is at least 500, 800, 1000, 1500, 2000, or 3000 bases long. 
     
     
         37 . The method of  claim 31 , wherein each subsequence is about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 or more bases long. 
     
     
         38 . The method of  claim 31 , wherein N is an integral and is 3, 4, 5, 6, 7, 8, or more. 
     
     
         39 . A plurality of starting nucleic acids to be assembled into a target nucleic acid, designed according to the method of  claim 31 . 
     
     
         40 . The plurality of starting nucleic acids of  claim 40 , each further comprising an engineered universal primer binding site for amplifying the plurality of starting nucleic acids therefrom. 
     
     
         41 . The plurality of starting nucleic acids of  claim 40 , each further comprising an engineered restriction enzyme recognition sequence. 
     
     
         42 . A system for assembling a target nucleic acid, the system comprising:
 a solid support for synthesizing the plurality of starting nucleic acids of  claim 31 , wherein each starting nucleic acid further comprises an engineered universal primer binding site and an engineered restriction enzyme recognition sequence;   a polymerase reaction unit for synthesizing complementary strands of the plurality of starting nucleic acids a polymerase-based reaction using a universal primer complementary to the universal primer binding site, thereby producing a plurality of blunt-end double-stranded nucleic acid fragments;   a digestion unit for producing a plurality of cohesive-end double-stranded nucleic acid fragments via enzymatic digestion of the plurality of blunt-end double-stranded nucleic acid fragments, wherein the plurality of cohesive-end double-stranded nucleic acid fragments each have two different and non-complementary overhangs; and   a ligation unit for ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase, wherein a first overhang of a first cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a second overhang of a second cohesive-end double-stranded nucleic acid fragment.   
     
     
         43 . A computer program product for designing a plurality of starting nucleic acids to be assembled into a target nucleic acid, said program residing on a hardware computer readable storage medium and having a plurality of instructions which, when executed by a processor, cause the processor to perform operations comprising:
 (a) obtaining a target sequence of a target nucleic acid;   (b) selecting a plurality of subsequences therein such that every two adjacent subsequences overlap with each other by N bases;   (c) storing the resulting overlapping N-base sequences in a memory;   (d) comparing the overlapping N-base sequences to one another to ensure that they differ from one another by at least one base; and   (e) repeating steps (b) to (d) until a plurality of satisfactory starting nucleic acids are obtained wherein any two adjacent starting nucleic acids uniquely overlap with each other by N bases.