Beruflich Dokumente
Kultur Dokumente
10
Abstract: Gene synthesis is an emerging field which has widespread implications in synthetic biology and molecular biology. The field is constantly evolving which has led to key advances in oligonucleotide synthesis and gene synthesis
technologies, with simplicity, cost effectiveness and high throughput. The miniaturization, multiplexing, microfluidic
processing and the integrated microchip engineering will drive down cost and increase productivity without compromising DNA synthesis fidelity, whereas the gigantic amount of genome information provides infinite source of DNA elements and genes as raw material for synthetic biology. This article describes some of the recent patents on oligonucleotide
synthesis and gene synthesis.
Keywords: Oligonucleotide synthesis, gene synthesis, miniaturization, multiplexing, microfluidic processing, microchip.
INTRODUCTION
Oligonucleotide synthesis and DNA synthesis are integral parts of the gene synthesis processes. Oligonucleotide
synthesis is the process of attaching short nucleotides into a
string, using chemical methods. It is essential in producing
primers for polymerase chain reaction (PCR) [1]. Typically,
oligonucleotides are synthesized mainly by means of phosphodiester synthesis, phosphotriester synthesis and so on.
However, the phosphoramidite method emerged to be the
most desirable and most popular approach to synthesize oligonucleotides [2].
In the post-genomic era, the large-scale scrutiny of genome and proteome, and the deliberation, comparison and
integration of mega data sets, became routine in biological
laboratories. DNA sequences from unknown proteins are
essential in exploring and building inventories on the functions of these proteins. The canonical way of gene cloning
from various sources is pretty straightforward, but the pertinent approaches have several limits: the expression of natural gene in heterologous systems like Escherichia coli or else
may not be ideal; vaccine attenuation may require codon deoptimization, and druggable proteins may need to be engineered for longer half-lives and more potent activities, etc.
Moreover, directed evolution may require de novo gene synthesis, and creation of artificial life forms requires mega
DNA synthesis. With rapid advances in synthetic biology
and molecular biology, the demand for synthetic nucleic
acids is on the rise. Besides, the techniques of microarrays
*Address correspondence to these authors at the School of Life Sciences,
Sun Yat-sen University, Guangzhou, 510275, P. R. China; Tel: 86-2084110296; E-mail: yts9009@sina.com; and The Key Laboratory of Gene
Engineering of Ministry of Education, Sun Yat-sen University, Guangzhou
510275, P. R. China; Tel: 86-20-84110296; Fax: 86-20-84036551;
E-mail: liuqiuyunzhongda@yahoo.com.cn;
a
Table 1.
11
Publication Number
Inventors
Publication
Date
US8008270
2011/08/30
US8008269
Antiviral oligonucleotides
2011/08/30
US8008468
2011/08/30
US8008005
2011/08/30
US20110196145A1
Manoharan, M.;
Jung, M. E.(+3)
2011/08/11
US7985565
2011/07/26
US20110177514A1
Integrated instrument performing synthesis and amplification, and a system and method thereof
2011/07/21
US7981871
Prestwich, G.D.;
Shu, X.Z.; (+1)
2011/07/19
US20110171649A1
Kutyavin, I.;
2011/07/14
US7972820
Mayer, P.
2011/07/05
US7964343
2011/06/21
US20110137021A1
Guzaev, A.P.;
2011/06/09
US7947659
Fougerolles, A.D.;
Kamenetsky, M.F.; (+3)
2011/05/24
US20110124524A1
Ermakov, S. V.
2011/05/26
US7939258
2011/05/10
US7939677
2011/05/10
US7939256
Williams, J.G.K.
2011/05/10
US20110097762A1
Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports
2011/04/28
WO2010134992A3
Synthesis labile base protected modified deoxy & modified ribonucleosides, corresponding phosphoramidites and supports and their use in high
purity oligonucleotide synthesis
2011/04/07
US7919612
2011/04/05
US7919473
2011/04/05
WO2011028218A1
2011/03/10
US7901892
Methods and compositions for determining the purity of chemically synthesized nucleic acids
2011/03/08
US20110046344A1
Kuimelis, R. G. ;
Mcgall, G. H.
2011/02/24
US20110045541A1
2011/02/24
US7893249
2011/02/22
Yu et al.
(Table 1) contd.
Publication Number
Inventors
Publication
Date
US7893036
2011/02/22
US7884086
Bennett, F. C.;
Mckay, R. (+6)
2011/02/08
US7875733
2011/01/25
US20110015382A1
2011/01/20
US7872121
Nanda, D.S;
Satya, K
2011/01/18
US20110009606A1
Synthesis of 2', 3'- and 3', 5' cyclic phosphate mono-and oligonucleotides
Laikhter, A;
Srivastava, S.C; (+1)
2011/01/13
US20110009294A1
Jones, K.W. ;
Shapero, M. H.; (+1)
2011/01/13
US7862820
Peters, R.T.;
Mezo, A.R.; (+3)
2011/01/04
US7858560
Koster, H.;
Siddiqi, S.; (+1)
2010/12/28
US7858311
Williams, J. G. K.
2010/12/28
US7851615
Manoharan, M.;
Rajeev, K. G.
2010/12/14
US7846733
Kurn, N.;
Palo, A.C.
2010/12/07
US7846666
Kurn, N.;
Palo, A.C.
2010/12/07
WO2010134992A2
Synthesis labile base protected modified deoxy & modified ribonucleosides, corresponding phosphoramidites and supports and their use in high
purity oligonucleotide synthesis
2010/11/25
US7838466
Gao, X.;
Zhou, X.(+1)
2010/11/23
US7834171
Leake, D.;
Reynolds, A.R.; (+2)
2010/11/16
EP2248820A1
2010/11/10
WO2010062404A3
2010/10/14
US7812149
Prakash, T. P.;
Baker, B. F. (+6)
2010/10/12
US7807807
Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports
Gao, X.;
Zhang, H.; (+5)
2010/10/05
US7803529
Cantor, C. R.;
Koster, H.; (+2)
2010/09/28
US7794945
Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
Hedgpeth, J.;
Afonina, I. A.; (+4)
2010/09/14
Fig. (1). The general structure of novel aryl-substituted 5-phenyl1H-tetrazoles as catalysts in the coupling reactions of the
phosphoramidite approach. At least one R contains a perfluoroalkyl
substituent and n is an integer selected from 1-5. Adapted from
reference [7].
13
ture, and improved therapeutic efficacy as compared to 5-105 MOE (2-O-methoxyethyl, 2-MOE or simply MOE) gapmer antisense oligonucleotides targeted at the same sequence. The gap region has 12 to 18 straight 2-deoxyribonucleosides, whereas the two wings have 1 to 4 2-O-(2methoxyethyl) ribonucleotides.
Table 2.
Reference No.
[5]
Yu et al.
Title
[6]
Chloral-free DCA in
oligonucleotide synthesis
[7]
Publication
Date
2010-10-14
2010-07-20
2011-03-01
Publication
Number
Inventors
Applications
Drawbacks
WO2010118264
Rinderknecht, D.;
Keen, R.;
Charib, M.
It produced oligonucleotides
that were virtually free of
chloral adducts consequently
led to better purity. Hence,
such oligos would be useful
in therapeutic approaches.
US7897758B2
Wolter, A.;
Leuck, M.
Bioavailability may be
slightly decreased due
to large molecular
weight.
US7759480
[8]
Enhanced antisense
oligonucleotides
2011-04-05
US7919472B2
Monia, B.P.;
Siwkowski, M.;
Bhanot, S.
[9]
2010-05-25
US7723528
Guzaev, A.P.;
US20050250116A1
Azhayev, A.;
Antopolskii, M.
WO2010094772
Stahler, P. F.;
Carapito, R.;
Stahler, C. F.;
(+4)
US2005/0272042A1
Gardner, S.N.;
MariellaMariella,
R.P.; Christian,
A.T.; Young,
J.A.; Clague,
D.S.
Robotics may be
needed for long sequence assemblies.
Assembly using short
oligomers may be very
inefficient at difficult
regions.
US2005/0255477A1
Carr, P.A.;
Chow, B.Y.;
Jacobson, J.M.;
Mosley, D.W.;
Emig, C.
US2008/0261833A1
Stemmer,
W.P.C.;
Crameri, A.
Efficient screening
method may be an
advantage to deal with
generated libraries.
[10]
[18]
[20]
Sequential addition of
short DNA oligos in
DNA-polymerasebased synthesis reactions
[21]
[22]
2005-11-10
2009-02-20
2005-12-08
2005-11-17
2008-10-23
15
(Table 2) contd.
Reference No.
Title
[23]
[24]
[25]
[26]
Gene synthesis
method
Publication
Date
Publication
Number
Inventors
Applications
Drawbacks
US7906282B2
Wood, K.V.;
Gruber, M.G.;
Zhuang, Y.;
Paguio, A.
EP1538206
Evans, G,A.
2011-05-26
US20110124049
It presented an integrated
microfluidic device for gene
assembly, purification and
error correction.
2009-05-11
WO2010132019
2011-03-15
2005-06-08
Previous universal supports that use neighboring aminomethyl or diamino-ethyl groups to assist the elimination
reaction have been reported by Andrei [9]. All common types
of oligonucleotides can be prepared with these supports. Oligomers with base labile nucleoside units could be synthesized as well. Yet, basic volatile reagents at high temperatures and long treatment period were required in the process.
Such features render these supports somewhat unpopular for
industrial purposes.
In brief, an ideal universal support would allow efficient
cleavage and dephosphorylation at low temperatures and
under relatively mild reaction conditions. The supports described by Alex et al. do not have a pre-attached nucleoside,
and therefore are compatible with any DNA or RNA synthesis. Furthermore, the supports described permit fast cleavage
and dephosphorylation under mild conditions (e.g., using a
2M ammonia in methanol reagent).
In the formula described in Fig. (2), hydrogen, alkyl,
aryl, or a polymeric or silica base material could be selected
for Substituent A, whereas acyl, aroyl, or a polymeric or
silica base material constituted Substituent B. Substituent C
was chosen from a dimethoxytrityl group or a protecting
group removable under acidic or neutral conditions. The
polymeric or silica base material comprised one of Substituent A or B. An oligonucleotide was linked to the support at
substituent C.
PART II GENE SYNTHESIS
A Brief History of Gene Synthesis
Khorana and Itakura pioneered the field with the thenepic total synthesis of tRNA structural genes and the synthesis and expression of the somatostatin gene [11-13]. Polymerase cycling assembly (PCA) has been most popular among
all methods due to its inherent simplicity. Overlapping and
complementary oligonucleotides were denatured, annealed
and recursively extended with a thermal-stable DNA polymerase to give rise to a full-length sequence, which was then
amplified by conventional PCR. PCA, first reported for synthesis of the 303-bp HIV-2 Rev gene, has since evolved into
a widely recognized general method for synthesis of genes of
various sizes [14].
To produce a completely new molecule is both time consuming and expensive. Thus, there is room for a technology
that allows the creation of novel DNA molecules in a single
step without resorting to any existing recombinant or naturally-occurring DNA.
Stemmer et al. split the entire sequence of a gene, designed oligonucleotides of 40 base long that overlapped each
other by 20 bases. The full-length gene was generated in a
single reaction by iterative overlap extension PCR (OEPCR), followed by second PCR amplification with two outer
most primers [15]. The method of Stemmer et al. has since
been recognized for its simplicity and low cost.
Researchers have also been committed in synthesizing
relatively long genes. Lo-Chun Au et al. assembled short
oligonucleotides via ligase chain reaction (LCR) in high
stringency conditions to make unit fragments which were
then joined to give rise to a full-length gene sequence by
PCR, and a recombinant leptin gene was synthesized according to the codon bias of E. coli, so that the procedure did not
confer constraints on the nature of the sequence and length
[16]. Several softwares have been adopted in assisting oligo
design. Marcus Bode, Samuel Khor, Hongye Ye et al. introduced a computer program entitled TmPrime to design oligonucleotide sets for gene assembly by both ligase chain
reaction (LCR) and polymerase chain reaction (PCR) [17].
The program split the long target DNA sequence, and optimized the length of oligonucleotides to achieve homologous
melting temperatures. It presented no constraints on the gene
and oligonucleotide lengths. The list of gene synthesis patents is in Table 3.
Table 3.
Yu et al.
Publication Number
Title
Inventors
Publication
Date
US7932025
Methods for high fidelity production of long nucleic acid molecules with
error control
2011-04-26
US7879580
2011-02-01
US8008005
Belshaw, P. J. Sussman,
M.J.(+2)
2011-08-30
US7853410
2010-12-14
US7795030
2010-09-14
US7981614
2011-07-19
US7904249
2011-03-08
US7868138
2011-01-11
US7803934
2010-09-28
US7790381
2010-09-07
US20110190163A1
Genome-Wide Construction of Schizosaccharomyces Pombe Heterozygous Deletion Mutants Containing Gene-Specific Barcodes by the Methods of 4-round Serial or Block PCR, or Total Gene Synthesis Thereof
2011-08-04
US7957912
2011-06-07
US7873499
2011-01-18
US7851679
2010-12-14
US7816085
2010-10-19
WO2011102796A1
Nurmemmedov, E.
2011-08-25
WO2011066185A1
Chu, L.L.
2011-06-03
WO2010130824A3
2011-01-27
US20110124049A1
2011-05-26
US7993921
2011-08-09
US7985840
2011-07-26
US7985575
2011-07-26
US7959926
2011-06-14
US7928215
2011-04-19
US7919591
2011-04-05
US7897359
2011-03-01
17
(Table 3) contd.
Publication Number
Title
Inventors
Publication
Date
US7883866
2011-02-08
EP2285974A2
2011-02-23
EP2268808A1
Genome-Wide Construction of Schizosaccharomyces Pombe Heterozygous Deletion Mutants Containing Gene-Specific Barcodes by the Methods of 4-round Serial or Block PCR, or Total Gene Synthesis Thereof
2011-01-05
WO2010132019A1
2010-11-18
US7858344
2010-12-28
US7846689
2010-12-07
US 2010/0035768 A1
WO 2009/103027 A2
US7838265
2010-11-23
US7838219
2010-11-23
US7833759
2010-11-16
US7829310
2010-11-09
US7816320
Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35
2010-10-19
US7879580
2011-02-01
WO 2010/071602 A1
WO 2010/132019 A1
WO 2008/112683 A2
WO 2008/112683 A3R4
EP2190988A4
Daniel, G.G;
Venter J. C.;
Huang M.C.;
Li M.H.; (+2)
Li M.H.;
Jackie Y. Y.; (+2)
Duhee B.,
George M. C.
Duhee B.,
George M. C
Li M., Ying J.Y., Huang M., Pin
Ng, S.
2010-02-01
2009-08-20
2010-06-24
2010-11-18
2008-09-18
2008-09-18
2010-12-22
Yu et al.
A nanopore device was also adopted to monitor the deprotection of the 5 protecting groups to eliminate deletion
errors. In the single molecular synthesis system, the growing
chain could be shown to be deprotected when the wash was
flowed through the nanopore and fluorescence was detected.
The researchers also pointed out that a crosslinking between mismatch binding proteins with erroneous DNA
would prevent its amplification by DNA polymerases.
Methods for Generating Polynucleotides having Desired
Characteristics by Interative Selection and Recombination [22]
This patent describes the use of oligos in the conventional gene shuffling process. These oligos can have identity
to some regions and heterology to others. This provides unlimited diversity to the parental sequences for PCR shuffling
and assembly. Eventually affinity screening can be applied
to pull out positive clones.
Removal of Transcriptional Regulatory Sites and Gene
Expression [23]
This patent used gene synthesis to remove transcriptional
regulatory sites, including transcriptional binding sites, intron splicing sites, poly-adenylation sites, promoters and
enhancers, so the synthetic molecules had at least 3 fold less,
preferably 5 fold less regulatory sequences. Through a further optimization of codon usage via the use of favorite
codons, protein expression levels could be markedly enhanced.
Method for the Complete Chemical Synthesis and Assembly of Genes and Genomes [24]
It is a method designed by Glen A Evans for the complete chemical synthesis and assembly of genes and genomes. It provided an efficient way to generate nearly any
nucleic acid sequence. This approach is completely synthetic
by nature, so, there are no constraints, even for constructing
very long stretches of nucleic acid. Integrating gene parts
and functional elements via the use of simple computer
software, it is possible to construct a virtual polynucleotide
in a computer which is subsequently used as the blueprint for
DNA synthesis.
Different sequences are needed for transferring to different kinds of cells with corresponding origin of replication.
The genome sequence was broken down into a set of overlapping oligonucleotides of specified length by computer
software. To make sure a single mixture contained equal
concentrations of each, the concentrations of oligomers
needed to be balanced. Then the oligomers would be coannealed and a virtual gene or genome is constructed from
scratch. The DNA sequence was ligated via T4 DNA ligase
or topoisomerase 2, and then removed from the solid support
if attached. After a final ligation, the molecule would be
transferred into host cells.
Synthesis of Sequence-Verified Nucleic Acids [18]
The method documented by Stahler et al. could produce
synthetic nucleic acids from sequence-verified building
19
Yu et al.
Technologies
Challenges Ahead
Prompt editing may be required in the genome synthesis process to avoid delays
Metabolic engineering
Repetitive sequences
Efficient screening assays need to be envisaged as the odds for successful isolation of artificial proteins could be very low
[2]
Many researchers are now engaged in genome synthesizing, metabolic engineering, etc. (Table 4). Since errors could
be introduced to synthetic genomes, consequently editing is
required. Completely synthetic genomes are still some distance away. One future challenge will be the synthesis of
DNA with extremely skewed GC or AT contents which may
serve as functional elements in natural or synthetic genomes.
With the advances in integrated microchip technology, and
the boom of commercial venders, gene synthesis cost is
bound to plummet. Synthetic genomes or engineered metabolic pathways may be one day exploited to produce biofuels at industrial scale, and enable the manufacture of many
drugs, vaccines or valuable products.
ACKNOWLEDGMENTS
[1]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
Kodumal SJ, Patel KG, Reid R, Menzella HG, Welch M, Santi DV.
Total synthesis of long DNA sequences: Synthesis of a contiguous
32-kb polyketide synthase gene cluster. Proc Natl Acad Sci USA
2004; 101: 15573-8.
Stemmer WP, Crameri A, Ha KD, et al. Single-step assembly of a
gene and entire plasmid from large number of oligodeoxyribonucleotides. Gene 1995; 164: 49-53.
Au LC, Yang FY, Yang WJ, Lo SH, Kao CF. Gene synthesis by a
LCR-based approach: High-level production of Leptin-L54 using
synthetic gene in Escherichia coli. Biochem Bioph Res Co 1998;
248: 200-3.
Bode M, Khor S, Ye H, Li MH, Ying JY. TmPrime: fast, flexible
oligonucleotide designs software for gene synthesis. Nucl Acids
Res 2009; 37: 214-21.
Stahler PF, Carapito R, Stahler CF, et al. Synthesis of sequenceverified nucleic acids. WO2010094772, 2009.
Wu G, Wolf JB, Ibrahim AF, Vadasz S, Gunasinghe M, Freeland
SJ. Simplified gene synthesis: A one-step approach to PCR-based
gene construction. J Biotechnol 2006; 124: 496-503.
Gardner SN, Mariella RP, Christian AT, Young JA, Clague DS.
Sequential addition of short DNA oligos in DNA-polymerasebased synthesis reactions. US2005/0272042 A1, 2005.
Carr PA, Chow BY, Jacobson JM, Mosley DW, Emig C. Methods
for high fidelity production of long nucleic acid molecules.
US2005/0255477 A1, 2005.
Stemmer WPC, Crameri A. Methods for generating polynucleotides having desired characteristics by interactive selection and recombination. US2008/0261833 A1, 2008.
Wood KV, Gruber MG, Zhuang Y, Paguio A. Synthetic nucleic
acid molecule compositions and methods of preparation.
US7906282 B2, 2011.
Evans GA. Method for the complete chemical synthesis and assembly of genes and genomes. EP1538206, 2005.
Li, M, Ying JY, Huang M, Pin Ng S. Integrated microfluidic device
for gene synthesis US20110124049, 2011.
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
21