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  10  Recent Patents on DNA & Gene Sequences 2012,  6,  10-21  2212- 3431 /12 $100.00+.00 © 2012 Bentham Science Publishers Recent Patents on Oligonucleotide Synthesis and Gene Synthesis Tingsheng Yu a, *, Xiao Bao a , Wenxian Piao a , Jingli Peng a , Wei Li a , Cui Yang a , Meng Xing a ,Yiliu Zhang a , Jinhuan Qi a , Lei Xu a , Li Xu and Qiuyun Liu * The Key Laboratory of Gene Engineering of Ministry of Education and Biotechnology Research Center, The School of  Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China  Received: August 17, 2011 Revised: October 03, 2011 Accepted: October 24, 2011 Abstract: Gene synthesis is an emerging field which has widespread implications in synthetic biology and molecular bi-ology. 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 compromis-ing DNA synthesis fidelity, whereas the gigantic amount of genome information provides infinite source of DNA ele-ments 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 inte-gral 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 phos- phodiester synthesis, phosphotriester synthesis and so on. However, the phosphoramidite method emerged to be the most desirable and most popular approach to synthesize oli-gonucleotides[2]. In the post-genomic era, the large-scale scrutiny of ge-nome 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 func-tions of these proteins. The canonical way of gene cloning from various sources is pretty straightforward, but the perti-nent approaches have several limits: the expression of natu-ral gene in heterologous systems like  Escherichia coli  or else may not be ideal; vaccine attenuation may require codon de-optimization, and druggable proteins may need to be engi-neered for longer half-lives and more potent activities, etc. Moreover, directed evolution may require de novo gene syn-thesis, 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: 86-20-84110296; E-mail:; 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:; a These authors contributed equally to this work and polymerase chain reaction (PCR) also accelerated the widespread adoption of synthetic nucleic acid polymers[3]. Below we will review some of the recent patents on oligonu-cleotide synthesis and gene synthesis. PART I OLIGONUCLEOTIDE SYNTHESIS Phosphoramidite method, which was based on the work of Caruthers, is routinely used as a standard approach for  preparing synthetic nucleic acids. H-phosphonate[4], on the other hand, even though achieving the same goal of synthe-sizing the desired polymer, fails to attract due attention com- pared to the former one due to lower yield. To streamline the procedure and automate the processes, solid phases are commonly employed to allow the growing molecular chain to anchor on. Since the polymer has to be split off upon completion, suitable linkers, which can con-nect the polymer and its solid phase, are also indispensable[5]. In the 1990s, with the advent of in situ synthesis of mi-croarrays, which could load thousands of different sequences on a substrate on a single try, many more methods have flourished from it and synthetic nucleic acid research ad-vanced to another level[5](Table 1 ). Chloral-Free Dichloroacetic Acid in Oligonucleotide Syn-thesis[6]  Oligonucleotides and derivatives have at least a number of applications in research and medicine. Oligomers can serve as primers for PCR amplifications and probes for DNA and RNA hybridizations, as well as for next generation se-quencing by hybridizations. Oligonucleotide chips are widely used in the studies of transcriptome, epigenome, as well as genome hybridizations such as genome tiling. Oli-gonucleotides are also invaluable as therapeutics in the clinic.   Recent Patents on Oligonucleotide Synthesis and Gene Synthesis Recent Patents on DNA & Gene Sequences 2012  , Vol. 6, No. 1 11 Table 1. Patents Related to Oligonucleotide Synthesis Publication Number Ref. No. Title Inventors Publication Date US8008270 Antiviral oligonucleotides targeting viral families Vaillant, A.; Juteau, J.M. 2011/08/30 US8008269 Antiviral oligonucleotides Vaillant, A.; Juteau, J.M. 2011/08/30 US8008468 RNAi expression constructs with liver-specific enhancer/promoter Roelvink, P. W. ; Suhy, D. A.; (+3) 2011/08/30 US8008005 Method for the synthesis of DNA sequences Belshaw, P.J; Sussman, M.J.(+2) 2011/08/30 US20110196145A1 Process for desilylation of oligonucleotides Manoharan, M.; Jung, M. E.(+3) 2011/08/11 US7985565 Method of nucleic acid amplification Kawashima, E.H.; Farinelli, L.; (+1)2011/07/26 US20110177514A1 Integrated instrument performing synthesis and amplification, and a sys-tem and method thereof Froehlich, T.; Gutekunst, M.(+3) 2011/07/21 US7981871 Modified macromolescules and associated methods of synthesis and use Prestwich, G.D.; Shu, X.Z.; (+1) 2011/07/19 US20110171649A1 Detection of nucleic acids by oligonucleotide probes cleaved in presence of endonuclease V Kutyavin, I.; 2011/07/14 US7972820 Isothermal amplification of nucleic acids on a solid support Mayer, P. 2011/07/05 US7964343 Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture Hofstadler, S.A.; Cummins, L.L. 2011/06/21 US20110137021A1 Sulfur transfer reagents for oligonucleotide synthesis Guzaev, A.P.; 2011/06/09 US7947659 iRNA agents targeting VEGF Fougerolles, A.D.; Kamenetsky, M.F.; (+3) 2011/05/24 US20110124524A1 Fluid processing device for oligonucleotide synthesis and analysis Ermakov, S. V. 2011/05/26 US7939258  Nucleic acid amplification procedure using RNA and DNA composite  primers Kurn, N.; Wang, S. 2011/05/10 US7939677 Oligomeric compounds comprising 4 ' -thionucleosides for use in gene modulation Bhat, B.; Dandc, P.; (+4) 2011/05/10 US7939256 Composition and method for nucleic acid sequencing Williams, J.G.K. 2011/05/10 US20110097762A1 Linkers and co-coupling agents for optimization of oligonucleotide syn-thesis and purification on solid supports Gao, X.; Zhang, H.; (+5) 2011/04/28 WO2010134992A3 Synthesis labile base protected – modified deoxy & modified ribonucleo-sides, corresponding phosphoramidites and supports and their use in high  purity oligonucleotide synthesis Srivastava, S.C; Srivastava, N.P 2011/04/07 US7919612 2'-substituted oligomeric compounds and compositions for use in gene modulations Baker, B.F ; Eldrup, A.B. ; (+6) 2011/04/05 US7919473 IRNA agents targeting VEGF Fougerolles, A.D.; Kamenetsky, M.F.; (+3) 2011/04/05 WO2011028218A1 Process for triphosphate oligonucleotide synthesis Zlatev, I.; Morvan, F.; (+3) 2011/03/10 US7901892 Methods and compositions for determining the purity of chemically syn-thesized nucleic acids Agris, P. F. ; Mitchell, L. G.; (+1) 2011/03/08 US20110046344A1 Parallel preparation of high fidelity probes in an array format Kuimelis, R. G. ; Mcgall, G. H. 2011/02/24 US20110045541A1 Method of nucleic acid amplification Rudi, K.; Holck, A. 2011/02/24 US7893249 Deprotection and purification of oligonucleotides and their derivatives Bowman, K; Shaffer, C.; (+1) 2011/02/22  12  Recent Patents on DNA & Gene Sequences 2012  , Vol. 6, No. 1 Yu et al. (Table 1) contd…. Publication Number Ref. No. Title Inventors Publication Date US7893036  In vivo  production of small interfering RNAs that mediate gene silencing Zamore, P.D.; Juanita, M.; (+3) 2011/02/22 US7884086 Conjugates for use in hepatocyte free uptake assays Bennett, F. C.; Mckay, R. (+6) 2011/02/08 US7875733 Oligomeric compounds comprising 4'-thionucleosides for use in gene modulation Bhat, B.; Dandc, P.; (+4) 2011/01/25 US20110015382A1 Synthesis of N-FMOC protected deoxy nucleosides, ribo nucleosides, modified deoxy and ribo nucleosides, and phosphoramidites, and their use in oligonucleotide synthesis Srivastava, S.C.; Srivastava, N.P. 2011/01/20 US7872121 Process for the removal of exocyclic base protecting groups  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 Methods for genotyping selected polymorphism Jones, K.W. ; Shapero, M. H.; (+1) 2011/01/13 US7862820 Immunoglobulin chimeric monomer-dimer hybrids Peters, R.T.; Mezo, A.R.; (+3) 2011/01/04 US7858560 Capture compounds, collections thereof and methods for analyzing the  proteome and complex compositions Koster, H.; Siddiqi, S.; (+1) 2010/12/28 US7858311 Composition and method for nucleic acid sequencing Williams, J. G. K. 2010/12/28 US7851615 Lipophilic conjugated iRNA agents Manoharan, M.; Rajeev, K. G. 2010/12/14 US7846733 Methods and compositions for transcription-based nucleic acid amplifica-tion Kurn, N.; Palo, A.C. 2010/12/07 US7846666 Methods of RNA amplification in the presence of DNAKurn, N.; Palo, A.C. 2010/12/07 WO2010134992A2 Synthesis labile base protected –modified deoxy & modified ribonucleo-sides, corresponding phosphoramidites and supports and their use in high  purity oligonucleotide synthesis Srivastava, S.C.; Srivastava, N.P. 2010/11/25 US7838466 Device for chemical and biochemical reactions using photo-generated reagents Gao, X.; Zhou, X.(+1) 2010/11/23 US7834171 Modified polynucleotides for reducing off-target effects in RNA interfer-enceLeake, D.; Reynolds, A.R.; (+2) 2010/11/16 EP2248820A1 Universal supports for oligonucleotide synthesis Azhayev, A.; Antopolskii, M. 2010/11/10 WO2010062404A3 N-fmoc nucleosides and phosphoramidites, and oligonucleotide synthesis Srivastava, S.C.; Srivastava, N.P. 2010/10/14 US7812149 2'-Fluoro substituted oligomeric compounds and compositions for use in gene modulations Prakash, T. P.; Baker, B. F. (+6) 2010/10/12 US7807807 Linkers and co-coupling agents for optimization of oligonucleotide syn-thesis and purification on solid supports Gao, X.; Zhang, H.; (+5) 2010/10/05 US7803529 Solid phase sequencing of biopolymers Cantor, C. R.; Koster, H.; (+2) 2010/09/28 US7794945 Hybridization and mismatch discrimination using oligonucleotides conju-gated to minor groove binders Hedgpeth, J.; Afonina, I. A.; (+4) 2010/09/14   Recent Patents on Oligonucleotide Synthesis and Gene Synthesis Recent Patents on DNA & Gene Sequences 2012  , Vol. 6, No. 1 13  Antisense oligonucleotides are capable of silencing gene expression, thus reducing protein levels. It has held great  promise in pre-clinical research. Its mode of action is distinct from conventional therapeutic methods, which usually modulate protein activities through direct interaction be-tween putative drugs and drug targets-usually proteins in nature. A typical oligonucleotide synthesis using phosphoramid-ite chemistry (i.e. the amidite methodology) includes a solid  primer support. The 5-hydroxyl protecting groups in the nucleoside linked to the solid support need to be removed in the synthesis of oligonucleotides. The 4, 4-dimethoxy-triphenylmethyl (DMT) group has been frequently used at this position, and it can be cleaved with the use of dichloroacetic acid (DCA). However, DCA frequently contains trace amount of chloral or chloral hydrate, and even 1 % residue can generate a lot of chloral adducts on the oligos. DCA essentially free of impurities was prepared via vacuum distillation by patentees. The procedure can be adopted for producing oligonucleotides that are virtually free of chloral adducts (Table 2 ). Activators for Oligonucleotide and Phosphoramidite Synthesis [7] The use of aryl-substituted 5-phenyl-1H-tetrazoles, with  perfluoroalkyl groups on the aromatic ring as activators in the synthesis of oligonucleotides and nucleoside phosphora-midites, was documented in this patent. The activators were highly soluble and efficient, and the coupling time for DNA  phosphoramidites was about or less than 15 seconds. The coupling time for 2-O-tert-butyldimethylsilyl RNA phos- phoramidites was no more than 5 minutes. A nucleoside or oligonucleotide harbouring a hydroxyl group, could also react with a phosphitylating agent in the presence of the acti-vator mentioned above to generate a phosphoramidite. Fig. (1).  The general structure of novel aryl-substituted 5-phenyl-1H-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]. A spectrophotometric DMT assay showed that coupling efficiencies over 99.0% could be achieved in the synthesis of oligonucleotide. The purity of the crude oligonucleotides could peak to 98% under mild conditions. Enhanced Antisense Oligonucleotides [8]  The gap-widened antisense oligonucleotides, each 18 to 24 nucleotides in length, have a wing-gap-wing linear struc-ture, and improved therapeutic efficacy as compared to 5-10-5 MOE (2-O-methoxyethyl, 2-MOE or simply MOE) gap-mer antisense oligonucleotides targeted at the same se-quence. The gap region has 12 to 18 straight 2-deoxyribo-nucleosides, whereas the two wings have 1 to 4 2-O-(2-methoxyethyl) ribonucleotides. Fig. (2). Formula for a universal support. Adapted from reference [10]. Sulfur Transfer Reagents for Oligonucleotide Synthesis [9] Oligonucleotide phosphorothioates are oligonucleotide analogues whose non-bridging oxygen atoms of the  phosphate group are substituted by a sulfur atom. The enhanced nuclease resistance has increased their values in  pharmaceutical research. Sulfurization on the phosphite moiety is allowed after each coupling in the phosphoramidite approach. Patentee introduced sulfur transfer reagents capa- ble of converting P(  ) internucleosidic linkages to P(  ) phosphorothioate linkages in oligonucleotides. A widely used Beaucage reagent 1, 2-benzodithiol-3-one-1, 1-dioxide, and a compound 5-ethoxy-3H-1, 2, 4-dithiazole-2-one abbreviated as EDIT, both with poor hydro-lytic stability, were somewhat tricky to synthesize. Agents such as TETD (tetraethylthiuram disulfide) have displayed sluggish reaction kinetics hence are less convenient in indus-trial mass production.  N-formamidino-5-amino-3H-1, 2, 4-dithiazole-3-thiones were described as efficient sulfur-transfer reagents in this  patent, having clear edge over commercially available Beau-cage reagent and TETD. The novel sulfurizing agents were synthesized inexpensively via simple chemical methods, and were highly stable in solution. The sulfur transfer from these reagents was investigated in the solid-phase synthesis of oli-gonucleotide phosphorothioates via phosphoramidite meth-ods. The efficiency of the sulfur transfer reaction for 2'-deoxyoligonucleotides was determined to top 99.5%. Universal Supports for Oligonucleotide Synthesis[10]  Most oligonucleotide syntheses are conducted on sup- ports pre-attached with a nucleoside. Therefore four de-oxynucleoside and four ribonucleoside supports are required for routine DNA and RNA synthesis. Currently the use of universal supports for DNA and RNA synthesis is not popular in industry. One major hurdle is to find proper conditions to remove the terminal phosphate attached to the terminal hydroxyl group in the first phos- phoramidite reaction cycle.
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