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Gene Synthesis

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Formation of the N(2)-acetyl-2,6-diaminopurine oligonucleotide impurity caused by acetyl capping.
Bioorg Med Chem Lett. 2014 Jun 18;
Authors: Rodriguez AA, Cedillo I, Mowery BP, Gaus HJ, Krishnamoo…

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Phylogenomic Guided Identification of Industrially Relevant GH1 β-Glucosidases Through DNA Synthesis and Nanostructure-Initiator Mass Spectrometry.

ACS Chem Biol. 2014 Jul 1;

Authors: Heins RA, Cheng X, Nath S, Deng K, Bowen BP, Chivian DC, Datta S, Friedland GD, D’Haeseleer P, Wu D, Tran-Gyamfi M, Scullin CS, Singh S, Shi W, Hamilton MG, Bendall ML, Sczyrba A, Thompson J, Feldman T, Guenther JM, Gladden JM, Cheng JF, Adams PD, Rubin EM, Simmons BA, Sale KL, Northen TR, Deutsch S

Abstract
Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput Mass Spectrometry, and applied it to the systematic characterization of GH1 β-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using Nanostructure-Initiator Mass Spectrometry (NIMS), generating over 10,000 data points. Combined with HPLC-based sugar profiling we observed GH1 enzymes active over a broad temperature range, and towards many different di-saccharides in the β configuration. For some GH1s we also observed activity towards laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pre-treatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% [C2mim][OAc] over the course of a 24-hour saccharification reaction. Using our unbiased approach we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

PMID: 24984213 [PubMed - as supplied by publisher]

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Synthesis of Cross-Linked DNA Containing Oxidized Abasic Site Analogues.
J Org Chem. 2014 Jun 20;
Authors: Ghosh S, Greenberg MM
Abstract
DNA interstrand cross-links are an important family of DNA d…

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Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort.
ACS Synth Biol. 2014 Jun 12;
Authors: Ho…

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Error removal in microchip-synthesized DNA using immobilized MutS.

Nucleic Acids Res. 2014 May 14;

Authors: Wan W, Li L, Xu Q, Wang Z, Yao Y, Wang R, Zhang J, Liu H, Gao X, Hong J

Abstract
The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.

PMID: 24829454 [PubMed - as supplied by publisher]

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Efficient Functionalization of Oligonucleotides by New Achiral Nonnucleosidic Monomers.
Org Lett. 2014 May 12;
Authors: Kupryushkin MS, Nekrasov MD, Stetsenko DA, Pyshnyi DV
Abstract

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Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.

Tumour Biol. 2014 May 2;

Authors: Hu SJ, Jiang RX, Xie HH, Ma AL, Shi HL, Shen H, Hao ZM

Abstract
The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.

PMID: 24789430 [PubMed - as supplied by publisher]

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Scaffolding along Nucleic Acid Duplexes Using 2′-Amino-Locked Nucleic Acids.

Acc Chem Res. 2014 Apr 21;

Authors: Astakhova IK, Wengel J

Abstract
Conspectus Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined with advanced properties of the locked nucleic acids (LNAs) provide opportunities to develop new nanomaterials and devices like sensors, aptamers, and machines. In this Account, we describe recent research on preparation and investigation of the properties of LNA/DNA hybrids containing functionalized 2′-amino-LNA nucleotides. By application of different chemical reactions, modification of 2′-amino-LNA scaffolds can be efficiently performed in high yields and with various tags, postsynthetically or during the automated oligonucleotide synthesis. The choice of a synthetic method for scaffolding along 2′-amino-LNA mainly depends on the chemical nature of the modification, its price, its availability, and applications of the product. One of the most useful applications of the product LNA/DNA scaffolds containing 2′-amino-LNA is to detect complementary DNA and RNA targets. Examples of these applications include sensing of clinically important single-nucleotide polymorphisms (SNPs) and imaging of nucleic acids in vitro, in cell culture, and in vivo. According to our studies, 2′-amino-LNA scaffolds are efficient within diagnostic probes for DNA and RNA targets and as therapeutics, whereas both 2′-amino- and isomeric 2′-α-l-amino-LNA scaffolds have promising properties for stabilization and detection of DNA nanostructures. Attachment of fluorescent groups to the 2′-amino group results in very high fluorescent quantum yields of the duplexes and remarkable sensitivity of the fluorescence signal to target binding. Notably, fluorescent LNA/DNA probes bind nucleic acid targets with advantages of high affinity and specificity. Thus, molecular motion of nanodevices and programmable self-assembly of chemically modified LNA/DNA nanomaterials can be followed by bright fluorescence signaling from the functionalized LNA units. Another appealing aspect of the amino-LNA scaffolds is specific targeting of nucleic acids and proteins for therapeutic applications. 2′-Amino-LNA/DNA conjugates containing peptide and polyaromatic hydrocarbon (PAH) groups are promising in this context as well as for advanced imaging and diagnostic purposes in vivo. For imaging applications, photostability of fluorescence dyes is of crucial importance. Chemically stable and photostable fluorescent PAH molecules attached to 2′-amino functionality of the 2′-amino-LNA are potent for in vitro and in vivo imaging of DNA and RNA targets. We believe that rational evolution of the biopolymers of Nature may solve the major challenges of the future material science and biomedicine. However, this requires strong scientific progress and efficient interdisciplinary research. Examples of this Account demonstrate that among other synthetic biopolymers, synthetic nucleic acids containing functionalized 2′-amino-LNA scaffolds offer great opportunities for material science, diagnostics, and medicine of the future.

PMID: 24749544 [PubMed - as supplied by publisher]

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Procedures to view aberrations-A travel from protein to gene: Literature review.
Indian J Dent Res. 2014 Jan-Feb;25(1):91-4
Authors: Premalatha B, Ramesh V, Babu SK, Balamurali PD
Abstract

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Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

Bioorg Med Chem. 2014 Mar 29;

Authors: Kovaliov M, Segal M, Kafri P, Yavin E, Shav-Tal Y, Fischer B

Abstract
A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2′-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2′-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2′-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2′-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.

PMID: 24726303 [PubMed - as supplied by publisher]

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