Synthetic generation of influenza vaccine viruses for rapid response to pandemics.
Sci Transl Med. 2013 May 15;5(185):185ra68
Authors: Dormitzer PR, Suphaphiphat P, Gibson DG, Wentworth DE, Stockwell TB, Algire MA, Alperovich N, Barro M, Brown DM, Craig S, Dattilo BM, Denisova EA, De Souza I, Eickmann M, Dugan VG, Ferrari A, Gomila RC, Han L, Judge C, Mane S, Matrosovich M, Merryman C, Palladino G, Palmer GA, Spencer T, Strecker T, Trusheim H, Uhlendorff J, Wen Y, Yee AC, Zaveri J, Zhou B, Becker S, Donabedian A, Mason PW, Glass JI, Rappuoli R, Venter JC
During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.
PMID: 23677594 [PubMed - in process]
Rational Design of Orthogonal Libraries of Protein Coding Genes.
ACS Synth Biol. 2013 Mar 7;
Authors: Ryan D, Papamichail D
Array-based oligonucleotide synthesis technologies provide access to thousands of custom-designed sequence variants at low cost. Large-scale synthesis and high-throughput assays have become valuable experimental tools to study in detail the interplay between sequence and function. We have developed a methodology and corresponding algorithms for the design of diverse protein coding gene libraries, to exploit the potential of multiplex synthesis and help elucidate the effects of codon utilization and other factors in gene expression. Using our algorithm, we have computationally designed gene libraries with hundreds to thousands of orthogonal codon usage variants, uniformly exploring the design space of codon utilization, while demanding only a small fraction of the synthesis cost that would be required if these variants were synthesized independently.
PMID: 23654273 [PubMed - as supplied by publisher]
Rapid synthesis of defined eukaryotic promoter libraries.
ACS Synth Biol. 2012 Oct 19;1(10):483-90
Authors: Rajkumar AS, Maerkl SJ
Current gene synthesis methods allow the generation of long segments of dsDNA. We show that these techniques can be used to create synthetic regulatory elements and describe a method for the creation of completely defined, synthetic variants of the PHO5 promoter from the budding yeast Saccharomyces cerevisae. Overall, 128 promoters were assembled by high-temperature ligation, cloned into plasmids by isothermal assembly, maintained in E. coli, and consequently transformed into yeast by homologous recombination. Synthesis errors occurred at frequencies comparable to or lower than those achieved with current gene synthesis methods. The promoter synthesis method reported here is robust, fast, and readily accessible. Synthetically engineered promoter libraries will be useful tools for dissecting the intricacies of promoter input-output functions and may serve as tunable components for synthetic genetic networks.
PMID: 23656186 [PubMed - in process]
Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.
Gene. 2013 May 3;
Authors: Marzo M, Liu D, Ruiz A, Chalmers R
Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.
PMID: 23648487 [PubMed - as supplied by publisher]
A simple, universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis.
Gene. 2013 Apr 15;
Authors: Zhang P, Ding Y, Liao W, Chen Q, Zhang H, Qi P, He T, Wang J, Deng S, Pan T, Ren H, Pan W
Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialized experience.
PMID: 23597923 [PubMed - as supplied by publisher]
Aminosilane functionalizations of mesoporous oxidized silicon for oligonucleotide synthesis and detection.
J R Soc Interface. 2013;10(83):20130160
Authors: De Stefano L, Oliviero G, Amato J, Borbone N, Piccialli G, Mayol L, Rendina I, Terracciano M, Rea I
Direct solid phase synthesis of peptides and oligonucleotides (ONs) requires high chemical stability of the support material. In this work, we have investigated the passivation ability of porous oxidized silicon multilayered structures by two aminosilane compounds, 3-aminopropyltriethoxysilane and 3-aminopropyldimethylethoxysilane (APDMES), for optical label-free ON biosensor fabrication. We have also studied by spectroscopic reflectometry the hybridization between a 13 bases ON, directly grown on the aminosilane modified porous oxidized silicon by in situ synthesis, and its complementary sequence. Even if the results show that both devices are stable to the chemicals (carbonate/methanol) used, the porous silica structure passivated by APDMES reveals higher functionalization degree due to less steric hindrance of pores.
PMID: 23536541 [PubMed - in process]
From design to screening: a new antimicrobial Peptide discovery pipeline.
PLoS One. 2013;8(3):e59305
Authors: Guralp SA, Murgha YE, Rouillard JM, Gulari E
Antimicrobial peptides (AMPs) belong to a class of natural microbicidal molecules that have been receiving great attention for their lower propensity for inducing drug resistance, hence, their potential as alternative drugs to conventional antibiotics. By generating AMP libraries, one can study a large number of candidates for their activities simultaneously in a timely manner. Here, we describe a novel methodology where in silico designed AMP-encoding oligonucleotide libraries are cloned and expressed in a cellular host for rapid screening of active molecules. The combination of parallel oligonucleotide synthesis with microbial expression systems not only offers complete flexibility for sequence design but also allows for economical construction of very large peptide libraries. An application of this approach to discovery of novel AMPs has been demonstrated by constructing and screening a custom library of twelve thousand plantaricin-423 mutants in Escherichia coli. Analysis of selected clones by both Sanger-sequencing and 454 high-throughput sequencing produced a significant amount of data for positionally important residues of plantaricin-423 responsible for antimicrobial activity and, moreover, resulted in identification of many novel variants with enhanced specific activities against Listeria innocua. This approach allows for generation of fully tailored peptide collections in a very cost effective way and will have countless applications from discovery of novel AMPs to gaining fundamental understanding of their biological function and characteristics.
PMID: 23527157 [PubMed - in process]
Attachment of nucleosides and other linkers to solid-phase supports for oligonucleotide synthesis.
Curr Protoc Nucleic Acid Chem. 2013 Mar;Chapter 3:Unit3.2
Authors: Guzaev AP, Pon RT
Specific step-by-step instructions for conversion of 5'-O-(4,4'-dimethoxytrityl)- and base-protected nucleosides and other mono-O-(4,4'-dimethoxytrityl)-protected diols to their hemisuccinate esters and their coupling to CPG (controlled-pore glass) supports bearing aminopropyl or long chain aminoalkyl groups are presented. Additional guidelines are provided for selecting a coupling protocol and performing in-process control. Curr. Protoc. Nucleic Acid Chem. 52:3.2.1-3.2.23. © 2013 by John Wiley & Sons, Inc.
PMID: 23512695 [PubMed - in process]
Synthesis of Threose Nucleic Acid (TNA) Triphosphates and Oligonucleotides by Polymerase-Mediated Primer Extension.
Curr Protoc Nucleic Acid Chem. 2013 Mar;Chapter 4:Unit4.54
Authors: Zhang S, Yu H, Chaput JC
This unit describes the chemical synthesis of α-L-threofuranosyl nucleic acid (TNA) triphosphates for thymidine (T), guanosine (G), cytidine (C), and the diaminopurine (D) analog of adenosine and their incorporation into TNA oligonucleotides by enzyme-mediated polymerization of a DNA primer-template complex. Starting from suitably protected threofuranosyl nucleosides, TNA triphosphates are synthesized in a single-pot reaction and purified by ion-exchange and HPLC chromatography. Purified TNA triphosphates are diluted into stock solutions and used as substrates for the synthesis of TNA oligonucleotides. Oligonucleotide synthesis is accomplished using Therminator DNA polymerase, a commercial variant of the 9(o)N DNA polymerase bearing the A485L mutation. Curr. Protoc. Nucleic Acid Chem. 52:4.54.1-4.54.17. © 2013 by John Wiley & Sons, Inc.
PMID: 23512696 [PubMed - in process]
Characterization and expression profile of vitellogenin gene from Scylla paramamosain.
Gene. 2013 Mar 2;
Authors: Jia X, Chen Y, Zou Z, Lin P, Wang Y, Zhang Z
The full-length (7816bp) cDNA of Vitellogenin (Vtg) encoding 2560 aa with an estimated molecular mass of 287.743kDa was cloned from the green mud crab Scylla paramamosain. Semi-quantitative PCR (sq-PCR) revealed a specific expression pattern of Sp-vtg gene in ovaries and hepatopancreas. With the development of ovaries, the expression level of Sp-vtg gene showed an increasing trend both in ovaries and hepatopancreas, and the expression level of Sp-vtg gene in hepatopancreas and ovary was stable after stage IV. By in situ hybridization, the positive signals of Sp-vtg gene were detected in the cytoplasm of oocytes in stage I, in the follicle cell and the surrounding of the nucleus in stage III, and in the nucleus in stage V. Furthermore, the signals become stronger with the later development stages of ovary. Moreover, in situ hybridization analysis revealed that positive signals of Sp-vtg gene present in the hepatopancreatic tubule, and the signals increase during the development, becoming the strongest in stage V. Our results indicate that both ovaries and hepatopancreas are site of vitellogenin gene synthesis in S. paramamosain.
PMID: 23466977 [PubMed - as supplied by publisher]