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
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]
Expression of codon optimized genes in microbial systems: current industrial applications and perspectives.
Front Microbiol. 2014;5:21
Authors: Elena C, Ravasi P, Castelli ME, Peirú S, Menzella HG
The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent “silent” nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.
PMID: 24550894 [PubMed - as supplied by publisher]
Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.
MAbs. 2013 Dec 11;6(2)
Authors: Valdés-Alemán J, Téllez-Sosa J, Ovilla-Muñoz M, Godoy-Lozano E, Velázquez-Ramírez D, Valdovinos-Torres H, Gómez-Barreto RE, Martinez-Barnetche J
High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.
PMID: 24492293 [PubMed - as supplied by publisher]
A 6′-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF—HC Pseudohydrogen Bonds.
J Org Chem. 2014 Jan 14;
Authors: Dugovic B, Leumann CJ
The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6′ (6′F-bc-T) has been achieved. Key steps of the synthesis were an electrophilic fluorination / stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1-2 degC compared to thymidine and +1-3degC compared to a standard bc-T residue. Structural investigations of the 6′F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a non-classical CF-HC hydrogen bond. In contrast, structural investigations in solution by 1H- and 13C-NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. Based on these results we put forward the hypothesis that these weak CF-HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle chi of the 6′F-bcT nucleoside.
PMID: 24422513 [PubMed - as supplied by publisher]
[Progress in gene synthesis technology].
Sheng Wu Gong Cheng Xue Bao. 2013 Aug;29(8):1075-85
Authors: Feng M, Wang L, Tian J
Gene synthesis is the most fundamental and widely used tec…
[Preface for special issue on synthetic biology (2013)].
Sheng Wu Gong Cheng Xue Bao. 2013 Aug;29(8):1041-3
Authors: Chen G
Synthetic biology has developed quickly worldwide. In this …
Synthesis of nucleotide-amino acid conjugates designed for photo-CIDNP experiments by a phosphotriester approach.
Beilstein J Org Chem. 2013;9:2898-909
Authors: Abramova TV, Morozova OB, Silnikov VN, Yurkovsk…
A novel inducible expression system for the functional study of toxic gene in bacteria.
World J Microbiol Biotechnol. 2013 Dec 11;
Authors: Guo J, Jia R
The cloning and expression of toxic proteins in bacteria have posed a great challenge because of the leaky expression in inducible expression systems. Using artificial gene synthesis and clone screening methods, we identified a mutant T5 promoter, which significantly reduced leaky expression of lac operator. The mutant T5 promoter contains two T deletions at -35 region and may reduce promoter activity. A bacterial lethal gene, Φ174 lytic gene E, was successfully cloned in this system and expressed in the presence of isopropyl β-D-1-thiogalactopyranoside. The system is compatible with existing T5 inducible expression systems and can be used for the controlled expression of toxic proteins.
PMID: 24326910 [PubMed - as supplied by publisher]
COStar: A D-star Lite-based Dynamic Search Algorithm for Codon Optimization.
J Theor Biol. 2013 Dec 4;
Authors: Liu X, Deng R, Wang J, Wang X
Codon optimized genes have two major adva…