Commercial Oligonucleotide Research Articles

A Catalytic Approach for the Synthesis of Peptide-Oligonucleotides Conjugates in Aqueous Solution or On-Column.

Peptide-oligonucleotide conjugates (POCs) are covalent architectures composed of a DNA or RNA molecules linked to a peptide. These constructs have found widespread applications ranging from hybrid nanomaterials to gene-targeted therapies. Considering the important role of POCs, a new catalytic approach for their preparation is reported here, that could be applied either on solid support in anhydrous media, or post-synthetically in aqueous buffer. Single amino acids, peptides and cell penetrating peptides (CPPs) were conjugated to various oligo(ribo)nucleotides with high conversions and good isolated yields. The applicability of the method was demonstrated on more than 35 examples including an analogue of a commercial therapeutic oligonucleotide. Other conjugation partners, such as deoxycholic acid and biotin were also successfully conjugated to oligonucleotides. To highlight the potential of this catalytic approach, these conditions have been applied to iterative processes, which is of high interest for the development of DNA-Encoded Libraries.

Cross-contamination of CRISPR guides and other unrelated nucleotide sequences among commercial oligonucleotides.

Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used.

DNA Origami-Based Single-Molecule Force Spectroscopy and Applications.

Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered and explored using techniques such as atomic force microscopy and force spectroscopy using optical or magnetic tweezers. These experimental approaches are limited by thermal noise resulting from a physical connection of the studied biological system to the macroscopic world. To overcome this limitation, we recently introduced the DNA origami force clamp (FC) which is a freely diffusing nanodevice that generates piconewton forces on a DNA sequence of interest. Binding of a protein to the DNA under tension can be detected employing fluorescence resonance energy transfer (FRET) as a sensitive readout.This protocol introduces the reader to the working principles of the FC and provides instructions to design and generate a DNA origami FC customized for a protein of interest. Molecular cloning techniques are employed to modify, produce, and purify a custom DNA scaffold. A fluorescently labeled DNA suitable to detect protein binding via FRET is generated via enzymatic ligation of commercial DNA oligonucleotides. After thermal annealing of all components, the DNA origami FC is purified using agarose gel electrophoresis. The final section covers the interrogation of the FC using confocal single-molecule FRET measurements and subsequent data analysis to quantify the binding of a DNA-binding protein to its cognate recognition site under a range of forces. Using this approach, force-dependent DNA-protein interactions can be studied on the single-molecule level on thousands of molecules in a parallelized fashion.

Assessment of enzymatically synthesized DNA for gene assembly.

Phosphoramidite chemical DNA synthesis technology is utilized for creating de novo ssDNA building blocks and is widely used by commercial vendors. Recent advances in enzymatic DNA synthesis (EDS), including engineered enzymes and reversibly terminated nucleotides, bring EDS technology into competition with traditional chemical methods. In this short study, we evaluate oligos produced using a benchtop EDS instrument alongside chemically produced commercial oligonucleotides to assemble a synthetic gene encoding green fluorescent protein (GFP). While enzymatic synthesis produced lower concentrations of individual oligonucleotides, these were available in half the time of commercially produced oligonucleotides and were sufficient to assemble functional GFP sequences without producing hazardous organic chemical waste.

HLA Diversity in Saudi Population: High Frequency of Homozygous HLA Alleles and Haplotypes.

Human leukocyte antigens (HLA) diversity has a tremendous impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions. Here, we conducted a retrospective study of HLA class I and class II homozygosity at allelic and haplotype levels in unrelated individuals genotyped from 2012 to 2016 in a tertiary hospital in the capital of Saudi Arabia. Among 5,000 individuals, 2,773 individuals meet inclusion criteria and were retrospectively analyzed for HLA-A, -B, -C–DRB1, and -DQB1 homozygosity at allelic and haplotype levels. HLA molecular typing was performed using a commercial reverse sequence-specific oligonucleotide (rSSO) kit. We were able to identify 15 HLA-A, 20 HLA-B, 11 HLA-C, 13 HLA-DRB1, and five HLA-DQB1 homozygous alleles demonstrating a very low genetic diversity in the Saudi population. The highest homozygosity in HLA class I was found in locus C followed by A and B (20.3% > 16.1% > 15.5%; p < 0.001) where the most homozygote alleles were A*02 (9.2%), B*51 and B*50 (5.7% and 3.7%), and C*07, C*06, and C*15 (7.2%, 5.48%, and 3.3%) and in HLA class II, the highest homozygosity was found in locus DQB1 compared to DRB1 (31.71% > 19.2%; p < 0.001), with the most common homozygote alleles being DRB1*07 and DRB1*04 (5.33% and 4.2%) and DQB1*02, DQB1*06, and DQB1*03 (13.55%, 7.92%, and 7.64%). The frequency of finding an individual with one homozygote allele was (24.6%), two homozygote alleles (13.5%), three homozygote alleles (4.7%), four homozygote alleles (3.4%), and five alleles were (4.8%). The most frequent homozygote haplotypes are A*23∼C*06∼B*50∼DRB1*07∼DQB1*02 and A*02∼C*06∼B*50∼DRB1*07∼DQB1*02. This study shows low diversity of both class I and II alleles and haplotypes in the Saudi population, which would have a significant impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions.

Outcome Prediction in CLL Patients Based on a Disease-Specific Oligonucleotide Microarray.

Chronic Lymphocytic Leukemia (CLL) is characterized by a extreme variability in the clinical course; some patients survive only a few months whereas others have stable disease for more than 10 years. Individual outcome prediction in CLL is still a challenge, in spite of the existence of multiple newly described prognostic factors: cytogenetic alterations, mutational IgVH status, and expression of surrogate markers such as ZAP-70. The accuracy of outcome prediction in CLL may improve thanks to the inclusion of new additional biological variables identified by high-throughput molecular techniques. This more reliable risk-stratification of patients could allow to assign a risk-adapted individualized therapy. With this aim, we have analysed with oligonucleotide microarrays three different series of CLL patients in a three-step process.In a first-step, RNA extracted from tumour samples of a series of 26 CLL patients, was hybridised using commercial 22K oligonucleotide chips (Agilent Technologies). A total of 135 genes whose expression was associated with changes in survival probability were identified. An additional review of published data yielded a total number of 497 genes whose expression has been related with outcome in CLL. A CLL specific chip was then built in a second-step including, besides these 632 genes, control genes until a total of 1023 genes. This comprehensive CLL-chip was then hybridised with RNA extracted from peripheral-blood samples in a new series of consecutively diagnosed 100 CLL patients. Cox's univariate analysis was performed to look for molecular variables associated with Progression Free Survival (PFS) as endpoint. FDR significance levels were also used when appropriate, for adjusting the associated p-value. Expression of 94 genes was significantly associated with PFS. Profile patterns of the 94 resulting genes were clustered, and allow to identify 12 clusters. Based on biological function, we selected a representing gene of each cluster, to perform a multivariate Cox's analysis. This approach generated a model of 4 genes, implicated in BCR signalling, signal transduction and transcription regulation, that distinguishes four quartiles of CLL patients with PFS at 5 years of 95%, 70%, 44% and 8% respectively.These results have been validated in a new independent series of 60 consecutively diagnosed cases of CLL. If confirmed in additional studies, the availability of this CLL-specific chip could allow a more accurate prediction of the clinical course in CLL patients, this giving support to a stratification of patients into different risk-groups

Coumarin Amine-Reactive DYE C392STP: an Efficient Building Block to Synthesize Single Labeled Oligonucleotides with Application as Fish Probes

The demand of new reactive fluorophores with improved photophysical characteristics for labeling oligonucleotide probes is growing. Sodium (E/Z)-4-(4-(2-(6,7-dimethoxycoumarin-3-yl)vinyl)-benzoyl)-2,3,5,6-tetrafluoro-benzenesulfonate (C392STP) is an amine-reactive fluorescent dye that has recently shown interesting photophysical properties. In this work, we investigate the possibility of: i) synthesizing it cheaply and easily; ii) using this reactive dye to synthesize single-labeled fluorescent oligonucleotides; and iii) applying the resulting fluorescently labeled oligonucleotides as fluorescence in situ hybridization (FISH) probes. According to the results obtained in this study, C392STP: i) can be synthesized through a low cost and simple pathway; ii) is an efficient building block for mono-labeling of oligomers with an efficiency as high as that of an amine-reactive Alexa Fluor® 488 dye supplied in a commercial oligonucleotide labeling kit. Furthermore, the RNA-FISH assays revealed that C392-mono-labeled probes containing standard carbon linkers (C6) exhibited effective performance as RNA-FISH probes. These findings evidenced the applicability of C392STP in labeling of biomolecules and bioimaging. The results invite the researchers to continue exploring other uses of this reactive dye for exploiting its potential in biological applications.

Killer cell immunoglobulin-like receptor genotypes in the Turkish population

One hundred eighty-seven healthy and unrelated volunteers from various regions of Turkey were selected for the study. Killer-cell immunoglobulin-like receptors (KIR) genotyping was performed by polymerase chain reaction using commercial sequence-specific oligonucleotide probe (SSOP) kits. Gene frequencies of the Turkish population were determined by direct counting of the positive and negative loci. The genotype data is publicly available in the Allele Frequencies Net Database under the population name “Turkey KIR pop 3” number “3399”.

P224 Diversity of alleles and haplotypes of HLA class I and class II in Saudi population

Aim Human Leukocyte Antigens (HLA) play an important role in host immune responses to infectious pathogens, bone marrow and organ transplantation, cancer and autoimmune diseases. HLA association studies have shown that homozygosity was associated with worse clinical outcomes of transfusion associated-graft versus host disease (TA-GvHD). Here we conducted a retrospective study of HLA class I and class II genes homozygosity at allelic and haplotype levels in patients and healthy donors genotyped from 2012 to 2016 in a tertiary hospital at the capital of Saudi Arabia. Methods About 2773 cases were retrospectively analyzed for HLA-A, -B, -C -DRB1 and DQB1 homozygosity at allelic and haplotype levels. HLA molecular typing was performed using a commercial Sequence-Specific oligonucleotides (SSO) kit and following the manufacturer instructions (rSSO; One Lambda, Canoga Park, CA). Statistical analysis was done with Fisher’s exact test (two-tailed). The analysis was performed on Stata package. Results In contrast to other populations, we were able to identify only 46 HLA class I and 18 class II alleles demonstrating a very low genetic diversity in Saudi population. A total of 15 HLA-A, 20 HLA-B, 11 HLA-C, 13 HLA-DRB1 and 5 HLA-DQB1 alleles have been identified. In class I, the highest homozygosity is found in locus C followed by A and B (21.3% > 17.1% > 15.5%; p 20.3%; p Conclusions We found low genetic diversity of both class I and II alleles and haplotype in Saudi population which would have a significant impact in shaping the transplantation practices, transfusion associated graft versus host disease practices and host-pathogen interactions in the population.

Quantifying the influence of 5′-RNA modifications on RNA polymerase I activity

For ensemble and single-molecule analyses of transcription, the use of synthetic transcription elongation complexes has been a versatile and powerful tool. However, structural analyses demonstrate that short RNA substrates, often employed in these assays, would occupy space within the RNA polymerase. Most commercial RNA oligonucleotides do not carry a 5′-triphosphate as would be present on a natural, de novo synthesized RNA. To examine the effects of 5′-moities on transcription kinetics, we measured nucleotide addition and 3′-dinucleotide cleavage by eukaryotic RNA polymerase I using 5′-hydroxyl and 5′-triphosphate RNA substrates. We found that 5′ modifications had no discernable effect on the kinetics of nucleotide addition; however, we observed clear, but modest, effects on the rate of backtracking and/or dinucleotide cleavage. These data suggest that the 5′-end may influence RNA polymerase translocation, consistent with previous prokaryotic studies, and these findings may have implications on kinetic barriers that confront RNA polymerases during the transition from initiation to elongation.

P118 Association between the frequency of human Leukocyte antigen class I and class II genes in Saudi population and relative risk of developing nasopharyngeal carcinoma

Aim Genetic susceptibility and EBV infection have been reported as co-factors responsible for the risk of developing NPC. The incidence of NPC has a marked geographic variation, though rare in the west; it is high in countries such as Taiwan and China. While Human Leukocyte Antigen (HLA) have been associated with NPC predisposition in Asians, yet there have been no studies on HLA association to NPC in Saudis. Methods HLA-A, -B, -C and -DRB1 were examined for their association to NPC in case-control study of Saudi patients compared to healthy Saudi attending a tertiary hospital (n = 45 cases and n = 107 controls). HLA typing was molecularly performed using a commercial Sequence-Specific oligonucleotides (SSO) kit and following the manufacturer instructions (rSSO; One Lambda, Canoga Park, CA). Statistical analysis was done with Fisher’s exact test (two-tailed). The analysis was performed on Stata package. The genetic model was allele positivity corresponding to the dominant model. Results The results clearly demonstrated that while NPC protection was associated with HLA-DRB1∗11 allele (OR = 0.1412; p = 0.043), most NPC susceptibility was strictly associated with HLA-B∗08 (OR = 3.629; p = 0.017) and HLA-DRB1∗07(OR = 2.296; p = 0.041) alleles. Surprisingly and in contrast to the Taiwanese and Chinese studies, we could not find any significant association with any of the HLA-A or HLA-C alleles. Conclusions Our study show for the first time, that HLA-DRB1∗11 allele have protective association with NPC and HLA-B∗08 and HLA-DRB1∗07 have susceptibility association with NPC.

Killer-cell immunoglobulin-like receptors genotyping of 127 individuals from Terceira Island, Azores, Portugal

One hundred and twenty-seven unrelated Azorean individuals were randomly selected to study the gene frequencies of Killer-cell immunoglobulin-like receptors (KIR) in the Azorean (Terceira) population. KIR genotyping was performed by polymerase chain reaction using commercial sequence-specific oligonucleotide probe kits. All loci were in HWE, showing no locus-level deviations. The genotype data is available in the Allele Frequencies Net Database under the population name “Azores Terceira Island KIR”.

English

The management of colored cotton is an agricultural activity widely adopted by farmers located at Brazilian semiarid region. The fiber colors currently available are still limited to green and shades of brown, however, there is possibility to broaden the variability for this trait by using accessions from Gossypium Brazilian bank in breeding programs. Therefore, it is necessary to know the genetic diversity of available accessions in the collection. Here, the genetic divergence in colored fiber accessions was estimated in order to identify promising candidates for further use in hybridization procedures of cotton improvement. DNA of twelve accessions were extracted from leaves and used in inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) assays, using commercial oligonucleotides. The genetic divergence was estimated by clustering-unweighted pair group method with arithmetic mean (UPGMA) method. Five groups were clustered among them, three were contributive results for further use in hybridization procedures, including Brazilian cultivars and Peruvian accessions. Based on level of divergence, we suggest that lines generated from these materials could generate news shades of fiber colors in further use for selection procedures in cotton breeding. Key words: Gossypium, molecular marker, variability, genetic improvement.

Global gene expression profiling using heterologous DNA microarrays to analyze alterations in the transcriptome of Mus spretus mice living in a heavily polluted environment.

Microarray platforms are a good approach for assessing biological responses to pollution as they enable the simultaneous analyses of changes in the expression of thousands of genes. As an omic and non-targeted methodology, this technique is open to unforeseen responses under particular environmental conditions. In this study, we successfully apply a commercial oligonucleotide microarray containing Mus musculus whole-genome probes to compare and assess the biological effects of living in a heavily polluted settlement, the Domingo Rubio stream (DRS), at the Huelva Estuary (SW Spain), on inhabitant free-living Mus spretus mice. Our microarray results show that mice living in DRS suffer dramatic changes in gene and protein expression compared with reference specimens. DRS mice showed alteration in the oxidative status of hepatocytes, with activation of both the innate and the acquired immune responses and the induction of chronic inflammation, accompanied by metabolic alterations that imply the accumulation of lipids in the liver (hepatic steatosis). The identified deregulated genes may be useful as biomarkers of environmental pollution.

Transcriptional profiling reveals functional links between RasGrf1 and Pttg1 in pancreatic beta cells.

BackgroundOur prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those “pancreatic” phenotypes.ResultsHere, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins.Analysis of the mPttg1 promoter region identified specific recognition sites for numerous transcription factors which were also found to be differentially expressed in RasGrf1 KO pancreatic islets and are known to be relevant for Ras-ERK signaling as well as beta cell function. Reporter luciferase assays in BT3 insulinoma cells demonstrated the ability of RasGrf1 to modulate mPttg1 promoter activity through ERK-mediated signals. Analysis of the phenotypic interplay between RasGrf1 and Pttg1 in double knockout RasGrf1/Pttg1 mice showed that combined elimination of the two loci resulted in dramatically reduced values of islet and beta cell count and glucose homeostasis function which neared those measured in single Pttg1 KO mice and were significantly lower than those observed in individual RasGrf1 KO mice.ConclusionsThe specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1019) contains supplementary material, which is available to authorized users.

Heterologous Microarray Analysis of Transcriptome Alterations inMus spretusMice Living in an Industrial Settlement

This work demonstrates the successful application of a commercial oligonucleotide microarray containing Mus musculus whole-genome probes to assess the biological effects of an industrial settlement on inhabitant Mus spretus mice. The transcriptomes of animals in the industrial settlement contrasted with those of specimens collected from a nearby protected ecosystem. Proteins encoded by the differentially expressed genes were broadly categorized into six main functional classes. Immune-associated genes were mostly induced and related to innate and acquired immunity and inflammation. Genes sorted into the stress-response category were mainly related to oxidative-stress tolerance and biotransformation. Metabolism-associated genes were mostly repressed and related to lipid metabolic pathways; these included genes that encoded 11 of the 20 cholesterol biosynthetic pathway enzymes. Crosstalk between members of different functional categories was also revealed, including the repression of serine-protease genes and the induction of protease-inhibitor genes to control the inflammatory response. Absolute quantification of selected transcripts was performed via RT-PCR to verify the microarray results and assess interindividual variability. Microarray data were further validated by immunoblotting and by cholesterol and protein-thiol oxidation level determinations. Reported data provide a broad impression of the biological consequences of residing in an industrial area.

Genome-wide transcription analyses in Mycobacterium tuberculosis treated with lupulone.

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, still causes higher mortality than any other bacterial pathogen until now. With the emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR-TB) strains, it becomes more important to search for alternative targets to develop new antimycobacterial drugs. Lupulone is a compound extracted from Hops (Hurnulus lupulus), which exhibits a good antimicrobial activity against M. tuberculosis with minimal inhibitory concentration (MIC) value of 10 μg/mL, but the response mechanisms of lupulone against M. tuberculosis are still poorly understood. In this study, we used a commercial oligonucleotide microarray to determine the overall transcriptional response of M. tuberculosis H37Rv triggered by exposure to MIC of lupulone. A total of 540 genes were found to be differentially regulated by lupulone. Of these, 254 genes were upregulated, and 286 genes were downregulated. A number of important genes were significantly regulated which are involved in various pathways, such as surface-exposed lipids, cytochrome P450 enzymes, PE/PPE multigene families, ABC transporters, and protein synthesis. Real-time quantitative RT-PCR was performed for choosed genes to verified the microarray results. To our knowledge, this genome-wide transcriptomics approach has produced the first insights into the response of M. tuberculosis to a lupulone challenge.

138-P: HLA TYPING AMBIGUITY OF SEQUENCE-SPECIFIC OLIGONUCLEOTIDE TYPING PROTOCOLS

Using Shannon’s entropy, we objectively measured the HLA typing ambiguity of various commercial Sequence-Specific Oligonucleotide (SSO) protocols (groups of SSO kits used in tandem). We previously compared the HLA typing ambiguity obtained by serology, allele family level DNA-based typing, SSO, and single-pass sequence based typing (SBT). However, commercial companies have made significant advances in SSO technology in recent years, and those results only evaluated early generic versions of SSO kits. For several US populations, and for 5 HLA loci relevant to transplantation (HLA-A, -B, -C, -DRB1, -DQB1), we identify a set of commonly used SSO protocols. We use population-level haplotype frequency data to generate a cohort of simulated subjects, followed by 5-locus genotype imputation to generate a list of genotypes that would result from SSO typing each simulated subject. The imputation step computes the relative likelihood of each genotype, which is then used for entropy calculation. Distribution of entropies across populations and for each locus is generally similar, while the magnitudes differ. For example, entropy for locus HLA-A in African American population reaches 0.19, while the maximum entropy in Caucasians is 0.055. We generally observed that protocols comprised of newer kits had lower ambiguity across all populations. In certain populations, some newer kits had higher entropy than older ones. This may indicate those kits were designed to detect alleles uncommonly found in those populations, but resolving ambiguity in other populations. Ambiguity in SSO typing kits has reduced substantially since their introduction in the mid-1990s. Results also indicate a European focus toward selection of additional probes, which is suboptimal for typing diverse populations. We hope to use Shannon’s entropy next to evaluate group-specific sequence primer strategies with SBT. These results will guide selection of HLA typing methods by researchers and laboratories.

Solid-Phase Synthesis of DNA Chemical Sensor

Oligonucleotides are essential components of many applications in molecular biology. The synthesis chemistry is robust and commercial oligonucleotide synthesizers have taken advantage of the chemistry to provide oligonucleotides of high quality and purity. This paper established nucleic acid synthesis platform to carry out the synthesis of the labeled nucleic acid probes based on the DNA synthesizer and solid-phase synthesis technology. We chose to study the automated synthesis starting from DMT protected FAM labeled amidite attached to controlled pore glass (CPG) support and the standard trityl-off oligonucleotide synthesis cycle was performed, yielding the solid-supported oligonucleotide. The reported automated solid-phase oligonucleotide synthesis procedure successfully employs the common iterative synthesis, deblocking, activation, coupling, capping, oxidation, and isolation steps in standard oligonucleotide synthesis. The automated synthetic approach can also be applied to oligonucleotides of different length, composition of nucleotide, demonstrating the universality of the method. Moreover, the synthesis involved the use of commercially available, safe, stable, and inexpensive reagents, particularly advantageous and attractive for their use in automated solid-phase synthesis. The synthesis allows custom tailoring of their structure to the requirements of biological assays within hours, as opposed to traditional approaches that require weeks or months of work in the laboratory. Therefore it will become much easier to investigate biological interactions and optimize for objectives such as the receptor mediated targeting of oligonucleotides.

DNA Microarray Gene Expression Profile of Mycobacterium tuberculosis when Exposed to Osthole

Tuberculosis (TB), affecting one-third of the global population, kills an estimated two to three million people every year. The development of drug resistance is becoming a serious threat to any attempt to control this disease, which underscores the need for new agents targeting Mycobacterium tuberculosis (M. tuberculosis). Osthole (7-methoxy-8-isopentenoxycoumarin) is a coumarin derivative present in many medicinal plants. Previous studies have shown that osthole possesses antimycobacterial effects, however, the action mechanism of osthole is unclear. In the study, we used a commercial oligonucleotide microarray to determine the overall transcriptional response of M. tuberculosis H37Rv triggered by exposure to osthole. Analysis of the microarray data revealed that a total of 478 genes were differentially regulated by osthole. Of these, 241 genes were upregulated, and 237 genes were downregulated. Some of the important genes that were significantly regulated are related to different pathways such as fumarate reductase, class I peroxidase, cell wall, nitrate respiration, and protein synthesis. Real-time quantitative RT-PCR was performed for chosen genes to validate the microarray results. To our knowledge, this genome-wide transcriptomics approach has produced the first insights into the response of M. tuberculosis when exposed to osthole.