An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans

Backer, De; Nelissen, Bart; Logghe, Marc; Viaene, Jozef; Loonen, I; Vandoninck, Sandy; Hoogt, Ronald de; Dewaele, Stijn; Simons, FA; Verhasselt, Peter; Vanhoof, Greet; Contreras, Roland; Luyten, Walter

doi:10.1038/85677

Cited by 112 publications

(56 citation statements)

References 30 publications

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“…By combining these multiple diverse data sets, we can construct detailed functional networks, where relationships between genes is established by direct experimental manipulation of their expression. Related efforts using vector-based approaches have already been described for antisense and siRNA for use in either Candida albicans (De Backer et al, 2001) Antisense oligonucleotides are also a valuable technology to suppress gene expression in animals. Like tissue culture studies, the development of the 2 0 -MOEmodified antisense oligonucleotide has been of considerable value in facilitating our ability to generate potent and long-lasting agents to inhibit gene expression in animals.…”

Section: Antisense Oligonucleotide Drug Discovery and Target Validationmentioning

confidence: 99%

Antisense oligonucleotide-based therapeutics for cancer

2003

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There has been steady progress in antisense technology over the past 14 years. We now have a far better appreciation of the attributes and limitations of the technology. Antisense oligonucleotides have been used to selectively inhibit thousands of genes in mammalian cells, hundreds, if not thousands, of genes in rodents and other species and multiple genes in humans. There are over 20 antisense drugs currently in clinical trials, several of which are showing promising results. Like any other class of drugs in development, there will continue to be successes and failures in the clinic. Despite some disappointments with the technology, it appears to be a valid platform for both drug discovery and as an experimental tool for functionalizing genes. Advances in the medicinal chemistry and formulation of antisense oligonucleotides will further enhance their therapeutic and commercial potential.

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Section: Antisense Oligonucleotide Drug Discovery and Target Validationmentioning

confidence: 99%

Antisense oligonucleotide-based therapeutics for cancer

2003

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“…Such screens represent a massive investment of time and resources, and they are not always feasible. For instance, traditional essentiality screens are difficult in Candida albicans because of its partial-diploid nature, mating differences, and a lack of insertional mutagenesis methods (De Backer et al 2001). Furthermore, recent shotgun sequencing of microbial communities has suggested that <1% of species are amenable to laboratory culture (Riesenfeld et al 2004;Chen and Pachter 2005).…”

mentioning

confidence: 99%

Predicting essential genes in fungal genomes

Seringhaus¹,

Paccanaro²,

Borneman³

et al. 2006

Genome Res.

111

107

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Essential genes are required for an organism's viability, and the ability to identify these genes in pathogens is crucial to directed drug development. Predicting essential genes through computational methods is appealing because it circumvents expensive and difficult experimental screens. Most such prediction is based on homology mapping to experimentally verified essential genes in model organisms. We present here a different approach, one that relies exclusively on sequence features of a gene to estimate essentiality and offers a promising way to identify essential genes in unstudied or uncultured organisms. We identified 14 characteristic sequence features potentially associated with essentiality, such as localization signals, codon adaptation, GC content, and overall hydrophobicity. Using the well-characterized baker's yeast Saccharomyces cerevisiae, we employed a simple Bayesian framework to measure the correlation of each of these features with essentiality. We then employed the 14 features to learn the parameters of a machine learning classifier capable of predicting essential genes. We trained our classifier on known essential genes in S. cerevisiae and applied it to the closely related and relatively unstudied yeast Saccharomyces mikatae. We assessed predictive success in two ways: First, we compared all of our predictions with those generated by homology mapping between these two species. Second, we verified a subset of our predictions with eight in vivo knockouts in S. mikatae, and we present here the first experimentally confirmed essential genes in this species.

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“…HP1 is a mimic of a rRNA sequence in C. albicans that forms quaternary and tertiary contacts in both Escherichia coli (19,28) and Thermus thermophilus (25) ribosomes. 10 200 Ϯ 50 dT 20 600 Ϯ 150 dT 30 500 Ϯ 80 dT 40 650 Ϯ 104 *Intracellular concentrations of 2ЈOMe HP1 at pH 2, 3, 3.5, 4, and 4.5 are 180 Ϯ 60, 100 Ϯ 32, 56 Ϯ 20, 34 Ϯ 7, and 38 Ϯ 10 M, respectively, when the initial OD 540 is 0.01.…”

Section: Resultsmentioning

confidence: 99%

“…The C. albicans genome is being sequenced, and a functional genomics approach has identified essential genes in this organism (10), so the database of targetable sequence is increasing. Biochemical information discovered using related organisms, such as S. cerevisiae, should also facilitate rational design.…”

Section: Discussionmentioning

confidence: 99%

“…In vivo dimethyl sulfate (DMS) modification of rRNA and rates of protein synthesis suggest that the inhibitory effect may be due to targeting of the ribosome. These results lay the foundation for development of oligonucleotide therapeutics toward C. albicans and other fungi by exploiting the information from genome sequencing and functional genomics projects (10).…”

mentioning

confidence: 99%

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Uptake and antifungal activity of oligonucleotides in Candida albicans

Disney

Haidaris

Turner

2003

Proc. Natl. Acad. Sci. U.S.A.

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Candida albicans is a significant cause of disease in immunocompromised humans. Because the number of people infected by fungal pathogens is increasing, strategies are being developed to target RNAs in fungi. This work shows that oligonucleotides can serve as therapeutics against C. albicans. In particular, oligonucleotides are taken up from cell culture medium in an energydependent process. After uptake, oligonucleotides, including RNA, remain mostly intact after 12 h in culture. For culture conditions designed for mammalian cells, intracellular concentrations of oligonucleotides in C. albicans exceed those in COS-7 mammalian cells, suggesting that uptake can provide selective targeting of fungi over human cells. A 19-mer 2 OMe (oligonucleotide with a 2 -O-methyl backbone) hairpin is described that inhibits growth of a C. albicans strain at pH < 4.0. This pH is easily tolerated in some parts of the body subject to C. albicans infections. In vivo dimethyl sulfate modification of ribosomal RNA and the decreased rate of protein synthesis suggest that this hairpin's activity may be due to targeting the ribosome in a way that does not depend on base pairing. Addition of anti-C. albicans oligonucleotides to COS-7 mammalian cells has no effect on cell growth. Evidently, oligonucleotides can selectively serve as therapeutics toward C. albicans and, presumably, other pathogens. Information from genome sequencing and functional genomics studies on C. albicans and other pathogens should allow rapid design and testing of other approaches for oligonucleotide therapies.T he fungus Candida albicans infects humans and is particularly pernicious for immunocompromised hosts, such as cancer and AIDS patients. Because the number of humans infected by fungi and the occurrence of resistant strains are increasing, finding new compounds to treat these infections is important (1). In vitro results suggest that oligonucleotides are promising compounds for targeting RNA in fungal pathogens (2, 3).Oligonucleotides have several advantages for targeting RNA. Pairing rules between an oligonucleotide and RNA facilitate rational design. Gene expression can be reduced by RNase H cleavage of target RNA (4) or by blocking mRNA translation (5). Oligonucleotides can also serve as suicide inhibitors (2, 3) or misfold RNAs (6). Nucleic acids are easily synthesized (7), which allows rapid testing of oligonucleotides of different composition. Properties such as higher affinity and nuclease stability can be programmed into oligonucleotides by modifying the backbone, sugar, or base (8).The use of oligonucleotides in cell culture poses several challenges. For example, uptake into mammalian cells is not efficient and oligonucleotides are often degraded after entering cells (4). Transfection agents, small molecules, and peptides have been used to circumvent these problems in mammalian cells (9). Little is known, however, about oligonucleotide uptake into other organisms, such as fungi. Cell membranes and metabolism differ substantially between fungi and mam...

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An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans

Cited by 112 publications

References 30 publications

Antisense oligonucleotide-based therapeutics for cancer

Antisense oligonucleotide-based therapeutics for cancer

Predicting essential genes in fungal genomes

Uptake and antifungal activity of oligonucleotides in Candida albicans

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