Biomedical subjects

Sakari Kauppinen

Publications and source records attributed to Sakari Kauppinen.

12 recordsLinked to original sources

PubMed2006

LNA-modified oligonucleotides mediate specific inhibition of microRNA function.

microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been observed in many human cancers. Here we present a method for specific inhibition of miRNA function through interaction with LNA-modified antisense oligonucleotides and report the specificity of this application. We show that LNA-modified oligonucleotides can inhibit exogenously introduced miRNAs with high specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous miRNA in Drosophila melanogaster cells, leading to up-regulation of the cognate target protein. The method shows stoichiometric and reliable inhibition of the targeted miRNA and can thus be applied to studies of miRNA functions and validation of putative target genes.

Animals↗

PubMed2006

In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes.

MicroRNAs (miRNAs) are 20-23 nucleotide (nt) RNA molecules that regulate gene expression post-transcriptionally. A key step toward understanding the function of the hundreds of miRNAs identified in animals is to determine their expression during development. Here we performed a detailed analysis of conditions for in situ detection of miRNAs in the zebrafish embryo using locked nucleic acid (LNA)-modified DNA probes and report expression patterns for 15 miRNAs in the mouse embryo.

Animals↗

PubMed2006

Spatio-temporal accumulation of microRNAs is highly coordinated in developing plant tissues.

MicroRNAs (miRNAs) are an abundant class of small, endogenous non-protein-coding RNAs, approximately 21 nucleotides in length, that modulate the expression of animal and plant target genes at the post-transcriptional level. Recent work has shown that miRNA-based gene regulation plays a crucial role in pathways involved in plant growth and development. However, knowledge about the timing and spatial regulation of plant miRNA expression is still limited. Here we used in situ analysis to demonstrate that miRNAs accumulate spatially and temporally in a highly restricted manner in Nicotiana benthamiana and Arabidopsis thaliana. The presence of the seven investigated miRNAs was characteristic of the developing organs, implying a role in cell-fate establishment, differentiation and cell-cycle progression. Spatial analyses revealed that six of the studied miRNAs were present in vascular bundles, suggesting that mobile miRNAs in the phloem could contribute to the coordination of organogenesis and development. The obvious absence of miR167 in vascular bundles represented an exception to this observation, implying an active process in regulating the presence of miRNAs in the vascular system. Taken together, our results imply that the spatially and temporally organized accumulation of miRNAs plays a pivotal role in fine-tuning of target gene expression in plant development.

Arabidopsis↗

PubMed2005

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain.

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.

Brain↗

PubMed2005

MicroRNA expression in zebrafish embryonic development.

MicroRNAs (miRNAs) are small noncoding RNAs, about 21 nucleotides in length, that can regulate gene expression by base-pairing to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in situ hybridizations, using locked-nucleic acid-modified oligonucleotide probes. Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity.

Animals↗

PubMed2004

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes.

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.

Animals↗

PubMed2004

Expression profiling by oligonucleotide microarrays spotted on coated polymer slides.

We have developed a ready-to-spot polymer microarray slide, which is coated with a uniform layer of reactive electrophilic groups using anthraquinone-mediated photo-coupling chemistry. The slide coating reduces the hydrophobicity of the native polymer significantly, thereby enabling robust and efficient one-step coupling of spotted 5' amino-linked oligonucleotides onto the polymer slide. The utility of the coated polymer slide in gene expression profiling was assessed by fabrication of spotted oligonucleotide microarrays using a collection of 5' amino-linked 70-mer oligonucleotide probes representing 96 yeast genes from Operon. Two-colour hybridizations with labelled cDNA target pools derived from standard grown and heat-shocked wild type yeast cells could reproducibly measure heat shock induced expression of seven different heat shock protein (HSP) genes. Moreover, the observed fold changes were comparable to those reported previously using spotted cDNA arrays and high-density 25-mer oligonucleotide arrays from Affymetrix. The low hybridization signals obtained from the DeltaSSA4 mutant cDNA target, together with the high signal detected in two-colour hybridizations with heat-shocked wild type yeast relative to the DeltaSSA4 mutant strain implies that unspecific binding of cDNA target to the SSA4-specific 70-mer oligonucleotide probes is negligible. Combined, our results indicate that the coated polymer microarray slide represents a robust and cost-effective array platform for pre-spotted oligonucleotide arrays.

Adsorption↗

PubMed2004

Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture.

LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T-A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50-100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.

Actins↗

PubMed2003

OligoDesign: Optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling.

We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.

Animals↗

PubMed2002

Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases.

Innate immunity, an ancient form of defense against microbial infection, is well described for animals and is also suggested to be important for plants. Discrimination from self is achieved through receptors that recognize pathogen-associated molecular patterns (PAMPs) not found in the host. PAMPs are evolutionarily conserved structures which are functionally important and, thus, not subject to frequent mutation. Here we report that the previously described peptide elicitor of defense responses in parsley, Pep-13, constitutes a surface-exposed fragment within a novel calcium-dependent cell wall transglutaminase (TGase) from Phytophthora sojae. TGase transcripts and TGase activity are detectable in all Phytophthora species analyzed, among which are some of the most destructive plant pathogens. Mutational analysis within Pep-13 identified the same amino acids indispensable for both TGase and defense-eliciting activity. Pep-13, conserved among Phytophthora TGases, activates defense in parsley and potato, suggesting its function as a genus-specific recognition determinant for the activation of plant defense in host and non-host plants. In summary, plants may recognize PAMPs with characteristics resembling those known to trigger innate immune responses in animals.

Amino Acid Motifs↗

PubMed2002

LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E.

Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs are embedded in GC-rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) capture probes combined with LNA-enhancer oligonucleotides to obtain efficient and specific interrogation of SNPs in the apoE codons 112 and 158, respectively. The results demonstrate the usefulness of LNA oligonucleotide capture probes combined with LNA enhancers in mismatch discrimination. The assay was applied to a panel of patient samples with simultaneous genotyping of the patients by DNA sequencing. The apoE genotyping assays for the codons 112 and 158 SNPs resulted in unambiguous results for all patient samples, concurring with those obtained by DNA sequencing.

Apolipoproteins E↗

PubMed2002

A stepwise optimization of crystals of rhamnogalacturonan lyase from Aspergillus aculeatus.

Recombinant rhamnogalacturonan lyase from Aspergillus aculeatus has been crystallized by a stepwise procedure and X-ray diffraction data have been collected. The crystals were grown using hanging-drop vapour-diffusion and microseeding techniques. Crystals were obtained showing a flat plate morphology. The crystallization conditions were 20% PEG 4000, 9% PEG 400, 0.1 M (NH(4))(2)SO(4) and 0.1 M sodium acetate pH 4.4. These crystals diffracted to a resolution of 1.5 A. The unit-cell parameters are a = b = 77.0, c = 170.8 A with the possible space group P4(3)2(1)2 or P4(1)2(1)2. There is most likely to be one molecule in the asymmetric unit, leading to a calculated solvent content of approximately 47% for the crystals.

Aspergillus↗