Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 16248646

A versatile toolbox for variable DNA functionalization at high density.

Stefan Jäger·Goran Rasched·Hagit Kornreich-Leshem·Marianne Engeser·Oliver Thum·Michael Famulok
View original source ↗Export metadata ↓

Abstract

To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.

Explore related subjects

Base SequenceBiotechnologyCircular DichroismDNADNA-Directed DNA PolymeraseDeoxyribonucleotidesElectrophoresis, Polyacrylamide GelNanotechnologyPolymerase Chain ReactionPolyphosphatesTemplates, GeneticComparative StudyJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Stefan Jäger, Goran Rasched, Hagit Kornreich-Leshem, Marianne Engeser, Oliver Thum, Michael Famulok. 2005-11-02. A versatile toolbox for variable DNA functionalization at high density.. https://doi.org/10.1021/ja051725b

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2016

miR-503-3p promotes epithelial-mesenchymal transition in breast cancer by directly targeting SMAD2 and E-cadherin.

Although progress in clinical and basic research has significantly increased our understanding of breast cancer, little is known about the molecular mechanism underlying breast cancer metastasis. Identification of effective therapeutic targets to prevent breast cancer metastasis is urgently needed. The function of miR-503-3p has been investigated in other cancers, but its role in breast cancer remains undefined. Here, we found that miR-503-3p was overexpressed in breast cancer tissue and plasma compared with adjacent normal breast tissue and with plasma from healthy individuals. Moreover, we identified miR-503-3p to be an oncogene of breast cancer cell proliferation, migration and invasion. Upregulation of miR-503-3p in breast cancer cells inhibited expression of epithelial-mesenchymal transition (EMT)-related protein SMAD2 and the epithelial marker protein E-cadherin by directly binding to their mRNA 3' untranslated region, whereas increased expression of mesenchymal marker proteins, including vimentin and N-cadherin. Taken together, our findings support a critical role for miR-503-3p in induction of breast cancer EMT and suggest that plasma miR-503-3p may be a useful diagnostic biomarker for breast cancer.

Base Sequence↗
PubMed2006

Increased expression of telomere length regulating factors TRF1, TRF2 and TIN2 in patients with adult T-cell leukemia.

Here, we report that freshly isolated unstimulated adult T-cell leukemia (ATL) cells present high telomerase activity compared to asymptomatic carriers or normal donors. In spite of this high telomerase activity, ATL cells retained shorter telomeres compared to those of uninfected cells isolated from the same patients. Because the safeguarding of telomere length is critical to the unlimited proliferation of tumor cells, we investigated the underlying mechanism for short telomere maintenance in ATL cells. Transcriptional and posttranscriptional expression of telomere-binding proteins TRF1, TRF2, TIN2 and POT1, known to regulate telomere homeostasis and protection, were evaluated. We found that TRF1 and TRF2 are overexpressed in in vivo patient's samples from ATL but not asymptomatic carriers, while levels of POT1 expression did not specifically increase in ATL. To gain insights into the regulation of TRF genes in HTLV-I infected cells, we investigated the expression of TIN2, a regulator of these genes, and found an increase in TIN2 expression in ATL patients. Together our results underscore the importance of telomerase and telomere length regulating factors as novel markers for ATL disease progression and as potential therapeutic targets for the treatment of HTLV-I-associated malignancies.

Base Sequence↗
PubMed2006

Mutations within Wnt pathway genes in sporadic colorectal cancers and cell lines.

Wnt signaling pathway activation via mutation of genetic components, commonly adenomatous polyposis coli (APC), has a major role in colorectal cancer (CRC). Most components have not been assessed for mutation in sporadic CRC. We have analyzed AXIN2, CK1alpha, DKK1, GSK-3beta, SOX17, LRP6 and PPP2R1B, beta-catenin and APC in a collection of sporadic CRCs (n = 47) and CRC cell lines (CLs; n = 26). The CRC set was enriched for microsatellite unstable cancers (MSI+, 30%, 14/47). Somatic mutation was not found in CK1alpha, DKK1, LRP6, beta-catenin or GSK-3beta; but heterozygous frame-shift mutations, and an in-frame deletion mutation were detected in exon 7 of AXIN2 (CRCs, 11%, 5/47; CLs, 8%, 2/26). Our data refute a previous suggestion that a CRC-related mutational hot-spot occurred in the Huntington elongation A subunit TOR (HEAT) repeat 2 of PPP2R1B; this "hotspot" is, more likely, a rare germline polymorphism. An early investigation proposing a high mutational frequency in HEAT repeat 13 was not substantiated. A heterozygous SOX17 mutation (L194P) was also found in a cell line. APC gene mutations were identified in 64% (30/47) of cancers and 7% of these (2/30) had an additional mutation in another Wnt gene. Overall, 70% (33/47) of CRCs had a somatic mutation in a Wnt pathway gene. The number of tumors containing such a mutation was not significantly higher in MSI+ (57%, 8/14) compared to MSI- (76%, 25/33) cancers (p = 0.3, Fisher's exact test); APC mutation was significantly increased in the MSI- subgroup (p = 0.02, Fisher's exact test). Further, mutational screening of other Wnt pathway genes is warranted.

Base Sequence↗