New carbamate supports for the preparation of 3'-amino-modified oligonucleotides

A novel approach for the preparation of oligonucleotides carrying amino groups at the 3'-end is described. Several CPG supports having aminoalkyl groups and 3'-amino-2',3'-dideoxynucleosides linked through base-labile carbamate linkages such as 2-(2- nitrophenyl)ethoxycarbonyl and fluorenylmethoxycarbonyl were prepared using two different strategies. These supports are compatible to the standard solid phase phosphite-triester methodology and yield oligonucleotides containing amino groups at the 3'-end. Several properties of the 3'-amino oligonucleotides, such as nuclease resistance, hybridization, and preparation of oligonucleotide conjugates are discussed.Financial support from CICYT (PB92-0043) and E.E.C.C. Biomedicine and Health Programme (BMH1-CT93-1669) is gratefully acknowledged. We thank Drs P. Herdewijn, A. van Aerschot, T. Saison- Behmoaras, and W. Pfleiderer for their helpful suggestions. We are grateful to Marten Wiersma for his technical assistance.Peer reviewe

Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field

RNA-seq analysis in plant–fungus interactions

Many fungi are pathogens that infect important food and plantation crops, reducing both yield and quality of food products. Understanding plant–fungus interactions is crucial as knowledge in this area is required to formulate sustainable strategies to improve plant health and crop productivity. High-throughput RNA-sequencing (RNA-seq) enables researchers to gain insights of the mixed and multispecies transcriptomes in plant–fungus interactions. Interpretation of huge data generated by RNA-seq has led to new insights in this area, facilitating a system approach in unraveling interactions between plant hosts and fungal pathogens. In this review, the application and challenges of RNA-seq analysis in plant–fungus interactions will be discussed

Inhibition of Hha I DNA (cytosine-c5) methyltransferase by oligodeoxyribonucleotides containing 5-Aza-2′-deoxycytidine: Examination of the intertwined roles of co-factor, target, transition state structure and enzyme conformation

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between Hha I methyltransferase (M.Hha I) and oligo-deoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.Hha I. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.Hha I that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.Hha I.Partial support for this work was provided by the DAMD Breast Cancer Program (DAMD 17-98-1-8215) to JKC and fellowship support from the Graduate College at UNMC to ASB. We are also greatful to Drs. X. Cheng and S. Kumar for their generous gifts of purified M.HhaI.Peer reviewe

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2′-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.a European Molecular Biology Laboratory, D-69117 Heidelberg, Germany b Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 69198-4525, United States c UNMC/Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 69198-4525, United States d Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, United States e Institut de Biología Molecular de Barcelona, CSIC, Jordi Girona 18-26, E-08034 Barcelona, SpainN

The use of wideband filters in distinguish green fluorescent protein in roots of arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) fungi form arbuscules in the inner cortical cells of roots. Accumulation of autofluorescent materials within the roots, especially around senescent arbuscules, has hampered analyses of the localization and dynamics of green fluorescent protein (GFP)-fusion proteins in arbusculated cells. In this report, the author proposes an efficient method to distinguish GFP from autofluorescence. To detect GFP fluorescence, colonized cells were observed with wideband filters rather than GFP-specialized filters because the autofluorescence generally has a broad fluorescent spectrum that can easily be distinguished from GFP by color. Moreover, the autofluorescence of arbusculated cells could possibly contain strong green fluorescence that could not be excluded by GFP-specialized filters. The multicolor imaging and in vivo real-time observations suggested that the expression of autofluorescence in arbusculated cells did not overlap with the expression of OsPT11-GFP, a useful marker for active arbuscules, and that autofluorescent materials appeared after the initiation of senescence in infection units

Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus

International audienceOur view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance