NUCLEIC ACID SYNTHESIS
The aim of the Nucleic Acid Synthesis Laboratory is to deliver high-quality DNA- and RNA oligomers quickly and directly for the ongoing BRC projects. We produce a great variety of specifically modified nucleic acid sequences on demand by chemical synthesis. We are also committed to the research of new applications of chemically modified oligonucleotides as structural elements and as genomic tools in various living organisms. In order to realize this goal, we also take part in various scientific collaboration projects with a number of research laboratories.
1. Natural and modified sequences
Oligonucleotides with natural structure, the working horses of molecular biology are involved in many known procedures as primers, probes and even total synthetic genes. On the other hand, chemically modified structures enable new applications – labeling, visualization, stabilization, conjugation, enzyme- and structural studies. As a collaboration partner, we synthesize almost all types of oligonucleotide derivatives, elaborate new compounds and actively participate in developing novel methods in diverse fields of molecular biology.
2. Targeted inhibition of gene expression
Targeted inhibition of gene expression is perhaps the most exciting area of oligonucleotide research. These types of compounds are able to act directly on living cells and organisms as active compounds or drugs. Oligonucleotides bind to the nucleic acids of the cells in a sequence-selective fashion, and thereby cause specific inhibition of gene expression. Our own research project is devoted to the research of various applications of this phenomenon. The so-called antisense oligonucleotide (ODN) and small inhibitory RNA projects are focused mainly to plant systems, and the final goal is to develop a novel genomic research tool.
2.1. Inhibition of the gene expression of different chloroplast proteins with antisense oligonucleotides (gene function analysis)
Selective inhibition of gene expression by ODNs in plants are scarce. We have studied them in different plant species, optimizing their uptake (Figure X1), stability, and efficiency with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. In Arabidopsis (Arabidopsis thaliana) leaves was achieved up to 85% and 72% inhibition of psbA mRNA and protein levels, respectively (Figure X2).1
Figure X1. Cellular distribution of fluorescein-labeled antisense ODNs (green) and Chl autofluorescence (red) in tobacco (G–L) leaves. Arrows in D to I show chloroplast accumulation of antisense ODNs. The arrow in J shows a nucleus filled with fluorescent antisense ODNs. Bars = 10 mm. (Dinc et al. 2011)
Figure X2. Effects of psbA antisense ODNs on the Chl a Fm (A), the relative transcript level (B) and the amount of D1 protein in ODN-treated Arabidopsis leaves aftre 48 h of illumination (C). Control leaves were treated with random nonsense ODNs. The inset in A shows OJIP transients of control and psbA4-treated leaves. A representative western blot is shown in the inset in C. (Dinc et al. 2011).
2.2. A novel polyploidization approach: Inhibition of the gene expression of microtubule associated proteins with antisense oligonucleotide
(collaboration with group: LABORATORY OF PLANT ARCHITECTURE AND DEVELOPMENT, Topic II and Ferhan Ayaydin (BRC) and Zoltán Kupihár (University of Szeged))
Biomass of fast growing woody plants is one of the most significant renewable resources for energy production. The flexibility of the genome of higher plants frequently allows autopolyploidization of plant species and generally one can observe that the increased and stabilized ploidy level frequently results in enlarged organ size and faster growth rate also in woody species. This project aims to produce tetraploid variants of the short rotation willow transient silencing of genes encoding microtubule associated proteins. Based on sequence data base of Poplar, we clone cDNAs of different tubulin interacting proteins of willow and design various antisense molecules for uptake experiments. After optimizing uptake protocols the gene expression level and DNA content of treated cells will be determined.
3. Improving the efficiency of synthetic Oligonucleotide Directed Mutagenesis (ODM)
(collaboration with group LABORATORY OF PLANT ARCHITECTURE AND DEVELOPMENT, Topic II, Elfrida Fodor (BRC), Ferhan Ayaydin (BRC), and Zoltán Kupihár (University of Szeged))
Targeted genome editing has been developed as an alternative to classical mutation breeding and transgenic (GMO) methods to improve crop plants. The Oligonucleotide Directed Mutagenesis (ODM) as Targeted Nucleotide Exchange (TNE) by single stranded DNA oligonucleotides (SDOs) attracts special attention for use in both basic science and plant breeding. SDOs are short synthetic fragments that are complementary in sequence to the targeted DNA and carry the desired mutation which can be exchange, deletion or insertion of preferably one or possibly two nucleotide units. By invading the targeted site of the duplex DNA, SDOs are hybridized to the complementary strand through transient D-loop formation. (Fig. 1) Finally, the mutation is introduced to the DNA by the cellular repair or replication machinery [Liu, L. et al. Nat Rev Genet, 2003. 4(9): 679-89]. SDOs are expected to be degraded in cells but the induced mutations will be stably inherited. The most effective SDOs are: 30-60mers. Beside the phosphorothioate end-protection against exonucleases, they carry several modified structures like Locked Nucleic Acid (LNA), N-alkinyl nucleotides in order to increase duplex invading capability [Andrieu-Soler, C. et al. Nucleic Acids Res, 2005. 33(12): 3733-42.]. The targeting efficacy is still too low for general use at an absolute scale.
Novel chemical modifications in single stranded oligonucleotides used for ODM
We construct novel forms of SDO molecules having structural elements which were not applied yet in mutation generation in the plant systems. We aim to clarify the influence of the new structural factors on the mutation efficacy, and develop novel methods, which will boost the practical use of ODM technology.
Dinc, E., Tóth, Sz. Z., Schansker, G., Ayaydin, F., Kovács, L., Dudits, D., Garab, Gy., and Bottka, S. (2011). Synthetic Antisense Oligodeoxynucleotides to Transiently Suppress Different Nucleus- and Chloro-plast-Encoded Proteins of Higher Plant Chloroplasts. Plant Physiology 157: 1628-1641.