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Humboldt-Universität zu Berlin - Faculty of Mathematics and Natural Sciences - Bioorganic Synthesis

DNA/RNA directed reactions


Key words: abasic site, DNA catalysis, DNA-directed synthesis, DNA-directed transfer reactions, ligation assay, FRET, multiplex assay, native chemical ligation, PCR-less DNA detection, peptide nucleic acids, product inhibition, signal amplification, single base mutation, single base mutations, SNP, templates, template catalysis, turnover


1. DNA/RNA-controlled ligation


Ligation reactions that proceed under the control of a DNA-like template illustrate how chemical reactivities can be controlled by Watson-Crick base-pairing. We design chemical ligatioFigure_03n reactions such that product formation would prove indicative for a certain single base mutation in DNA or RNA. We frequently use the DNA-analogue peptide nucleic acid (PNA) which binds complementary DNA with higher affinity and sequence specificity than DNA itself. The ligation strategy is that of a peptide coupling.[4]

We favour the use of native chemical ligation, a very powerful reaction known in protein chemistry. One PNA-probe is equipped with a thioester, the other features an N-terminal cysteine or isocysteine residue. In presence of a complementary oligonucleotide-template a ternary complex forms and the peptide ligation becomes accelerated (Fig 3).

The native chemical PNA-ligation developed by us proceeds as rapidly and more selective than ligase mediated oligonucleotide ligations.[5] The DNA template can induce more than 40.000-fold rate acceleration and the sequence specificity can be higher than 4000-fold in discriminating matched from single mismatched DNA. This can facilitate the detection of early cancer onset. As an example we have shown that as little as 0.2% of single-base mutated DNA in presence of 99.8% wild-type DNA can be detected by mass-spectrometric analysis of the PNA-native chemical ligation. We have also demonstrated the application of the PNA-ligation chemistry to double stranded DNA-templates produced by PCR.

We are currently taking advantage of the extreme sequence specificity of native chemical PNA ligation in the single base mutation analysis of “real world” DNA.Figure_04 We interfaced DNA-directed chemistry with PCR. By using this method attomolar amounts of template DNA are sufficient to instruct the formation of functional molecules while maintaining the extraordinary sequence specificity provided by DNA-templated reactions. However, the ‘extreme’ conditions are a challenge. Chemoselectivity has to be maintained in the presence of proteins and nucleic acids up to 95°C without increasing off-template reactions, hydrolysis and/or affecting PCR efficiency. We designed reaction scheme shown in Figure 5. The labelling with fluoresceine (F1) and rhodamine (F2) enables real-time monitoring of product formation due to fluorescence resonance energy trFigure_05ansfer.


It was of particular importance to adjust the reactivity of the thioster, which tends to hydrolyse under the conditions of PCR. We found that a ß-alanine thioester led to a significant stabilization without affecting the rate of the template reaction. The reaction on matched template was 970-fold faster than the background reaction in absence of template (Fig. 6A). We introduced the reactive probes into PCR mixes used to amplify a segment of human genomic DNA, which harbors a certain point mutation in the BRaf gene. The amplification curves of a dilution series show that 2.5 aM template could be distinguished from the no-template control (Figure 6B).





[4]  A. Mattes, O. Seitz, Angew. Chem. Int. Ed. 2001, 40, 3178-+.

[5] a) S. Ficht, A. Mattes, O. Seitz, J. Am. Chem. Soc. 2004, 126, 9970-9981; b) S. Ficht, C. Dose, O. Seitz, ChemBioChem 2005, 6, 2098-2103; c) C. Dose, S. Ficht, O. Seitz, Angew. Chem. Int. Ed. 2006, 45, 5369-5373; d) C. Dose, O. Seitz, Bioorg. Med. Chem. 2008, 16, 65-77.




2. DNA/RNA-triggered transfer reaction


Amplification of product signals is desired when the DNA or RNA is present at low concentration. We design reactions that are amenable to catalysis by non-structured DNA. It is our long-term goal to obtain catalytic efficiencies that may allow intracellular applications and "PCR-less" DNA-detection.

Template-controlled ligation reactions suffer from an increased affinity of the product to the DNA template. This causes product inhibition and prevents high catalytic activity of the nucleic-acid template. Our design of a reaction in which DNA acts as a catalyst involves a transfer reaction (Fig. 7).[6] For example, a DNA/RNA-triggered acyl transfer can be used to transfer a thioester-linked dabcyl reporter group (dark red, Fig 7) from the donor conjugate Figure_07(orange) to the accepting probe (blue), which bears an N-terminal iCys capable of attacking the thioester in a native chemical ligation-like fashion. Prior to transfer the FAM fluorescence of the donating probe is quenched by dabcyl. Transfer of dabycl results in an increase of FAM fluorescence. Simultaneously, the fluorescence intensity of TAMRA decreases, owing to the transfer of Dabcyl to accepting probe. This reaction scheme allows for ³ 400 turnovers and has been applied to the transfer of biotin, pyrene and Cy3.[6]

Very recently, we developed a benzylidene transfer reaction, which provides for signal amplification based on de novo fluorophore synthesis.[7] Here a gain in the signal is provided by two consecutive amplification mechanisms: a) a Wittig transfer reaction which facilitates turnover, because the probes after the reaction offer the same number of nucleotides for base pairing with the template as the probes before the reaction, and b) selective binding of the newly formed fluorophore to a receptor (Fig. 8). This consecutive approach leads to very low background, delivers a more than 100-fold enhancement in the fluorescence resulting in a more than 300-fold gain of the signal compared to a reactions in the absence of a suitable DNA template.Figure_08

The concept of consecutive signal amplification system is based on the formation of double bonds and additionally of host–guest chemistry for the detection of DNA.



[6]  a) T. N. Grossmann, O. Seitz, J. Am. Chem. Soc. 2006, 128, 15596-15597; b) T. N. Grossmann, L. Röglin, O. Seitz, Angew. Chem. Int. Ed. 2008, 47, 7119-7122; c) T. N. Grossmann, O. Seitz, Chem. Eur. J. 2009, 15, 6723-6730.

[7]  X. H. Chen, A. Roloff, O. Seitz, Angew. Chem. Int. Ed. 2012, 51, 4479-4483.