Falk Rohrbach

Light-controlled tools.

Spatial and temporal control over chemical and biological processes plays a key role in life, where the whole is often much more than the sum of its parts. Quite trivially, the molecules of a cell do not form a living system if they are only arranged in a random fashion. If we want to understand these relationships and especially the problems arising from malfunction, tools are necessary that allow us to design sophisticated experiments that address these questions. Highly valuable in this respect are external triggers that enable us to precisely determine where, when, and to what extent a process is started or stopped. Light is an ideal external trigger: It is highly selective and if applied correctly also harmless. It can be generated and manipulated with well-established techniques, and many ways exist to apply light to living systems--from cells to higher organisms. This Review will focus on developments over the last six years and includes discussions on the underlying technologies as well as their applications.

Label-free impedimetric aptasensor for lysozyme detection based on carbon nanotube-modified screen-printed electrodes

We report on the direct electrochemical detection of aptamer-protein interactions, namely between a DNA aptamer and lysozyme (LYS) based on electrochemical impedance spectroscopy (EIS) technique. First, the affinity of the aptamer to LYS and control proteins was presented by using filter retention assay. An amino-modified version of the DNA aptamer-recognizing lysozyme was covalently immobilized on the surface of multiwalled carbon nanotube-modified screen-printed electrodes (MWCNT-SPEs), which were employed for measurements and have improved properties compared with bare SPEs. This carbon nanotube setup enabled the reliable monitoring of the interaction of lysozyme with its cognate aptamer by EIS transduction of the resistance to charge transfer (R(ct)) in the presence of 2.5 mM [Fe(CN)₆]³⁻/⁴⁻. This assay system provides a means for the label-free, concentration-dependent, and selective detection of lysozyme with an observed detection limit of 12.09 μg/ml (equal to 862 nM).

Monitoring of plasma levels of activated protein C using a clinically applicable oligonucleotide-based enzyme capture assay

Summary.  Background: Human‐activated protein C (APC) is a serine protease with anticoagulant, anti‐inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular‐inflammatory biomarker.Objective: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions.Methods/patients: Based on the APC‐binding ssDNA aptamer HS02‐52G we developed an oligonucleotide‐based enzyme capture assay (OECA) that quantifies aptamer‐captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre‐analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery.Results and conclusion: A combination of APC–OECA with an aprotinin‐based quenching strategy allowed APC analysis with a limit of detection as low as 0.022 ± 0.005 ng mL−1 (0.39 ± 0.10 pmol L−1) and a limit of quantification of 0.116 ± 0.055 ng mL−1 (2.06 ± 0.98 pmol L−1). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC–OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12 ng mL−1 (214 pmol L−1). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn‐around time from blood sampling to generation of test results of < 7 h, the APC–OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions.

Chemical maturation of a bivalent aptamer by single domain variation

Aptamers are short nucleic acids that bind with high affinity and specificity to diverse target molecules. They are identified in vitro by specific selection schemes from combinatorial nucleic acid libraries. Aptamers represent a promising class of therapeutics as they potently inhibit the biological functions of their targeted molecules. To improve aptamers’ bioavailability and pharmacokinetic profiles, it is often necessary to improve aptamers’ stability, half-life in biological media, affinity, and activity. The last two of these have been achieved mainly through the partial mutation of aptamers and suitable reselection strategies. We recently made use of the modular character of aptamer domains and assembled a bivalent aptamer, HD1-22, which concurrently targets and inhibits both exosites of thrombin, but with a higher activity than its monovalent precursor subdomains. We now describe a chemical approach towards aptamer maturation that ultimately leads to a bivalent aptamer variant with superior properties in respect to affinity and the inhibition of thrombin-mediated coagulation. Despite its chemical modification, complementary antidote molecules can still efficiently control the activity of the improved aptamer, thus making it a potent but also safe drug candidate for anticoagulant therapy. Our hypothesis was that improving one subunit of a modularly assembled bivalent aptamer by chemical modification (Scheme 1 A) and subsequent reassembly of the analogous bivalent variant might yield a novel multivalent aptamer with superior overall activities (Scheme 1 B). Terminal modification or incorporation of polycyclic aromatic hydrocarbons (PAHs), such as pyrene molecules, into DNA strands was reported to provide intercalating moieties for assembling DNA nanoarchitectures or excimer probes for the detection of ligand-induced conformational changes of DNA. Improved conformational stability and, thus, activity of (R)-1-(4-(1-pyrenylethynyl)phenylmethyl)glycerol (TINA) bearing G-quadruplex DNA molecules has been described recently. However, neither of these PAHs have been used systematically to modulate and improve the functional properties of aptamers. To address this challenge we synthesized variants of the HD1 subdomain of HD1-22 with different PAHs at various positions (Table 1) and examined their biological and biophysical properties. We synthesized aptamer variants with either insertions or replacements of nucleotides by using three different PAHs, namely ortho-TINA (O) ((R)-3-((2-(1-pyrenylethynyl)benzyl)oxy)propane-1,2-diol), TINA (P) ((R)-3-((4-(1-pyrenylethynyl)benzyl)oxy)propane-1,2-diol) and Amany (Y) ((S)-4-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)phenoxy)butane-1,2-diol) (Scheme 1 A). We then assessed their inhibitory potential by measuring their impact on routine plasma-based coagulation assays, such as the thrombin time and the activated partial thromboplastin time. Most of the variants that had insertions of either TINA or ortho-TINA PAH revealed a decrease in affinity and inhibitory potential (Figure S1 in the Supporting Information). However, insertions in the TGT-loop region (nucleotides 7–9) of HD1 were tolerated to some extent. ortho-TINA moieties in the TGTloop region (HD1 i8O and HD1 i9O) led to a decrease in aptamer properties, whereas some variants with TINA residues in this region (HD1 i8P and HD1 i9P) revealed a higher affinity but almost unchanged anticoagulant properties (Figure S1). Overall, the insertion of PAHs into the oligonucleotide sequence did not result in reasonable aptamer improvement. Nevertheless, these data are in accordance with previous findings that define the TGT-loop region as a potential site for modifications without alteration of aptamer properties. We next investigated variants with PAH nucleotide replacements rather than insertions in the TGT-loop region. As shown in Figure 1, an improvement in the anticoagulant activities of some variants that bear ortho-TINA, TINA, or Amany moieties within the TGT-loop region of the HD1 aptamer was observed. The strongest improvement was obtained by replacing the nucleotide G8 (HD1 r8P) by a TINA or an Amany moiety (Figures 1 and S2). As already evident from the studies with the insertion of PAHs, replacement of G8 by TINA was superior to replacement by ortho-TINA. Importantly, the modulation of aptamer activity was also observed to be site selective. Precisely, replacement of nucleotide T7 was quite well tolerated, whereas substitution of nucleotide T9 by TINA or Amany led to a significant loss of aptamer activity. These data are in good agreement with results obtained from affinity measurements (Figure 2 A and Table 2) and data from circular dichroism (CD) spectroscopy (Figure 2 B–D). Replacement of nucleotide G8 with TINA or Amany PAHs led to lower KD values than when T7 or T9 was substituted (Figure 2 A and Table 2). In contrast, replacement of any position in the TGT-loop residues by ortho-TINA moieties had only a moderate impact on the determined dissociation constants (Figure 2 A and Table 2). Similarly, the HD1 variants with improved activity and affinity (HD1 r8P and HD1 r8Y) maintained G-quadruplex structures (Figure 2 B). Again CD spectra of variants with ortho[a] F. Rohrbach, Prof. G. Mayer Life and Medical Sciences Institute, University of Bonn Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany) E-mail : gmayer@uni-bonn.de [b] M. I. Fatthalla, Prof. M. Petersen, Prof. E. B. Pedersen Nucleic Acids Center, University of Southern Denmark Campusvej 55, 5230 Odense M (Denmark) [c] T. Kupper, Prof. B. Pçtzsch, Dr. J. M ller Institute for Experimental Hematology and Transfusion Medicine University Hospital Bonn Sigmund-Freud-Strasse 25, 53127 Bonn (Germany) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201200015.

Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer

Aptamers are valuable tools that provide great potential to develop cost-effective diagnostics and therapies in the biomedical field. Here, we report a novel DNA aptamer that folds into an unconventional G-quadruplex structure able to recognize and enter specifically into human Burkitts lymphoma cells. We further optimized this aptamer to a highly versatile and stable minimized version. The minimized aptamer can be easily equipped with different functionalities like quantum dots, organic dyes, or even a second different aptamer domain yielding a bi-paratopic aptamer. Although the target molecule of the aptamer remains unknown, our microscopy and pharmacological studies revealed that the aptamer hijacks the clathrin-mediated endocytosis pathway for its cellular internalization. We conclude that this novel class of aptamers can be used as a modular tool to specifically deliver different cargoes into malignant cells. This work provides a thorough characterization of the aptamer and we expect that our strategy will pave the path for future therapeutic applications.

Plug and Play with RNA

Retooling RNA: RNA aptamers are high-affinity ligands that can be assembled with other structures to yield multivalent molecules. These properties have been addressed in two recent studies: One describes a GFP-like RNA reporter used to study the dynamics of endogenous RNA; the other study reports on an aptamer-templated assembly of multi-enzyme complexes in bacteria for the controlled production of secondary molecules (see picture).