New Trends in Nucleic Acids Based Biosensors

25-28 October 2003
Firenze, Italy

Organisers

Scientific Committee
Prof. Marco Mascini (University of Florence, Italy)
Prof. Ulf Landegren (University of Uppsala, Sweden)
Dr. Ettore Luzi (University of Florence, Italy)
Dr. Maria Minunni (University of Florence, Italy)

Local organising committee(Biosensors Laboratory of the Department of Chemistry, University of Florence)
Prof. Marco Mascini
Dr. Ettore Luzi
Dr. Maria Minunni
Prof. Giovanna Marrazza
Dr. Sara Tombelli
Dr. Serena Laschi

Location of the Workshop:
Main Lecture Hall
"Polo Scientifico di Sesto Fiorentino"
University of Florence
Via Bernardini 6
50019 Sesto Fiorentino, Firenze, Italy
Phone: +39 055 4573283 - Fax: +39 055 4573384

Introduction

The first workshop on New Trends in Nucleic Acids Based Biosensors was held in the "Polo Scientifico of Sesto Fiorentino", Florence (Italy), from 25th to 28th October 2003. The Workshop was sponsored by the European Science Foundation (Programme on Integrated Approaches for Functional Genomics) and by the University of Florence.
The workshop featured on all aspects of the highly interdisciplinary area of nucleic acid-based biosensors with emphasis on new trends in nucleic acid research such as developments of aptamers and aptazymes as affinity ligands and potential coupling to transduction technologies.

Biosensors are analytical devices that use a biologically derived material immobilised at a physicochemical transducer to measure one or more analytes. These can be ions, small organic molecules, proteins, nucleic acids, drug, toxins, etc.
Recently, nucleic acid sensors have been developed, using DNA or RNA as recognition affinity element, coupled to different types of transducer (electrochemical, piezoelectric, optical) and applied to genomic studies and, more recently, to proteomics.
This powerful technology not only identifies binding compounds to a target molecule but also provides, depending on the transduction principle employed, real-time quantitative data on binding kinetics, affinity and specificity as well as the concentration of active molecules in a sample.
Regarding DNA studies, the progress of the Human Genome Project has generated substantial interest in the use of nucleic acid hybridisation technologies to detect and identify organisms and mutations. Biosensors and micro-array chips that are based on detection of hybridisation/interaction of short strands of nucleic acids offer platforms for applications such as screening of genomes, detection of pathogenic organisms, and efficient searching of compound libraries for detection of potential therapeutic agents.
Among nucleic acids, aptamers represent a new promising recognition element for biosensor development.
Recent understanding of structure-function of nucleic acids, specifically RNA, has opened new perspective in the development of new analytical and diagnostic methods. In vitro evolution from random sequence libraries makes it possible to build nucleic acids that specifically recognise and bind to virtually any kind of target, such as ions, metabolites, drugs, toxins, peptides and proteins.
The quickly growing area of genomics, ribonomics, proteomics and metabolomics requires the development of high-throughput and massive-parallel analysis of biological samples. At this regard, the biosensor technology coupled to aptamers could represent a successful approach to the functional genomic area.

The main objectives of this workshop have been:
- To bring together experts from biosensing and nucleic acid research areas for optimising the DNA or RNA biosensor tool
- Progress in understanding immobilisation of ssDNA and RNA to allow the design of analytical sensing surfaces that can be used for very rapid and quantitative determination of nucleic acid hybridisation
- Recent developments on SELEX technology for the rapid production of aptamers with improved affinity and stability
- Optimisation of analytical biosensors for studying RNA-protein interactions.

Report

1st day: In vitro Selection of Aptamers as New Affinity Ligands (SELEX; In vitro selection of modified ligands; New strategies in immobilisation of nucleic acids)

This part of the workshop was aimed at giving a general overview of how select, stabilise and use aptamers.
Many interesting applications of aptamers concern the inhibition of proteins playing central role in the cellular metabolism in order to develop diagnostic, therapeutic and imaging strategies. Dr. Hamm from University of Torino selected an RNA aptamer that inhibits specifically CREB phosphorylation by MSK1 by showing a magnesium-ion dependent conformational changes of the kinase. The high specificity of the RNA aptamer can be used to label it for imaging studies.
Prof. U. Landegren (university of Uppsala, Sweden) presented the proximity ligation as new aptamer based detection method for post-genomics study. This strategy is more sensitive than standard ELISA and can be multiplexed and combined with different selected aptamers in order to screen different analytes.
Since DNA and RNA aptamer have to be used in cell culture or in vivo, Prof. F. Eckstein from Max Plank Institut for Experimentelle Medizin of Gottingen discussed how to improve the sensitivity of these molecules to degradation by nucleases. Different strategies were used (post-SELEX, modifications introduced during transcription and synthesis) making the RNAs totally resistant to degradation.
In the afternoon, Prof. JJ. Toulmé (University of Bordeaux INSERM, France) showed as SELEX can be used to select in vitro RNA aptamers which recognise folded tertiary shape rather than the primary sequence of RNA target. Dr. D. Libri of CNRS (Gif sur Yvette, France) in his invited lecture showed how SELEX can be done ex-vivo in order to select RNA aptamers that inhibit oncogene suitable for diagnostic and therapeutic applications.
More conventional problems of biosensor development were exposed on two different talks.
New biosensors based on streptavidin aptamers have been discussed by Dr. B. Strehlitz (Centre for Environmental Research Leipzig-Halle GmbH, Germany) and new immobilisation strategies have been presented by Dr. C. Preininger (ARC Seibersdorf research, Austria)) for the development of biochips.

All the lessons stimulated interesting discussions between experts of different fields.

2nd day: State of Art and Future Trends in DNA/RNA (Evolution of nucleic acid sensors; analytical and diagnostic advantages)

In this session 7 invited speakers gave a thorough overview on the development of biosensors mainly based on DNA and with different transducers. In particular, Dr. B.S. Persson (Biacore AB, Sweden) reported on different applications of the Surface Plasmon Resonance (SPR) technology for several DNA-related research areas, such as detection of genetically modified organisms, DNA repair analysis and binding site identification for novel DNA-binding proteins.
The other technology taken into consideration related to quartz crystal microbalance (QCM) measurements: Dr. F. Hook (Chalmers University of Technology, Sweden) presented results on combined frequency and the energy dissipation QCM for investigations of various coupling strategies of single stranded DNA.
The other invited speakers of the session illustrated different applications and future developments of electrochemical DNA-based biosensors, from "Ultrasensitive and photonic DNA detection" (Prof. Willner, The Hebrew University of Jerusalem, Israel) to allele-specific genotyping (Prof. Ozsoz, Ege University, Turkey) and study of DNA association interactions and damage by DNA-modified screen-printed electrodes (Prof. J. Labuda, Slovak Technical University, Slovakia).
Moreover, Prof. Palecek (Academy of Sciences of the Czech Republic) reported on several electrochemical DNA-based biosensors which have been developed by his research group: use of mercury electrodes or electroactive markers, coupling of the electrochemical detection with an optical one, double-surface (DS) technique in which hybridisation is performed at one surface and electrochemical detection at another surface.

Other electrochemical DNA-based biosensors have been introduced in the oral presentations: Dr. A. Narvaez (University of Alcalà, Spain) described a new electrochemical configuration based on the use of self-assembled multilayers for the detection of hybridisation events. A field-effect transistor device for labelfree, fully electronic DNA detection was the work discussed by Dr. Ingebrandt (Institute for Thin Films & Interfaces, Juelich, Germany).
A key step of the design of DNA-based biosensors is the immobilisation procedure: Prof. A.M. Oliveira Brett (University of Coimbra, Portugal) presented an interesting work on AFM and electrochemical studies on DNA oligonucleotides immobilised on electrodes.
Another potential target of biosensors based on DNA is the detection and study of drugs: Prof. E. De Pauw (University of Liege, Belgium) used ESI-MS to determine sequence and structural recognition of drugs (telomestatin) with unusual DNA structures, such as DNA duplex, triplex, quadruplex and I-motif structures.
Potential mutagenic factors and sulfonamide and antracycline medicines were taken into consideration and a method for their detection was reported by Prof. G.A. Evtugyn (Kazan state University, Russia), who developed and amperometric DNA sensor with enzymatic amplification of the signal.
Related to the increasing interest in developing techniques which could represent valid alternatives to fluorescent detection in DNA sensing, an interesting work was presented by Dr. S.C. Hillier (University of Bath, UK): novel ferrocenylated molecules have been custom synthesised and attached to oligonucleotide probes allowing detection using differential pulse voltammetry at extremely low concentrations.
Finally, aptamers have again been considered in the presentation of Dr. K.I. Papamichael (University of cork, Ireland), who investigated an aptamer receptor specific for IgE (allergy) and others for bile acids as part of a mechanism for detection of colon and breast cancer.

All the presentations have been widely discussed and all the speakers had the opportunity to answer questions and clarify doubts of the audience.

3rd day: RNA based biosensors: aptasensors and aptazymes (Selection of novel allosteric catalytic RNA; b) arrays; affinity biosensors)

The invited lectures of this day covered all the different subjects of the 3rd session. Allosteric catalytic RNA theme was illustrated by Prof. A. Jaeschke (University of Heidelberg, Germany) whose presentation concerned the Diels-Alder reaction which is the only one known to be accelerated by a ribozyme acting as a catalyst. The possibility of generation of signal cascades in biosensor application has been examined by studying the capability of the ribozyme to recognise two small molecules and to promote their cycloaddition.
The subject related to development of arrays was covered by Prof. F.F. Bier (Fraunhofer institute of Biomedical Engineering, Germany) who demonstrated the real time measurement of binding events and enzymatic activities in several hundreds dots simultaneously. The examples regarded DNA-enzyme-interaction of restriction enzymes and polymerases.
Finally, Prof. F.W. Scheller (University of Potsdam, Germany) stated that "the borderline between recognition tools that are biological in nature and synthetic (organic) receptor molecules, is no longer definable". He presented an overview on molecular recognition elements in biosensing particularly focused on biomimetic sensors based on aptamers and molecularly imprinted polymers.

The area of RNA/DNA arrays or microarrays have been explored also in other oral presentations: Dr. T.T. Bachmann reported on the possible genotying of beta-lactamases studying the microbial antibiotic resistance by DNA-microarrays. He demonstrated that DNA-microarrays allow rapid and accurate genotyping of biological samples.
Arrays based on label-free electrochemical detection for routine use in life sciences and diagnostics and for DNA-hybridisation detection have been proposed by Dr. H. Hillebrandt (FRIZ Biochem GmbH, Germany) and Dr. F. Turcu (Ruhr Universitat Bochum, Germany), respectively.
In the field of RNA biosensors, Dr. L. Tedeschi (CNR, Pisa) described an integrated approach for the design, synthesis and connection of RNA probes to the transducing surface of a microgravimetric biosensor.
Finally, two aptasensors have been presented by Dr. C.K. O'Sullivan (Universitat Rovira i Virgili, Tarragona, Spain) and by Dr. S. Tombelli (Università di Firenze, Italy). The first one regarded the use of an anti-thrombin aptamer used to set up an initial model of aptasensor. Different models were developed based on an ELONA (enzyme linked oligonucleotide assay) format and the best one was evaluated using SPR and electrochemistry.
The second aptasensor presented used an aptamer specific for the HIV-1 viral regulatory trans-activator of transcription protein (Tat). The interaction between the immobilised aptamer and the protein was observed in real time and without labels using QCM and SPR techniques.

Each presentation ended with an open discussion on the presented subjects.

During the whole period of the workshop, 23 posters were showed to participants. The fields covered in the posters, from different research groups, ranged from SELEX, proximity ligation and therapy to luminescent probes, diagnostics applications and electrochemical or piezoelectric DNA-based biosensors.