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IUPAC Prize for Young Chemists - 2000
Honorable Mention

 

 

Harri Hakala receives one of five Honorable Mention awards associated with the IUPAC Prize for Young Chemists, for his Ph.D. thesis work entitled "Preparation, Categorization and Hybridization Properties of Oligodeoxyribonucleotide Coated Microparticles"

Current address (at the time of application)

Wallac Oy, P.O.Box 10
FIN-20101 Turku, Finland

Tel.: +358 2 2678693
Fax: +358 2 2678380
E-mail: harri.hakala@perkinelmer.com

Academic degrees

  • Ph.D., University of Turku, 28.6.1999, Org. Chemistry
  • M.Sc., University of Turku, 16.1.1992, Physical Chemistry

Ph.D. Thesis

Title Preparation, Categorization and Hybridization Properties of Oligodeoxyribonucleotide Coated Microparticles
Advisers Prof. Harri Lönnberg
Thesis Committee Prof. Wojciech Markiewicz (Inst. of Bioorganic Chemistry, Polish Academy Of Sciences, Poznan, Poland); Prof. Hartmut Seliger (University of Ulm, Germany) and Prof. Wlodzmierz Krzyzosiak (Inst. of Bioorganic Chemistry, Polish Academy Of Sciences, Poznan, Poland)

Essay

Solid supports bearing immobilized oligodeoxyribonucleotide probes are a useful tool in analyzing the base sequence of nucleic acids. This kind of mixed-phase assays combined with efficient amplification techniques, such as polymerase chain reaction (PCR), has gained increasing popularity in clinical diagnosis. These assays are usually based on arrays of allele-specific oligonucleotides attached to a solid support, hybridization of fluorescently or radioactively labeled PCR-amplified sequences to the support-bound probes, and detection of the hybridized sequence by location of the label spots on support. In my thesis I developed an alternative approach for DNA-diagnostic based on the following principle. A mixture of microscopic particles (Ć=50mm), each of which bears a given allele-specific oligonucleotide and a reporter group defining the particle category, is used as the solid phase. After hybridization, individual particles are separately subjected to two parallel measurements: one identifies the particle category (reporter group on the particle), and the other quantifies the fluorescently tagged oligonucleotide hybridized to the particle-bound probes. The fluorescent markers employed for labeling of oligonucleotides are photoluminescent lanthanide chelates, which have several advantages over traditional organic fluorophores. Firstly, their long-lived fluorescence allows the usage of the time-resolved mode in the measurement, and this, in turn, allows efficient elimination of the prompt background fluorescence. Secondly, the difference between the wavelength of excitation and emission bands is large, and thirdly, the emission bands are narrow. These two latter properties eliminate concentration quenching, which constrains the usefulness of organic fluorophores.

In this thesis several postsynthetic methods to immobilize oligonucleotides covalently to microparticles were introduced, and the hybridization efficiency and kinetics were found to be dependent on the linker employed. Particles obtained by direct solid phase assembly of oligonucleotides were surprisingly found to exhibit almost as high hybridization efficiency as the best particles obtained by the postsynthetic method. In fact, if expressed as signal-to-noise ratios, they were better than any of those obtained postsynthetically. For this reason, the particles obtained by in situ -synthesis were chosen as method-of-choise.

The properties of prepared particles were studied using hybridization of labeled target to the particle-bound oligonucleotide. Kinetics of hybridization between labeled oligonucleotide and immobilized oligonucleotide were found to be independent of the concentration of labeled target and the loading of immobilized oligonucleotides, but to depend on the number of particles. More precisely, the kinetics of hybridization is accelerated by increasing the number of particles in a given volume. On the other hand, the hybridization efficiency does not depend on the density of particles and hence, if the density of particles is increased the signal measured from a single particle is decreased. In other words, the kinetics of hybridization can not be accelerated by increasing the density of immobilized oligonucleotides on the solid surface but can be accelerated by increasing the density of particles, which, in turn, means that one have to make a compromise between kinetics and sensitivity. The hybridization efficiency remains constant over the target concentration of 5 orders of magnitude. This exceptionally wide dynamic range is due to good properties of lanthanide chelates and the high capacity of used porous polymer particles.

In the next step of the studies the sandwich type hybridization assay was examined, in which the labeled probe hybridized to target and the resulting duplex hybridized further to the particle-bound oligonucleotide. The dependence of the efficiency of sandwich hybridization on target concentration differs from that observed for direct hybridization of labeled target. The efficiency of sandwich hybridizations increases with the increasing concentration of target. Near the detection limit (0.05 amol/particle), the efficiency is only 16% and at the other end of the 5 orders of magnitude wide dynamic range the hybridization efficiency is 85%. The kinetics are very similar to those found in direct hybridization of the target. This evidently means that hybridization of the target to labeled probe is fast compared to the hybridization of the resulting duplex to the solid phase. The optimal length of the particle-bound allele specific probe was found to be dependent on the oligonucleotide loading on the particle. The lower the loading of immobilized oligonucleotide (2-10 mmol g-1) is, the longer the probe can be to achieve still sufficient discrimination between fullmatches and mismatches.

To allow multiparametric assay, the particles were categorized using two organic prompt fluorophores, dansyl and fluorescein. Both labels were observed to give different intensities at the 3'- and 5'-end, and for that reason labels were always incorporated in the middle of the linker tethered to the 3'-end of the oligonucleotide, i.e. near the particle. Both labels were diluted prior to the synthesis to prevent concentration quenching. For a model system, six categories were created but the number of possible categories could be about one dozen using those two labels and dozens of categories could be created by using more labels.

The usefulness of multiparametric assay was examined with six categories, each of which bore an allele specific probe, mimicing different types of gene mutations. It was observed that each particle category worked independently in the reaction mixture; neither the presence of particles belonging to another category, nor the presence of additional targets, had any effect on the efficiency and kinetics of hybridization. Furthermore, when samples prepared by mixing synthetic oligonucleotides were analyzed, it was observed that the results obtained gave not only yes/no answer, but also the amount of target in sample when the predetermined signal/[target] relationship was applied.

The multiparametric approach developed in this thesis is still at an early stage of development. Successful miniaturization, automatization and particle transport system are prerequisites for clinical applications. Nevertheless, the data obtained shows that chemical basis of the multiparametric assay is sound. Though further development and optimization of the assay format are still needed, some underlying features of the system are superior to those of excisting assay systems. Above all, the exceptionally wide range of detection allows more accurate quantitative determination of several oligonucleotide concentration from a single sample than the existing methods.


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