Ph.D. Thesis
Title Preparation, Categorization and Hybridization
Properties of Oligodeoxyribonucleotide Coated Microparticles
Advisers Prof. Harri L�nnberg
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 (�=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.
