7 research outputs found
Size Exclusion Chromatography with Multi Detection in Combination with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry as a Tool for Unraveling the Mechanism of the Enzymatic Polymerization of Polysaccharides
Determination of the size distributions of natural polysaccharides
is a challenging task. More advantageous for characterization are
well-defined synthetic (hyper)-branched polymers. In this study we
concentrated on synthetic amylopectin analogues in order to obtain
and compare all available data for different distributions and size
dependence of molecular weights. Two groups of well-defined synthetic
branched polysaccharides were synthesized via an in vitro enzyme-catalyzed
reaction using the enzyme phosphorylase <i>b</i> from rabbit
muscle and <i>Deinococcus geothermalis</i> glycogen branching
enzyme. Synthetic polymers had a tunable degree of branching (2%–13%
determined via <sup>1</sup>H NMR) and a tunable degree of polymerization
(30–350 determined indirectly via UV spectrometry). The systems
used for separation and characterization of branched polysaccharides
were SEC-DMSO/LiBr and multi detection (refractive index detector,
viscosity detector, and multi angle light scattering detector) and
SEC-water/0.02% NaN<sub>3</sub>; and SEC-50 mM NaNO<sub>3</sub>/0.02%
NaN<sub>3</sub> and multi detection. Additionally the side chain length
distribution of enzymatically debranched polysaccharides was investigated
by matrix-assisted laser desorption ionization-time-of-flight mass
spectrometry (MALDI-TOF MS) analysis. With this combination of characterization
techniques, we were able not only to characterize the amylopectin
analogues but also to solve parts of the molecular mechanism of their
enzymatic polymerization. Moreover our materials showed potential
to be standards in the field of natural polysaccharides characterization
Nucleic Acid Chemistry in the Organic Phase: From Functionalized Oligonucleotides to DNA Side Chain Polymers
DNA-incorporating
hydrophobic moieties can be synthesized by either
solid-phase or solution-phase coupling. On a solid support the DNA
is protected, and hydrophobic units are usually attached employing
phosphoramidite chemistry involving a DNA synthesizer. On the other
hand, solution coupling in aqueous medium results in low yields due
to the solvent incompatibility of DNA and hydrophobic compounds. Hence,
the development of a general coupling method for producing amphiphilic
DNA conjugates with high yield in solution remains a major challenge.
Here, we report an organic-phase coupling strategy for nucleic acid
modification and polymerization by introducing a hydrophobic DNA–surfactant
complex as a reactive scaffold. A remarkable range of amphiphile–DNA
structures (DNA–pyrene, DNA–triphenylphosphine, DNA–hydrocarbon,
and DNA block copolymers) and a series of new brush-type DNA side-chain
homopolymers with high DNA grafting density are produced efficiently.
We believe that this method is an important breakthrough in developing
a generalized approach to synthesizing functional DNA molecules for
self-assembly and related technological applications
Functionalization of Fatty Acid Vesicles through Newly Synthesized Bolaamphiphile–DNA Conjugates
The surface functionalization of
fatty acid vesicles will allow
their use as nanoreactors for complex chemistry. In this report, the
tethering of several DNA conjugates to decanoic acid vesicles for
molecular recognition and synthetic purposes was explored. Due to
the highly dynamic nature of these structures, only one novel bola-amphiphile
DNA conjugate could interact efficiently with or spontaneously pierce
into the vesicle bilayers without jeopardizing their self-assembly
or stability. This molecule was synthesized via a CuÂ(I)-catalyzed
[3 + 2] azide–alkyne cycloaddition (click reaction), and consists
of a single hydrocarbon chain of 20 carbons having on one end a triazole
group linked to the 5′-phosphate of the nucleic acid and on
the other side a hydroxyl-group. Its insertion was so effective that
a fluorescent label on the DNA complementary to the conjugate could
be used to visualize fatty acid structures
Photoswitching of DNA Hybridization Using a Molecular Motor
Reversible control
over the functionality of biological systems
via external triggers may be used in future medicine to reduce the
need for invasive procedures. Additionally, externally regulated biomacromolecules
are now considered as particularly attractive tools in nanoscience
and the design of smart materials, due to their highly programmable
nature and complex functionality. Incorporation of photoswitches into
biomolecules, such as peptides, antibiotics, and nucleic acids, has
generated exciting results in the past few years. Molecular motors
offer the potential for new and more precise methods of photoregulation,
due to their multistate switching cycle, unidirectionality of rotation,
and helicity inversion during the rotational steps. Aided by computational
studies, we designed and synthesized a photoswitchable DNA hairpin,
in which a molecular motor serves as the bridgehead unit. After it
was determined that motor function was not affected by the rigid arms
of the linker, solid-phase synthesis was employed to incorporate the
motor into an 8-base-pair self-complementary DNA strand. With the
photoswitchable bridgehead in place, hairpin formation was unimpaired,
while the motor part of this advanced biohybrid system retains excellent
photochemical properties. Rotation of the motor generates large changes
in structure, and as a consequence the duplex stability of the oligonucleotide
could be regulated by UV light irradiation. Additionally, Molecular
Dynamics computations were employed to rationalize the observed behavior
of the motor–DNA hybrid. The results presented herein establish
molecular motors as powerful multistate switches for application in
biological environments
Photoswitching of DNA Hybridization Using a Molecular Motor
Reversible control
over the functionality of biological systems
via external triggers may be used in future medicine to reduce the
need for invasive procedures. Additionally, externally regulated biomacromolecules
are now considered as particularly attractive tools in nanoscience
and the design of smart materials, due to their highly programmable
nature and complex functionality. Incorporation of photoswitches into
biomolecules, such as peptides, antibiotics, and nucleic acids, has
generated exciting results in the past few years. Molecular motors
offer the potential for new and more precise methods of photoregulation,
due to their multistate switching cycle, unidirectionality of rotation,
and helicity inversion during the rotational steps. Aided by computational
studies, we designed and synthesized a photoswitchable DNA hairpin,
in which a molecular motor serves as the bridgehead unit. After it
was determined that motor function was not affected by the rigid arms
of the linker, solid-phase synthesis was employed to incorporate the
motor into an 8-base-pair self-complementary DNA strand. With the
photoswitchable bridgehead in place, hairpin formation was unimpaired,
while the motor part of this advanced biohybrid system retains excellent
photochemical properties. Rotation of the motor generates large changes
in structure, and as a consequence the duplex stability of the oligonucleotide
could be regulated by UV light irradiation. Additionally, Molecular
Dynamics computations were employed to rationalize the observed behavior
of the motor–DNA hybrid. The results presented herein establish
molecular motors as powerful multistate switches for application in
biological environments
Non-covalent Monolayer-Piercing Anchoring of Lipophilic Nucleic Acids: Preparation, Characterization, and Sensing Applications
Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid–DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid–DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid–DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid–DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid–DNA. The formation of the lipid–DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity
Filling the Green Gap of a Megadalton Photosystem I Complex by Conjugation of Organic Dyes
Photosynthesis
is Nature’s major process for converting
solar into chemical energy. One of the key players in this process
is the multiprotein complex photosystem I (PSI) that through absorption
of incident photons enables electron transfer, which makes this protein
attractive for applications in bioinspired photoactive hybrid materials.
However, the efficiency of PSI is still limited by its poor absorption
in the green part of the solar spectrum. Inspired by the existence
of natural phycobilisome light-harvesting antennae, we have widened
the absorption spectrum of PSI by covalent attachment of synthetic
dyes to the protein backbone. Steady-state and time-resolved photoluminescence
reveal that energy transfer occurs from these dyes to PSI. It is shown
by oxygen-consumption measurements that subsequent charge generation
is substantially enhanced under broad and narrow band excitation.
Ultimately, surface photovoltage (SPV) experiments prove the enhanced
activity of dye-modified PSI even in the solid state