Samenvatting
Summary nanobiotechnology (NWI-MOL078)
- Instelling
- Radboud Universiteit Nijmegen (RU)
Samenvatting van de stof voor het vak nanobiotechnology.
[Meer zien]Voorbeeld 4 van de 54 pagina's
Voorbeeld 4 van de 54 pagina's
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Voorbeeld van de inhoud
Summary nanobiotechnology,Chapter 1 introduction to
nanobiotechnology
Nanobiotechnology forms a foundation for nanomedicine, the curing of people with the aid of
nanodevices. In order to have a proper understanding of nanobiotechnology, it is important to have
an idea of what the nanoscale is, such that the technical devises properly match the biological
dimensions → the one arm end to the other arm end of an antibody is 10 nm
• dimensions are important for e.g. entry routes of the nanodevice, as well as its proper
working (think of the IgG opsonised beats eaten by macrophages)
• One can make nanomachines that are biomimetic → to which degree can biology be
mimicked? → as the interface with the biosystem must be as good as possible
− to accomplish an as good as possible interface with the biological system, as well as
to be able to modify the properties of the biomimetic nanomachine, one must have
thorough understanding of the biosystem and technology
,Chapter 2 Chemistry for
nanobiotechnology: the chemical toolbox
For nanobiotechnology, we need to investigate the proteins, nucleic acids and nanodevices used. In
order to do so, numerous commercially available kits have been developed. An thorough
understanding of the principles underlying these kits, though, is required if one wants or has to
adjust the conditions and thus to properly operate them.
Chemistry in nanobiotechnology thus is used in biotechnical kits to for example:
• immobilise biomolecules for assays (ELISA, microarray → no DNA entanglement so good
read out)
• labelling biomolecules for imaging (e.g. FRET → visualisation/tracing, interactors, fishing out,
control activity)
• regulating protein activity
• protection of biomolecules → e.g. PEGgylation in nanomedicine to avoid immune responses
• construction of hybrid materials → ECM mimicry, cell culturing
• stabilisation/crosslinking of protein-based materials
Note that for all these kits, certain molecules have to be attached/cross-linked to the biomolecules.
The chemistry underlying these kits happens directly at the proteins involved. Note that these kits
can either operate in vivo or in vitro with the isolated protein. In both circumstances though, a
limited number of reactions can be used
• Biomolecules are very sensitive → minor environmental changes cause structural changes
and loss of function
• In vivo conditions are hard to control
The underlying chemistry
The underlying chemistry for these kits can exploit three different strategies:
• chemical covalent attachment
• enzyme covalent attachment
• non-covalent attachment
requirements
In order to make these kits useful for research, the chemistry underlying them must fulfil certain
requirements. These are:
• The chemistry must not interfere with the biomolecule activity
• The chemistry must be selective → otherwise one would get e.g. false positive results,
inaccurate quantities, unreplicable results etc.
− the label can react with different types of conjugation handles on the same molecule
→ especially a problem if label attachment reaction is quite “trivial”
− the label can react with the same type of conjugation handle but at the wrong
position → especially a problem if
o the conjugation handle is an amino acid that is incorporated frequently
o a problem for cysteins which are not frequently incorporated but if they are,
they are often positioned in the active site/as disulfide bridges
− the label can react with other off-target molecules → especially in vivo
• The chemistry must be efficient → all chemical reactions underlying these kits operate in an
aqueous environment, meaning that these reactions of the agent with the biomolecule
, competes with reactions of water with the biomolecule and the agent with water. The latter
renders the agent inactive.
− If the reaction of the agent with the biomolecule proceeds faster than with water
(which is an equilibrium), the majority of the agent molecules and biomolecules have
undergone the desired reaction → this can be accomplished by making the reaction
of (mainly) the agent with the biomolecule VERY thermodynamically favourable
and kinetically easy to proceed
o pushing the reaction toward the products of the agent + biomolecule
reaction → by e.g. good leaving group, very electrophilic, very strained
(reactants high in free energy), enzymatic, etc.
Chemical covalent attachment
In order to covalently attach an agent to a protein, the
protein must expose certain sites on the surface to which
molecules e.g. the labels can attach. These are known as
conjugation handles: active functional groups for
conjugation → as proteins are present in an aqueous
environment, these are polar/charged amino acid side
chains:
• Methionine, Cysteine, Serine, Threonine, Asparagine,
Glutamine
• Histidine, Lysine, Arginine
• Aspartate, Glutamate
So the available conjugation handles are:
• amine functionalities: basic (thermo), nucleophilic
(kinetic)
− alkylation
− acylation
− (thio) urea formation
− reductive alkylation
− glutaraldehyde aided collagen crosslinking
• thiol functionalities: basic, nucleophilic
− Michael addition: with maleimide
− Disulfide exchange: with disulfide
− Native chemical ligation (EPL): with C-
terminal thioesters
• acid functionalities
• hydroxy functionalities
Amine functionalities
We find amine groups at the side chain of lysine residues
and at the N-terminus of the oligopeptide. Note that these two groups are not chemically equivalent.
In these reactions, an -NH2 group from the protein reacts with a functional group with functionality
-R-X
• To understand what reactions are possible at the amine conjugation handles, one must know
the chemical properties of amine groups. These are:
− NH2 is a good nucleophile and is basic
− it has a lone pair
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