the data are analyzed and interpreted (curve fit model, identification and treatment of
outliers, statistical relevance).
Another essential factor leading to differences in K D values are the operator’s
skills and experience in planning and optimizing the experimental setup, in
performing the actual experiment, and in analyzing experiment data. Psychological
components are often neglected: the tendency to proclaim any initially determined
binding affinity as the sole benchmark for all later experiments, the ability to resist
external pressure to achieve highest possible affinities, or the desire to reach benchmark affinities from literature.
Last but not least, the laboratory infrastructure and the applied scientific/industrial
standards for aptamer-binding experiments (biophysical measurement device model,
device maintenance, consumable quality, software version, availability of precise
and reproducible liquid handling systems, and application of specific assay validation standards) can considerably influence the robustness and reliability of determined steady-state affinity values and will hence directly influence comparability of
binding data.
5 Summary and Outlook
Biophysical characterization of aptamer-target interactions is an essential aspect in
aptamer development. Various biophysical methods are available, each possessing
specific requirements, strengths, and disadvantages. A well-planned characterization
strategy is key for the success of the final application of an aptamer. Knowledge
which technology to apply, how to use the technology, and how to combine and
compare technologies to get the best picture of the aptamer-target interaction is the
basis of a successful characterization strategy.
Current biophysical analytical methods are centered around classical experimental conditions in well-established and historically developed artificial buffer systems.
Those may be beneficial for the stability of biological systems and simplify the
experimental setup, as well as the interpretation of binding data. However these
conditions lack biological relevance. Molecular interactions in nature do simply not
occur in artificial buffer systems. The use of artificial buffer systems was historically
fueled by the inability of biophysical methods to study molecular interactions in
bioliquids such as serum, cell lysates, or environmental samples. Nevertheless, latest
successful developments in the use of different biophysical methods such as microscale thermophoresis and biolayer interferometry indicate that a change in this
paradigm is possible.
As stated in this chapter, proper characterization of aptamer-target interactions
requires extensive expertise in the use of various biophysical methods, which is
sometimes simply not available within the team of an aptamer development project.
Lack of this expertise has already led and will result in unreliable binding data of
aptamers, casting a cloud not just over a specific scientist/group, but in long term
also influences the reputation of the aptamer community with all consequences.
Biophysical Characterization of Aptamer-Target Interactions
13
outliers, statistical relevance).
Another essential factor leading to differences in K D values are the operator’s
skills and experience in planning and optimizing the experimental setup, in
performing the actual experiment, and in analyzing experiment data. Psychological
components are often neglected: the tendency to proclaim any initially determined
binding affinity as the sole benchmark for all later experiments, the ability to resist
external pressure to achieve highest possible affinities, or the desire to reach benchmark affinities from literature.
Last but not least, the laboratory infrastructure and the applied scientific/industrial
standards for aptamer-binding experiments (biophysical measurement device model,
device maintenance, consumable quality, software version, availability of precise
and reproducible liquid handling systems, and application of specific assay validation standards) can considerably influence the robustness and reliability of determined steady-state affinity values and will hence directly influence comparability of
binding data.
5 Summary and Outlook
Biophysical characterization of aptamer-target interactions is an essential aspect in
aptamer development. Various biophysical methods are available, each possessing
specific requirements, strengths, and disadvantages. A well-planned characterization
strategy is key for the success of the final application of an aptamer. Knowledge
which technology to apply, how to use the technology, and how to combine and
compare technologies to get the best picture of the aptamer-target interaction is the
basis of a successful characterization strategy.
Current biophysical analytical methods are centered around classical experimental conditions in well-established and historically developed artificial buffer systems.
Those may be beneficial for the stability of biological systems and simplify the
experimental setup, as well as the interpretation of binding data. However these
conditions lack biological relevance. Molecular interactions in nature do simply not
occur in artificial buffer systems. The use of artificial buffer systems was historically
fueled by the inability of biophysical methods to study molecular interactions in
bioliquids such as serum, cell lysates, or environmental samples. Nevertheless, latest
successful developments in the use of different biophysical methods such as microscale thermophoresis and biolayer interferometry indicate that a change in this
paradigm is possible.
As stated in this chapter, proper characterization of aptamer-target interactions
requires extensive expertise in the use of various biophysical methods, which is
sometimes simply not available within the team of an aptamer development project.
Lack of this expertise has already led and will result in unreliable binding data of
aptamers, casting a cloud not just over a specific scientist/group, but in long term
also influences the reputation of the aptamer community with all consequences.
Biophysical Characterization of Aptamer-Target Interactions
13