Parallel Force Assay for Protein-Protein Interactions

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay

Parallel Force Assay for Protein-Protein Interactions

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay

A Force-Based, Parallel Assay for the Quantification of Protein-DNA Interactions

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA),parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction

Conservation Learning Initiative: Learn from evidence. Improve Conservation

The conservation community needs smarter and more successful actions to improve the impact of its work. For example, it is not always clear how to create training programmes that improve performance in a lasting way, or what the ingredients of a successful conservation partnership are, or how donors can set up funding so that grantees can work in a strategic and sustainable way.One way of designing successful, effective actions is through using insights from evidence-based learning. Recent years have seen significant steps forward in developing concepts for defining and using evidence in conservation. In late 2021, the MAVA Foundation, Foundations of Success (FOS), and Conservation Evidence joined forces in an initiative to build further on this work.Combining the strengths of their approaches with MAVA's treasure of nearly 30 years of conservation data, they set out to formulate assumptions and collect evidence to answer key learning questions. The results of this joint work are now available on the Conservation Learning Initiative website (https://conservation-learning.org/) and in a consolidated report.The website and report present:A practical 5-step approach for evidence-based learning in conservation, designed for combining different sources of evidence, dealing with differences in reliability and relevance, and drawing conclusions.Valuable insights based on data regarding four widely used conservation strategies: capacity-building, forming partnerships and alliances, providing flexible funding, and research and monitoring.The lessons learned will help conservationists fine-tune their work or investment to increase their conservation impact. By applying the approach on their own data, they can learn from evidence to make better decisions and improve strategies over time

A force-based, parallel assay for the quantification of protein-DNA interactions.

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction

Transfer of Cy5-labeled DNA to the glass slide.

<p>After the contact and separation process, the fluorescence intensity of Cy5 on the glass slide is determined. Histograms of selected areas (without prior background subtraction) show a very modest signal slightly above the background signal (1000–2000 counts) for the DNA harboring the no binding sequences for the protein in question. DNA with a high affinity sequence did bind the protein in question and a transfer signal is clearly visible. The images are optimized in contrast to make the transfer of the no binding sequence as well as the difference in fluorescence signal between the no binding sequence and high affinity motif visible. A first assessment of the binding strength is possible by varying the reference bond. The weaker reference of 20 bp shows a higher fluorescence intensity of 17000 counts compared to the stronger reference of 40 bp with 13000 counts.</p