Name: nmr-based fragment screening in a minimum sample but maximum automation mode
Uploaded: 2021-06-04T15:00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode Video at JoVE.com

This work has been supported by iNEXT-Discovery, project number 871037, funded by the Horizon 2020 program of the European Commission.

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+poised+fragment+library+enables+rapid+synthetic+expansion+yielding+the+first+reported+inhibitors+of+PHIP(2),+an+atypical+bromodomain.">A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain.</a> Chemical Science. 7, 2322-2330 (2016).</li><li>Hwang, T. L., Shaka, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+Suppression+That+Works.+Excitation+Sculpting+Using+Arbitrary+Wave-Forms+and+Pulsed-Field+Gradients.">Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients.</a> Journal of Magnetic Resonance, Series A. 112, 275-279 (1995).</li><li>Gossert, A. D., Jahnke, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+in+drug+discovery:+A+practical+guide+to+identification+and+validation+of+ligands+interacting+with+biological+macromolecules.">NMR in drug discovery: A practical guide to identification and validation of ligands interacting with biological macromolecules.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 97, 82-125 (2016).</li><li>Sklenar, V., Bax, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+water+suppression+technique+for+generating+pure-phase+spectra+with+equal+excitation+over+a+wide+bandwidth.">A new water suppression technique for generating pure-phase spectra with equal excitation over a wide bandwidth.</a> Journal of Magnetic Resonance. 75, 378-383 (1987).</li><li>Binas, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=19F+NMR-Based+Fragment+Screening+for+14+Different+Biologically+Active+RNAs+and+10+DNA+and+Protein+Counter-Screens.">19F NMR-Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter-Screens.</a> ChemBioChem. , (2020).</li></ol>

Versatility of the NMR-based fragment/drug screening. BMRZ has successfully implemented state-of-the-art automated NMR instrumentation as well as STD-NMR, waterLOGSY and relaxation experiments to identify fragments within a wide range of affinity regime for drug discovery. The installed hardware includes a high-throughput sample preparation robot and high-throughput sample storage, changer and data acquisition unit associated to a 600 MHz spectrometer. A recently purchased cryogenic probe for 1H, 19F, 13C and 15N ensures the required sensitivity for the proposed measurements and allows 1H (1) decoupling during 19F detection. This probe is connected to the latest generation of NMR console that offers the possibility to use the advanced software tools from Bruker, including CMC-q, CMC-assist, CMC-se and FBS (included in TopSpin). The fragment-based screening (FBS) tool is included in the latest version of TopSpin and helps to analyse the high-throughput data comprising of STD, waterLOGSY, T2/T1r-relaxation experiments. The liquid 1D 1H sample collection can be filled into the NMR-tubes in an automated manner by using the sample filling robot. Typically, a block of 96 tubes (3 mm) are filled in approximately two hours. The 96-well-plate-racks are directly positioned in the HT sample changer, which reads the barcode of the block and assigns the NMR tubes to the experiments controlled by the automation software (IconNMR). Five 96-well-plate-racks can be stored and programmed in the HT sample changer&#160;at the same time. The temperature of each of the individual racks can be controlled and regulated separately. Additionally, each individual sample can be preconditioned (preheating and tube drying for removal of condensed humidity) to the desired temperature before the measurement.
Suitability for wide range of applications. One of the broad applications of this automated NMR-based screening is to identify and develop novel ligands binding to a biomacromolecular target (DNA/RNA/Proteins). These ligands can include orthosteric and allosteric inhibitors that typically bind non-covalently. Further, FBDD by NMR is typically used as a first step to select promising compounds, the requirements to be met are availability of the biomolecular target in sufficient quantities. This objective is divided into two major tasks.
Task one is to develop and characterize an in-house fragment library for the following reasons: initial and periodic quality control, characterization, and quantification of more than 1000 fragments; determination of solubility of the fragments in buffers optimized for each target, in particular for protein targets; and the establishment of several libraries to accommodate diverse scaffolds and extending towards other macromolecule classes. Task two is to integrate workflows for fragment-based drug design (FBDD) by NMR using: automated 1D-ligand observed screening (1H and 19F observed); automated replacement assays (competition experiments with (natural) ligand) to differentiate orthosteric and allosteric binding; automated secondary screenings with multiple fragments; automated 2D-protein screening, and secondary screening of a set of derivatives around an initial hit making use of the EU-OPENSCREEN library or any other library; and re-profiling screening of FDA-library against the chosen targets.
Additionally, metabotyping of various cell lines (disease relevant) can be conducted in order to unravel the regulatory mechanisms that link cell cycle control and metabolism. Also, there is functional characterization of RNA/DNA/protein regulation elements in vivo and in vitro for optimization of construct/domain optimization (stability optimization for structural investigations (Buffer, pH, temperature, and salt screening), and an extension of NMR-based fragment screening to membrane proteins and intrinsically disordered proteins, which are generally inaccessible to other techniques.
Limitations. Use of 19F and 1H fragments libraries have their pros and cons, few of which will be mentioned in the following. The largest benefit of 19F versus 1H measurements is the speed of both the actual measuring time and the subsequent analysis, as the mixtures contain almost double the number of fragments and fewer experiments must be conducted. The follow up analysis is also easier for 19F screening, as there is no interference from buffers and additionally offers a broader chemical shift range with almost no signal overlap for an optimally designed fragment mixture. The spectra themselves are greatly simplified, usually only having one or two signals per fragment, depending on the number of fluorine atoms. The analysis of these spectra can therefore be automated, again cutting down on time. This comes at the cost of chemical diversity, at least for the library used in this study. As only ~13% of the library contains 19F, but naturally all of them are useable in 1H screening, the diversity of the 19F screening fragments will be lower. This could be circumvented using specifically designed 19F libraries with more fragments and bigger chemical diversity. Another disadvantage for 19F screening is the low number of signals per fragment. Fragments generally are composed of more than one hydrogen atom. Therefore, 1H observed screening experiments can rely on different signals for the same fragment for detecting binding. This gives a higher degree of confidence when identifying hits for the 1H screening, whereas the 19F screening must rely on the one or two signals given per fragment.
A detailed account on the modern automated NMR-based fragment screening instrumentation, software and analysis methods and protocols thereof has been presented. The installed hardware includes a high-throughput sample preparation robot and a high-throughput sample storage, changer and data acquisition unit associated to a 600 MHz spectrometer. A recently installed cryogenic probe head for 1H, 19F, 13C and 15N ensures the required sensitivity for the proposed measurements and allows 1H decoupling during 19F detection. Further, the latest generation of NMR console offers the possibility to use advanced analytical software for aiding acquisition and on-the-fly analysis. The above discussed technology, workflows, and the described protocols should foster remarkable success to users pursuing FBS by NMR.

Fragment-based screening is now a commonly used method for identifying rather simple and low molecular weight molecules (MW &#60;250 Da) that show weak binding to macromolecular targets including proteins, DNA and RNA. Initial hits from primary screens serve as the basis to conduct a secondary screen of commercially available larger analogues of the hits and then to utilize chemistry-based fragment growth or linking strategies. For a successful fragment-based drug discovery (FBDD) platform, in general, a robust biophysical method is required for detecting and characterizing weak hits, a fragment library, a biomolecular target and a strategy for follow-up chemistry. Four commonly applied biophysical methods within the drug discovery campaigns are thermal shift assays, surface plasmon resonance (SPR), crystallography and nuclear magnetic resonance spectroscopy (NMR).
NMR spectroscopy has displayed varied roles within the different stages of the FBDD. Apart from ensuring the chemical purity and solubility of the fragments in a fragment library dissolved in an optimized buffer system, ligand observed NMR experiments can detect fragment binding to a target with low affinity and the target observed NMR experiments can delineate the binding epitope of the fragment, thus enabling detailed structure-activity relationship studies. Within epitope mapping, NMR-based chemical shift changes cannot only identify the orthosteric binding sites but also allosteric sites that might be cryptic and only accessible in so called excited conformational states of the biomolecular target. If the biomolecular target already binds an endogenous ligand, the identified fragment hits can be easily classified as allosteric or orthosteric by performing NMR-based competition experiments. Determining the dissociation constant (KD) of the ligand-target interaction is an important aspect in the FBDD process. NMR-based chemical shift titrations, either ligand or target observed can be readily performed to determine the KD. A major advantage of NMR is that the interaction studies are performed in solution and near to physiological conditions. Thus, all conformational states for the analysis of ligand/fragment interaction with its target can be probed. Further, NMR-based approaches are not only restricted towards screening of well-folded soluble proteins, but also are being applied to accommodate larger target space including DNA, RNA, membrane bound and intrinsically disordered proteins1.
Fragment libraries are an indispensable part of the FBDD process. In general, fragments act as the initial precursors which eventually become part (substructure) of the new inhibitor developed for a biological target. Several drugs (Venetoclax2, Vemurafenib3, Erdafitinib4, Pexidartnib5) have been reported to have started as fragments and are now successfully used in the clinics. Typically, fragments are low molecular weight (&#60;250 Da) organic molecules with a high aqueous solubility and stability. A carefully crafted fragment library containing typically a few hundred fragments, can already promise efficient exploration of chemical space. The general composition of fragment libraries has evolved overtime and most often were derived by dissecting known drugs into smaller fragments or designed computationally. These diverse fragment libraries mainly contain flat aromatic or heteroatoms and adhere to the Lipinski Rule of 5 6, or to the current commercial trend Rule of 3 7, but avoid reactive groups. Some fragment libraries were also derived or composed of highly soluble metabolites, natural products and or their derivatives8. A general challenge posed by most of the fragment libraries is ease of downstream chemistry.
The Center for Biomolecular Magnetic Resonance (BMRZ) at the Goethe-University Frankfurt, is a partner of the iNEXT-Discovery (Infrastructure for NMR, EM and X-rays for Translational research-Discovery), a consortium for structural research infrastructures for all European researchers from all fields of biochemical and biomedical research. Within the previous initiative of iNEXT which ended in 2019, a fragment library comprising of 768 fragments was crafted with the aim of &#34;minimum fragments and maximum diversity&#34; covering a large chemical space. Further, unlike any other fragment library, the iNEXT fragment library was also designed based on the concept of &#34;poised fragments&#34; with the aim to ease downstream synthesis of complex, high affinity ligands and henceforth known as in-house library (Diamond, Structural Genomic Consortium and iNEXT).
Establishing FBDD by NMR requires manpower, knowledge, and instrumentation. At the BMRZ, optimized workflows to support technical assistance to fragment screening by NMR have been developed. These include quality control and solubility assessment of the fragment library 9, buffer optimization for the chosen targets, 1H or 19F- observed 1D-ligand based screening, competition experiments to differentiate between orthosteric and allosteric binding, 2D-based target observed NMR experiments for epitope mapping, and for characterizing the interaction with secondary set of derivatives of the initial fragment hits. BMRZ has established automated routines for the analysis, as also previously discussed in the literature 10,11, of small molecule-protein interactions and has in place all the necessary automated infrastructure for NMR-based fragment screening. It has implemented saturation transfer difference NMR (STD-NMR), water-ligand observed via gradient spectroscopy (waterLOGSY), and Carr-Purcell-Meiboom-Gill-based (CPMG-based) relaxation experiments to identify fragments within a wide range of affinity regimes as well as state-of-the-art automated NMR instrumentation and software for drug discovery. While NMR-based fragment screening is well established for proteins, this approach is less commonly used for finding new ligands interacting with RNA and DNA. BMRZ has established proof of concept for new protocols enabling the identification of small molecule-RNA/DNA interactions. In the following sections of this contribution, application of screening routines on protein and RNA samples is reported to make aware of the established protocols for a broad user base in biomacromolecular research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bruker Avance III HD</td>
			<td></td>
			<td>Bruker</td>
			<td>600 MHz NMR Spectrometer</td>
		</tr>
		<tr>
			<td>Matrix Clear Polypropylene 2D Barcoded Open-Top Storage Tubes</td>
			<td>3731-11 0.75ML V-BOTTOM TUBE/LATCH RACK</td>
			<td>ThermoFisher Scientific</td>
			<td>Barcoded Tubes</td>
		</tr>
		<tr>
			<td>Matrix SepraSeal und DuraSeal&#38;</td>
			<td>4463 Cap Mat, SeptraSeal 10/CS</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>SampleJet</td>
			<td></td>
			<td>Bruker</td>
			<td>HT Sample Changer</td>
		</tr>
		<tr>
			<td>SamplePro Tube</td>
			<td></td>
			<td>Bruker</td>
			<td>Pipetting Robot</td>
		</tr>
	</tbody>
</table>

nmr-based fragment screening in a minimum sample but maximum automation mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Quality control of fragment library 
The fragments from the in-house library were delivered as 50 mM stock solutions in 90% d6-DMSO and 10% D2O (10% of D2O ensures minimization of compound degradation due to repeated freeze-thaw cycles14). Single compound samples consisted of 1 mM ligand in 50 mM phosphate buffer (25 mM KPi pH 6.2 + 50 mM KCl + 5 mM MgCl2), pH 6.0 in 90% H2O/9% D2O/1% d6-DMSO. 1H-NMR experiments of fragments from the iNEXT library were measured on a 500/600 MHz NMR spectrometer. This data was further used for identifying the single compounds in 1H screening campaigns using the CMC-q software which allows the user to fully acquire spectra in an automated manner and the analysis addon CMC-a the quality (solubility and integrity) of fragments was assessed. The results from the automated analysis from CMC-a are shown as a graphical output similar to what is represented in Figure 3. The graphical output shows a representation of a 96-well plate. A red colored circle means that this fragment shows inconsistency in structure or concentration. Green colored wells indicate that the fragment is consistent.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62262/62262fig03.jpg" /> 
Figure 3: Quality control of fragment library. Schematic representation of CMC-a based automated output. Fragment properties such as concentration and structural integrity are assessed. Green stands for consistent, orange in this case stands for inconsistent. Inconsistent fragments are revised manually following the shown workflow. <a href="https://www.jove.com/files/ftp_upload/62262/62262fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Approximately, 65% and 35% of the fragments were classified as consistent and in-consistent, respectively, in both DMSO and buffer. Further, 30% of the inconsistent classified ligands turned consistent after a careful manual inspection of the spectra9.
19F Mixture design 
103 fragments containing one or several fluorine groups from the in-house library were divided into 5 mixes (A, B, C, D, E). Each mix has 20 to 21 fragments. In this case the mixtures had to be carefully designed to avoid signal overlap. 19F transverse relaxation experiments were measured for each mixture that apply CPMG pulse trains. These experiments can be modified by varying the relaxation delays. The 19F chemical shift of mixes A-E can be seen in Figure 4.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62262/62262fig04.jpg" /> 
Figure 4: 19F 1D-NMR spectra of mixture samples from the in-house library. <a href="https://www.jove.com/files/ftp_upload/62262/62262fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sample preparation 
The sample preparation in the 19F screening procedure was either done manually or with automated pipetting using a pipetting&#160;robot. The fragments in each mixture had a concentration of 2.5 mM in 90% d6-DMSO and 10% D2O. The final volume of a screening sample was 170 &#181;L with 5% D2O as a locking agent. Each mixture was pipetted two times, one in a buffer containing solution (without target) and one into a target containing buffer solution. The ratio of target and fragment was set to 1:1, resulting in a final target/ligand concentration of 50 &#181;M. Additionally control samples are the target biomolecule in screening buffer without a mixture to ensure target integrity as well as a control sample with only buffer and D2O to ensure buffer quality.
NMR screening data of 19F-1D and 19F-CPMG-T2 were&#160;measurements as described in section 3.1. For example, in the case of RNA a jump-return echo sequence (pp = zggpjrse,15) was acquired for the single target sample in buffer.
Data Analysis 
The 19F screening procedure was applied to the TPP riboswitch thiM from E. coli and protein tyrosine kinase (PtkA) from M. tuberculosis among several other targets16. The 19F screening library has 103 fragments that are divided into 5 Mixes labelled from Mix A to E. Preparation of screening samples can be performed manually without the use of a sample pipetting robot. 40 &#181;M thiM RNA containing solution (buffer conditions) was mixed with 3.2 &#181;L from the mixtures. Further control samples were prepared consisting of buffer only, buffer with 5% of DMSO (previously ensure the stability of the biomacromolecule in the presence of the desired DMSO concentration) and buffer with RNA. These 13 screening samples were prepared and transferred to 3 mm NMR-tubes. Barcodes of NMR tubes are scanned and each mixture in the presence and absence of RNA, as well as control samples were measured according to the aforementioned 19F NMR experiments performed at 298 K. Screening of thiM RNA against the in-house library was performed by conducting T2 measurements with CPMGs of 0 ms and 200 ms for each different sample. Proper shimming and water suppression were monitored after finishing the measurements by comparing all DMSO peaks in terms of line broadening and intensity loss of additionally measured 1H 1D experiments for all samples. Processing of obtained CPMG T2&#160;19F relaxation spectra was performed using a previously prepared and automated macro in TopSpin, respectively. Data analysis was performed following the instructions in the protocol section. The integral data obtained from TopSpin (following the instructions in the protocol) can be evaluated quickly and easily using a pre-made spreadsheet or any similar program, by setting the correct conditions and thresholds. As described previously, thresholds are useful in defining binder, weak binder, or non-binder. Figure 5 shows typical results of CPMG spectra of thiM RNA and PtkA, respectively. In some cases, further expert revision was needed.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62262/62262fig05.jpg" /> 
Figure 5: Cut out of 19F CPMG NMR spectra showing the intensity changes obtained from different delay times of CPMG based experiments. (A) Representation of a binder (hit) and a non-binder in 19F fragment-based screening performed on TPP riboswitch thiM RNA from E. coli. (B) Representation of a binder and a non-binder in 19F fragment-based screening performed on PtkA from M. tuberculosis. <a href="https://www.jove.com/files/ftp_upload/62262/62262fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1H Screening
Mixture design 
The used in-house library is so diverse that for 1H screening purposes no mixture design was performed. This means that 64 mixes were prepared by randomly choosing 12 to be mixing in one mixture.
Sample Preparation 
For the 1H screening of an exemplary SARS-CoV-2 RNA, automated pipetting using a pipetting robot was performed to prepare the samples. The fragments in each mixture had a concentration of 4.2 mM in 90% d6-DMSO and 10% D2O. The final volume of a screening sample was 200 &#181;L with 5% D2O as a locking agent. 64 samples each containing a different mixture in 25 mM KPi, 50 mM KCl at pH 6.2 were pipetted without target RNA. Respectively, 64 samples were pipetted with target RNA, each containing a different mixture. The RNA:Ligand ratio was set to 1:20, resulting in an RNA concentration of 10 &#181;M and a ligand concentration of 200 &#181;M.
Data Analysis 
For the 1H analysis, the FBS tool in TopSpin was used. To determine if a fragment is a hit, 1D chemical shift, waterLOGSY, and T2 relaxation experiments were conducted. For T2 relaxation, a decrease in intensity greater than 30% was counted as a hit, while for the chemical shift a shift of greater than 6 Hz was the cut-off. The waterLOGSY had to show a significant signal change (from negative to positive in this case). If any two of these three criteria were positive, a fragment was counted as a hit. Two examples for this can be seen in Figure 6.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62262/62262fig06.jpg" /> 
Figure 6: 1H screening performed on an exemplary SARS-CoV-2 RNA showing hit determination criteria. Acquisition of three different experiments (1H T2 CPMG (5/100 ms), waterLOGSY, and 1D 1H).&#160;<a href="https://www.jove.com/files/ftp_upload/62262/62262fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Hit-1 shows a T2 decrease of ~50% and a CSP &#8805; 6 Hz. The waterLOGSY does not show a significant enough change in signal to also be counted as positive. As two out of three experiments are positive, this fragment is counted as a hit. For Hit-2, the T2 shows a decrease of ~80% signal intensity and a clear signal change can be seen for the waterLOGSY. The CSP is not enough in this case, but as the two previous criteria are positive it is still counted as a hit.

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Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

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As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Real-time Tracking of DNA Fragment Separation by Smartphone Video (Video) | JoVE

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions Video (Video) | JoVE

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans Video (Video) | JoVE

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering Video (Video) | JoVE

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes Video (Video) | JoVE

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors Video (Video) | JoVE

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis Video (Video) | JoVE

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin Video (Video) | JoVE

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline Video (Video) | JoVE

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...