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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<li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+pharmacology+of+a+highly+specific+dual+FMS+and+KIT+kinase+inhibitor%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 5689-5694 (2013).</li>
<li>Lipinski, C. A., Lombardo, F., Dominy, B. W., Feeney, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+and+computational+approaches+to+estimate+solubility+and+permeability+in+drug+discovery+and+development+settings%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.</a> Advanced Drug Delivery Reviews. 23, 3-25 (1997).</li>
<li>Congreve, M., Carr, R., Murray, C., Jhoti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+%27Rule+of+Three%27+for+fragment-based+lead+discovery%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">A 'Rule of Three' for fragment-based lead discovery.</a> Drug Discovery Today. 8, 876-877 (2003).</li>
<li>Chávez-Hernández, A. L., Sánchez-Cruz, N., Medina-Franco, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Fragment+Library+of+Natural+Products+and+its+Comparative+Chemoinformatic+Characterization%5BTitle%5D)+AND+%22Molecular+Informatics%22%5BJournal%5D)">A Fragment Library of Natural Products and its Comparative Chemoinformatic Characterization.</a> Molecular Informatics. 39, 2000050(2020).</li>
<li>Sreeramulu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NMR+quality+control+of+fragment+libraries+for+screening%5BTitle%5D)+AND+%22Journal+of+Biomolecular+NMR%22%5BJournal%5D)">NMR quality control of fragment libraries for screening.</a> Journal of Biomolecular NMR. , 00327-00329 (2020).</li>
<li>Gao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+NMR+Fragment+Based+Screening+Identified+a+Novel+Interface+Blocker+to+the+LARG%2FRhoA+Complex%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Automated NMR Fragment Based Screening Identified a Novel Interface Blocker to the LARG/RhoA Complex.</a> PLoS One. 9, 88098(2014).</li>
<li>Peng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+and+Efficient+Fragment-Based+Lead+Generation+by+Fully+Automated+Processing+and+Analysis+of+Ligand-Observed+NMR+Binding+Data%5BTitle%5D)+AND+%22Journal+of+Medicinal+Chemistry%22%5BJournal%5D)">Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data.</a> Journal of Medicinal Chemistry. 59, 3303-3310 (2016).</li>
<li>Cox, O. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+poised+fragment+library+enables+rapid+synthetic+expansion+yielding+the+first+reported+inhibitors+of+PHIP%282%29%2C+an+atypical+bromodomain%5BTitle%5D)+AND+%22Chemical+Science%22%5BJournal%5D)">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/pubmed?term=((Water+Suppression+That+Works.+Excitation+Sculpting+Using+Arbitrary+Wave-Forms+and+Pulsed-Field+Gradients%5BTitle%5D)+AND+%22Journal+of+Magnetic+Resonance%2C+Series+A%22%5BJournal%5D)">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/pubmed?term=((NMR+in+drug+discovery%3A+A+practical+guide+to+identification+and+validation+of+ligands+interacting+with+biological+macromolecules%5BTitle%5D)+AND+%22Progress+in+Nuclear+Magnetic+Resonance+Spectroscopy%22%5BJournal%5D)">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/pubmed?term=((A+new+water+suppression+technique+for+generating+pure-phase+spectra+with+equal+excitation+over+a+wide+bandwidth%5BTitle%5D)+AND+%22Journal+of+Magnetic+Resonance%22%5BJournal%5D)">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/pubmed?term=((19F+NMR-Based+Fragment+Screening+for+14+Different+Biologically+Active+RNAs+and+10+DNA+and+Protein+Counter-Screens%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">19F NMR-Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter-Screens.</a> ChemBioChem. , (2020).</li>
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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><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&amp;amp;</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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NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

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.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

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Video: NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

Cancer Research

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Environment

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Genetics

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Immunology and Infection

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

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

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

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

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

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen.
Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification.
XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.

This paper describes the complete XChem process for crystal-based fragment screening, starting from applying for access and all subsequent steps to data dissemination.

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify ...

Chemistry

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

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