DNA-Encoded Chemical Libraries: Methods and Protocols

✍ Scribed by David Israel (editor), Yun Ding (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 225
• Series: Methods in Molecular Biology, 2541
• Edition: 1
• Category: Library

✦ Synopsis

This volume discusses protocols that cover synthesis, screening by selection, and analysis of DNA-encoded chemical libraries (DEL). Chapters in this book focus on methods used to practice DEL technology and include solution phase library synthesis using a variety of chemistries; DNA encoding of chemical structure; design, preparation and analysis of target proteins and tool compounds; screening of soluble protein targets by affinity selection; DEL qPCR, preparative PCR and DNA sequence analysis; computational methods used to analyze selections and choose compounds for resynthesis; and analysis of hit compounds. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and comprehensive, DNA-Encoded Chemical Libraries: Methods and Protocols is a valuable resource for scientists interested in DEL technology for drug discovery, and will contribute to the continued advancement in this important field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Library Synthesis: Building Block Selection, Handling, and Tracking
1 Introduction
2 Materials
2.1 Building Block Selection
2.2 Building Block Handling and Tracking
3 Methods
3.1 Building Block Selection
3.2 Building Block Handling and Tracking
4 Notes
References
Chapter 2: Library Synthesis: Building Block Validation
1 Introduction
2 Materials
2.1 Validation Chemistry
2.2 LC-MS Analysis
3 Methods
3.1 Validation Chemistry
3.2 LC-MS
3.3 Validation Data Analysis
4 Notes
References
Chapter 3: Library Synthesis and Analytics
1 Introduction
2 Materials
2.1 DNA Ligation
2.2 Chemistry
2.3 Analytics
2.3.1 Gel Electrophoresis
2.3.2 LC/MS
2.4 Purification
3 Methods
3.1 Enzymatic Ligation
3.2 Ligation QC
3.3 Chemistry
3.4 Chemistry QC
3.5 Pooling and Purification
4 Notes
Reference
Chapter 4: Chemical Ligation of Oligonucleotide Tags to Support Encoded Chemical Library Synthesis
1 Introduction
2 Materials
3 Methods
3.1 Triazole Ligation Junctions Prepared from 5′-Azido and 2′-Ribo-3′-Propargyl Oligonucleotides
3.2 Triazole Ligation Junctions Prepared from 5′-Azido and 2′-Deoxy-3′-Propargyl Oligonucleotides
3.3 Template-Dependent Polymerization Using a Triazole-Linked Oligonucleotide Template and the Klenow Fragment of DNA Polymera...
3.4 Phosphorothioate Ligation Junctions Prepared from 5′-Iodo and 2′-Deoxy-3′-Thiophosphate Oligonucleotides
3.5 Phosphodiester Ligation Junctions Prepared from 5′-Hydroxy and 2′-Deoxy-3′-Phosphate Oligonucleotides Using Cyanoimidazole...
4 Notes
References
Chapter 5: On-DNA Reductive Amination and Alkylation
1 Introduction
2 Materials
2.1 Chemistry
2.2 Reaction Equipment
2.3 Chemistry Analytics
3 Methods
3.1 Reductive Amination (see Fig. 1)
3.2 Reductive Alkylation (see Fig. 2)
3.3 Chemistry QC and Product Recovery
4 Notes
References
Chapter 6: On-DNA-1,2,3-Triazole Formation via Click Reaction
1 Introduction
2 Materials
2.1 Chemistry
2.2 Reaction Equipment
2.3 Chemistry Analytics
3 Methods
3.1 Copper-Catalyzed Azide-Alkyne Cycloaddition
3.2 Cu Scavenging and QC
3.2.1 Procedure for LC/MS Sample Preparation for QC
3.2.2 Procedure for Cu Scavenging and EtOH Precipitation
4 Notes
References
Chapter 7: On-DNA Suzuki-Miyaura Cross-Coupling
1 Introduction
2 Materials
2.1 Chemistry
2.2 Reaction Equipment
2.3 Chemistry Analytics
3 Methods
3.1 Suzuki-Miyaura Cross-Coupling
3.2 Pd Scavenging and QC
3.2.1 Procedure Prior to LC/MS Sample Preparation
3.2.2 Procedure Prior to EtOH Precipitation
4 Notes
References
Chapter 8: On-DNA Pd and Cu-Promoted C-N Cross-Coupling Reactions
1 Introduction
2 Materials
2.1 Chemistry
2.2 Reaction Equipment
2.3 Chemistry Analytics
2.4 Purification
3 Methods
3.1 Pd-Promoted on-DNA C-N Cross-Coupling for Aromatic Amines (see Fig. 1)
3.2 Cu-Promoted On-DNA C-N Cross-Coupling for Amino Acids (see Fig. 2)
3.3 Cu-Promoted On-DNA C-N Cross-Coupling for Aliphatic Amines (see Fig. 3)
4 Notes
References
Chapter 9: Palladium-Promoted On-DNA Heck Reactions
1 Introduction
2 Materials
2.1 Chemistry
2.2 Reaction Equipment
2.3 Chemistry Analytics
2.4 Purification
3 Methods
3.1 DNA-Conjugated Styrene Preparation (see Fig. 1)
3.2 Heck Reaction Between DNA-Conjugated Styrene and Aryl Halides (see Fig. 2)
3.3 Heck Reaction Between DNA-Conjugated Styrene and Aryl Borates (see Fig. 3)
3.4 DNA-Conjugated Acrylamide Preparation (see Fig. 4)
3.5 Heck Reaction Between DNA-Conjugated Acrylamide and Aromatic Halides (see Fig. 5)
3.6 Heck Reaction Between DNA-Conjugated Acryl Amide and Aromatic Borates (see Fig. 6)
3.7 DNA-Conjugated Aromatic Iodide Preparation (see Fig. 7)
3.8 Heck Reaction Between DNA-Conjugated Aromatic Iodide and Styrene (see Fig. 8)
4 Notes
References
Chapter 10: Ruthenium-Promoted On-DNA C-H Activation Reaction
1 Introduction
2 Materials
2.1 Chemistry
2.2 Reaction Equipment
2.3 Chemistry Analytics
2.4 Purification
3 Methods
3.1 C-H Activation Reaction (see Fig. 1)
3.2 Chemistry QC
4 Notes
References
Chapter 11: DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles
1 Introduction
2 Materials
2.1 DNA Headpiece
2.2 General Chemicals and Reagents
2.3 Borate Buffer
2.4 HEPES Buffer
2.5 TBE Buffer
2.6 Ethidium Bromide Stain
2.7 5M NaCl
2.8 Analytical HPLC
3 Methods
3.1 Ethanol Precipitation and DNA Reconstitution
3.2 Reaction Conversion Assessment by LC-MS
3.3 General Procedure for Acylation
3.4 General Procedure for SNAr Reaction
3.5 General Procedure for Nitro Reduction
3.6 General Procedure for Formation of Benzimidazoles
3.6.1 For the One-Pot Synthesis of Benzimidazole
3.6.2 For the Stepwise Procedure
4 Notes
References
12: Condensation of DNA-Conjugated Imines with Homophthalic Anhydride for the Synthesis of Isoquinolones On-DNA
1 Introduction
2 Materials
2.1 Chemistry
2.2 Chemistry Analytics
3 Methods
3.1 Isoquinolone Formation (see Fig. 1)
3.2 Chemistry QC (see Fig. 2)
4 Notes
References
Chapter 13: DNA Encoding of Natural Products
1 Introduction
2 Materials
2.1 UP LC-MS Analysis
2.2 DNA Quantification
3 Methods
3.1 Synthesis of D1
3.2 Photo Cross-Linking of NPs
3.3 Final Workup and QC
4 Notes
References
Chapter 14: Initiating DNA-Encoded Library Synthesis with a Hexathymidine DNA Oligonucleotide
1 Introduction
2 Materials
2.1 Synthesis of Hexathymidine DNA Conjugates
2.2 Purification of Oligonucleotides
2.3 Characterization of Oligonucleotides
2.4 Ligation of DNA Oligonucleotides with T4 Ligase
3 Methods
3.1 Deprotection of the MMt-Protected 5′-Amino Linker-Modified Hexathymidine DNA on Solid Support
3.2 Synthesis of the Hexathymidine-5′-PEG Conjugate 7
3.3 HexT-Conjugated β-Carbolines by Acid-Catalyzed Pictet-Spengler Reaction
3.4 Au(I)-Mediated Pyrazole Synthesis
3.5 Ligation of DNA Strands with T4 DNA Ligase
4 Notes
References
Chapter 15: One Bead-One Compound (OBOC) Peptidomimetic-Encoded Library Synthesis via Split-and-Pool Methods
1 Introduction
2 Materials
2.1 Resin
2.2 Reaction Equipment
2.3 Chemicals
2.4 Cleavage Solution
2.5 QC Materials
2.6 Buffers
2.7 Azido Headpiece DNA Synthesis, Purification, and Characterization.
3 Methods
3.1 Synthesis of Functionalized Linker
3.1.1 Resin Preparation
3.1.2 Installation of Methionine as First Residue
3.1.3 Submonomer Peptoid Synthesis:
3.2 DNA Conjugation via Click Chemistry
3.3 Library Synthesis
3.3.1 Cycle 1
3.3.2 Cycle 2
3.4 Structure Elucidation by Tandem Mass Spectrometry
4 Notes
References
Chapter 16: DNA Conjugates as Tool Compounds for DEL Selections
1 Introduction
2 Materials
2.1 DNA Conjugate
2.2 Binding Assays and Selections
3 Methods
3.1 Design the DNA Conjugate
3.2 Synthesize and Purify the Conjugate
3.3 Demonstrate that the Conjugate Binds to the Target Protein
3.4 Optimize a Selection Protocol Using the Conjugate in a Binding Assay
3.5 Conduct a Selection Using the Conjugate as a Positive Control Spiked into the DEL Library
4 Notes
References
Chapter 17: Quantitation of DNA-Encoded Libraries by qPCR
1 Introduction
2 Materials
3 Methods
3.1 qPCR Standard Preparation for 7-Point Curve Covering 500 pM to 0.5 fM
3.2 qPCR Sample Preparation
3.3 qPCR Instrument Setup and Data Analysis
4 Notes
References
Chapter 18: Target Protein Design and Preselection Analysis
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 Other Reagents
3 Methods
3.1 Design the Target Protein Construct
3.2 Express and Purify the Target Protein
3.3 Biochemical and Biophysical Characterization of the Target Protein
3.4 Protein Immobilization Test
3.5 Activity Test for Immobilized Target Protein
3.6 Activity of a Tool Compound on Immobilized Protein
4 Notes
References
Chapter 19: DEL Selections Against a Soluble Protein Target
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 DEL Library
3 Methods
3.1 Immobilization Mode of Selection
3.2 In-Solution Mode of Selection
3.3 Quality Control (QC)
4 Notes
References
Chapter 20: Selection Method of DNA-Encoded Chemical Library for Irreversible Covalent Binders
1 Introduction
2 Materials
2.1 Equipment
2.2 Chemical Library
2.3 On-DNA Tool Compounds (Optional)
2.4 Target Protein
2.5 Selection Buffer
2.6 IMAC Affinity Resin Tips
2.7 IMAC Magnetic Beads
2.8 PAGE Gel
2.9 Agencourt AMPure XP SPRI Beads
3 Methods
3.1 Capture Test
3.1.1 Capture Test - Affinity Resin Tip
3.1.2 Capture Test - Magnetic Bead
3.2 Selection Method (DEL Selection Can Be Performed in Either In-Solution or Immobilized Mode; In-Solution Mode Is Described ...
3.2.1 Selection Method - Affinity Resin Tip
3.2.2 Selection Method - Magnetic Bead
4 Notes
References
Chapter 21: Next Generation Sequencing of DNA-Encoded Libraries
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 PCR Optimization
3.2 Preparative PCR
3.3 Purification of PCR Product
3.4 Gel Extraction of DNA Fragments
3.5 Bead Purification
3.6 Quantification of Purified DNA
3.7 Preparation of Sequencing Template
3.8 Load Templates and Sequence
4 Notes
References
Chapter 22: Translation of DNA Sequence to Chemical Structure in DNA-Encoded Libraries
1 Introduction
2 Materials
3 Methods
3.1 Modeling DEL Structure Files to Artificial Chromosomes
3.2 Sequencing Quality Control and Reads Preprocess
3.3 Sequence Decoding
3.4 Translate Codons into Chemical Structures
3.5 Post Analysis Quality Control (Optional)
4 Notes
References
Chapter 23: Analysis of DNA-Encoded Library Screening Data: Selection of Molecules for Synthesis
1 Introduction
2 Materials
2.1 Data File
2.2 Software for Data Visualization
3 Methods
3.1 Calculating Enrichment
3.2 Truncates
3.3 Mathematical Description of Truncates
3.3.1 Pre-definitions
3.3.2 Enrichment Calculations
3.4 Generate Line Plot
3.5 Generate 3D Scatter Plot
3.6 Generate 2D Scatter Plot to Inspect Truncates
4 Removing Background Signal
5 Notes
References
Chapter 24: High-Throughput Binder Confirmation (HTBC)
1 Introduction
2 Materials
2.1 Chemistry
2.2 Analytics
2.3 Spin Filter
2.4 Equipment
3 Methods
3.1 Cleavable HP Preparation
3.1.1 Photocleavable HP (see Fig. 2)
3.1.2 THP HP (see Fig. 3)
3.2 Chemistry of Target Compounds
3.3 OD and QC
3.4 Cleavage
3.4.1 PCL Cleavage (see Fig. 4)
3.4.2 THP Cleavage (see Fig. 5)
4 Notes
References
Chapter 25: High-Throughput Binder Confirmation Using Affinity Selection Mass Spectrometry
1 Introduction
2 Materials
2.1 Compound Solutions
2.2 Consumables
2.3 Equipment
2.4 Software
3 Methods
3.1 Preparation of Reference Samples
3.2 Incubation Before Loading onto SEC Columns
3.3 Separation of Bound from Unbound Compounds
3.4 Identification of Binders by LC/MS
3.5 Data Analysis and HTBC Hit Identification
3.5.1 AS-MS Data Analysis
3.5.2 HTBC Hit Identification
3.6 Quantitation of Protein Recovery After AS-MS Assay
4 Notes
References
Untitled
Index