Peptide Nucleic Acids: Methods and Protocols (Methods in Molecular Biology, 2105)

Preface
Contents
Contributors
Chapter 1: Fmoc-Based Assembly of PNA Oligomers: Manual and Microwave-Assisted Automated Synthesis
	1 Introduction
	2 Materials
		2.1 Chemicals
		2.2 Solutions and Eluents
		2.3 Instruments
	3 Methods
		3.1 Preparations
		3.2 Assembly of PNA Oligomers and Final Cleavage
			3.2.1 Manual Synthesis of H-CTCATACCTTG-NH2
			3.2.2 MW-Assisted Solid-Phase Synthesis of H-CTCATACCTTG-NH2 Using Biotage Syro Wave (at 45 C)
		3.3 Purification and Characterization
	4 Notes
	References
Chapter 2: A Robust Method for Preparing Optically Pure MiniPEG-Containing Gamma PNA Monomers
	1 Introduction
	2 Materials
	3 Methods
		3.1 Synthesis of O3(Tetrahydropyran-2-yl)-l-Serine Methyl Ester Hydrochloride (4)
		3.2 Synthesis of N-(9-(4-Bromophenyl)-9-Fluorenyl)-O3-(Tetrahydropyran-2-yl)-Serine Methyl Ester (5)
		3.3 Synthesis of 2-N-(9-(4-Bromophenyl)-9-Fluorenyl)-3-Hydroxyl-N-Methoxy-N-Methylpropanamide (8)
		3.4 Synthesis of 2-N-(9-(4-Bromophenyl)-9-Fluorenyl)-3-(2-(2-(tert-Butoxy)Ethoxy)Ethoxy)-N-Methoxy-N-Methylpropanamide (9)
		3.5 Synthesis of 3-(2-(2-(tert-Butoxy)Ethoxy)Ethoxy)-N-Methoxy-N-Methyl-2-N-(9-(4-Morpholinophenyl)-9-Fluorenyl)-Propanamide (...
		3.6 Synthesis of 9-(4-Bromophenyl)-9-Fluorenol (12)
		3.7 Synthesis of ((2-(2-(tert-Butoxy)Ethoxy) Ethoxy)Methyl)Benzene (15)
		3.8 Synthesis of 2-(2-(tert-Butoxy)Ethoxy)Ethyl-4-Methylbenzene-Sulfonate (17)
		3.9 Synthesis of 3-(2-(2-(tert-Butoxy)Ethoxy)Ethoxy-2-N-(9-(4-Morpholinophenyl)-9-Fluorenyl))-Serine-Psi [CH2N]Gly-O-Allyl (19)
		3.10 A General Procedure for Coupling Nucleobase Acetic Acid with Backbone (19)
		3.11 A General Method to Convert MPhF-Protected Monomer Allyl Ester 20a-d to Fmoc-Protected Monomer Allyl Ester 22a-d
		3.12 A General Method for Converting Fmoc-Protected Monomer Allyl Ester 22a-d to the Final Monomer 1a-d
		3.13 Determination of Optical Purities
	4 Notes
	References
Chapter 3: Synthesis of Pyrrolidinyl PNA and Its Site-Specific Labeling at Internal Positions by Click Chemistry
	1 Introduction
	2 Materials
		2.1 General
		2.2 Synthesis of Pyrrolidinyl PNA Monomers
			2.2.1 cis-4-Hydroxy-d-Proline (1) and N-Boc-cis-4-Hydroxy-d-Proline (2)
			2.2.2 Diphenyldiazomethane (3)
			2.2.3 N-Boc-cis-4-Hydroxy-d-Proline Diphenylmethyl Ester (4)
			2.2.4 N-Boc-trans-4-Hydroxy-d-Proline Diphenylmethyl Ester (5a)
			2.2.5 N-Boc-trans-4-Tosyloxy-d-Proline Diphenylmethyl Ester (5b)
			2.2.6 N-Boc-cis-4-(N3-Benzoylthymin-1-yl)-d-Proline Diphenylmethyl Ester (6-TBz)
			2.2.7 N-Boc-cis-4-(N6-Benzoyladenin-9-yl)-d-Proline Diphenylmethyl Ester (6-ABz), N-Boc-cis-4-(N4-Benzoylcytosin-9-yl)-d-Proli...
			2.2.8 N-Fmoc PNA Monomers (Free Acid) (T-OH, ABz-OH, CBz-OH, G-OH)
			2.2.9 N-Fmoc PNA Monomers (Pentafluorophenyl Ester) (T-OPfp; ABz-OPfp; CBz-OPfp)
			2.2.10 N-Fmoc ACPC Pentafluorophenyl Ester (ACPC-OPfp)
			2.2.11 N-Fmoc APCTfa Pentafluorophenyl Ester (APCTfa-OPfp)
		2.3 Preparation of Stock Solutions for PNA Synthesis
		2.4 Preparation of Stock Solutions for Post-Synthetic Labeling of Pyrrolidinyl PNA by Sequential Reductive Alkylation-Click Ch...
	3 Methods
		3.1 Pyrrolidinyl PNA Synthesis
		3.2 Post-Synthetic Labeling of Pyrrolidinyl PNA by Sequential Reductive Alkylation-Click Chemistry
		3.3 Cleavage and Purification
	4 Notes
	References
Chapter 4: Synthesis of PNA-Peptide Conjugates as Functional SNARE Protein Mimetics
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Chemicals and Solutions
	3 Methods
		3.1 Automated SPPS of the SNARE Linker and Transmembrane Domains
		3.2 Manual SPPS of the PNA Recognition Unit
		3.3 Automated SPPS of a PNA-Peptide Conjugate
	4 Notes
	References
Chapter 5: Lipid-Modified Peptide Nucleic Acids: Synthesis and Application to Programmable Liposome Fusion
	1 Introduction
	2 Materials
		2.1 LiPNA Synthesis
		2.2 Liposomes and Fusion Experiments
	3 Methods
		3.1 LiPNA Synthesis and Purification
			3.1.1 Solutions for Solid-Phase Automated Peptide Synthesis (0.025 mmol Scale)
			3.1.2 Startup and Coupling Cycle for Automated Peptide Synthesis
			3.1.3 Cleavage and Deprotection of LiPNAs
			3.1.4 HPLC Purification of LiPNAs
		3.2 Stock Solutions for Liposome Fusion Experiments
			3.2.1 LiPNA Stock Solutions
		3.3 Liposomes
			3.3.1 Determine Concentrations of DOPC and DOPE Stock Solutions
			3.3.2 Lipid Film Preparation
			3.3.3 Liposome Preparation and Extrusion
			3.3.4 Size Exclusion of SRB-Filled Liposomes
		3.4 Liposome Characterization Using NTA
		3.5 Content Mixing Experiments
			3.5.1 Preparation of 1 μM LiPNA Solutions
			3.5.2 Engraftment of LiPNAs on Liposomes
			3.5.3 Measurement of Content Mixing Including Leakage and Leakage Only
			3.5.4 Data Treatment and Calculation of CM
	4 Notes
	References
Chapter 6: Facile Preparation of PNA-Peptide Conjugates with a Polar Maleimide-Thioether Linkage
	1 Introduction
	2 Materials
		2.1 Chemicals
		2.2 Solutions and Buffers
		2.3 Instruments
	3 Methods
		3.1 Preparations
		3.2 PNA Conjugation
		3.3 Purification and Characterization
	4 Notes
	References
Chapter 7: PNA-Encoded Synthesis (PES) and DNA Display of Small Molecule Libraries
	1 Introduction
	2 Materials
		2.1 Chemicals and Abbreviations
		2.2 Codon Systems for the Libraries
			2.2.1 Codon System for the 62,500 Combinatorial Assembly of Two PNA-Encoded Fragment Libraries (See Note 1)
			2.2.2 Codon System for the 10,000 Focus PNA-Encoded Library
		2.3 Synthesis of PNAs
		2.4 Microarray Analysis
	3 Methods
		3.1 Library Synthesis
			3.1.1 PES: PNA-Encoded Synthesis Using Split and Mix Techniques
				First Amino Acid Coupling on Resin with Loading Reduction to 0.2 mmol/g
				Fmoc Deprotection on 5 mg of Resin
				Mtt Deprotection on 5 mg of Resin
				Coupling of Carboxylic Acids: Small Molecule Amino Acids and PEG Spacer Using HOBt/DIC Activation
				Coupling of Amines Using an Activate Carbonate
				Coupling of Anilines Using Diglycolic Anhydride
				Coupling of Alcohols
				Azide Reduction on Resin (this should be moved after \'Coupling of Alcohols\' and before \'PNA synthesis\')
				PNA Synthesis
				Cleavage from the Resin
		3.2 Microarray Hybridization and Screening
			3.2.1 Library Hybridization
			3.2.2 Screening of the Libraries´ Interactions with Proteins
	4 Notes
	References
Chapter 8: Transcription Inhibition by PNA-Induced R-Loops
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of DNA Substrates
		3.2 PNA Invasion into DNA
		3.3 Monitoring PNA Invasion into DNA
		3.4 In Vitro Transcription Reaction
			3.4.1 In Vitro Transcription Reaction with T7 RNAP
			3.4.2 In Vitro Transcription Reaction with RNAP II in HeLa Nuclear Extract
	4 Notes
	References
Chapter 9: Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA
	1 Introduction
	2 Materials
		2.1 Peptide Synthesis
		2.2 PNA Synthesis
		2.3 HPLC Purification
		2.4 Isothermal Titration Calorimetry
	3 Methods
		3.1 Peptide Synthesis
		3.2 PNA Synthesis
		3.3 Labeling PNA with HiLyte Fluor 488 (HF488)
		3.4 PNA Cleavage from Solid Support and Deprotection
		3.5 HPLC Purification of Synthetic PNA
		3.6 Quantification of Synthetic PNA
		3.7 Preparation of the RNA Target
		3.8 Isothermal Titration Calorimetry
		3.9 Confocal Fluorescence Microscopy
	4 Notes
	References
Chapter 10: In Vitro Cellular Delivery of Peptide Nucleic Acid (PNA)
	1 Introduction
		1.1 Cellular Delivery of PNA by Cell-Penetrating Peptide (CPP)
		1.2 Cellular Delivery of PNA Conjugates by Cationic Lipids
		1.3 Improvement of PNA Cellular Delivery by Endosomolytic Treatment
	2 Materials
		2.1 Cell Culture
		2.2 PNA Transfection
		2.3 Improvement of PNA Cellular Delivery Efficacy by Endosomolytic Treatment
		2.4 PNA Synthesis
		2.5 PNA-Cholic Acid Conjugate Synthesis
	3 Methods
		3.1 Cell Culture
		3.2 PNA Transfection
			3.2.1 Cellular Delivery of CPP-PNA Conjugates in Serum-Free Medium
			3.2.2 Cellular Delivery of CPP-PNA Conjugates in Serum-Containing Medium
			3.2.3 Cationic Lipid-Mediated Delivery of PNA Conjugates
		3.3 Improvement of PNA Cellular Delivery Efficacy by Endosomolytic Treatment
			3.3.1 Chloroquine (CQ) Treatment
			3.3.2 Photochemical Internalization (PCI) Treatment
		3.4 Synthesis of PNA Conjugates
			3.4.1 Preparation of the MBHA Resin for PNA Synthesis
			3.4.2 Synthesis
			3.4.3 Cleavage/Deprotection
		3.5 PNA-Cholic Acid Conjugate Synthesis
	4 Notes
	References
Chapter 11: Reactive Quantum Dot-Based FRET Systems for Target-Catalyzed Detection of RNA
	1 Introduction
	2 Materials
		2.1 Solid-Phase Synthesis of PNA
		2.2 Immobilization of PNA Probes on Carboxyl-Functionalized QDs
		2.3 Synthesis of Label Donor PNA Using Thioesterification in Solution
		2.4 RNA-Templated Reaction
	3 Methods
		3.1 Solid-Phase PNA Synthesis
		3.2 Buffer Exchange for a QD Stock Solution
		3.3 Functionalization of ITK605 with Cycloalkyne Groups (Fig. 2)
		3.4 Immobilization of LAPNA on a QD
		3.5 RNA-Templated Reaction in a Microtiter Plate (Fig. 3)
	4 Notes
	References
Chapter 12: Peptide Nucleic Acids for MicroRNA Targeting
	Abbreviations
	1 Introduction
		1.1 MicroRNAs as Key Posttranscriptional Regulators of Gene Expression
		1.2 MicroRNA Therapeutics
		1.3 Peptide Nucleic Acids and MicroRNA Therapeutics
		1.4 Aim of the Article
	2 Materials
		2.1 Peptide Nucleic Acids (PNAs)
		2.2 Cell Culture
		2.3 RNA Extraction
		2.4 Real-Time Quantitative PCR of MicroRNAs
		2.5 TaqMan RT-qPCR for Gene Expression Analysis
		2.6 Western Blotting
		2.7 Viability and Apoptosis Assay
		2.8 Statistics
	3 Methods
		3.1 Human Cell Lines, PNA Treatment, and Culture Conditions
		3.2 RNA Extraction and Real-Time Quantitative Analysis of MiRNA Expression
		3.3 Studies on Alteration of Gene Expression: RT-qPCR Analysis
		3.4 Studies on Alteration of Gene Expression: Western Blotting
		3.5 Studies on Vitality and Apoptosis Effects
	4 Notes
	References
Chapter 13: Detection of Microorganisms by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid
	1 Introduction
	2 Materials
		2.1 Probe Design
		2.2 FISH Procedure
	3 Methods
		3.1 Probe Design
		3.2 Fluorescence In Situ Hybridization (FISH) Procedure
			3.2.1 Preparation of Samples
				Pure Cultures or Enriched Samples
				Histological Samples
				Biofilm or Complex Microbial Consortium in Solid Supports
			3.2.2 Fixation and Permeabilization
				Procedure for Cells Adhered to Surfaces (or to Histological Tissues)
				Procedure in Suspension
			3.2.3 Hybridization
				Procedure for Cells Adhered to Surfaces (or to Histological Tissues)
				Procedure in Suspension
		3.3 Analysis of the Samples by Epifluorescence Microscopy
	4 Notes
	References
Chapter 14: PNA Antisense Targeting in Bacteria: Determination of Antibacterial Activity (MIC) of PNA-Peptide Conjugates
	1 Introduction
	2 Materials
		2.1 PNA Preparation
		2.2 Bacterial Culture
		2.3 Minimal Inhibitory Concentration Measurement
	3 Methods
		3.1 Peptide-PNA Conjugate Stock Preparation
		3.2 Bacterial Growth
		3.3 MIC Assay (Fig. 1)
		3.4 Kill Curves
	4 Notes
	References
Chapter 15: In Vivo Administration of Splice Switching PNAs Using the mdx Mouse as a Model System
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Injection Solution
		3.2 I.m. Administration in Tibialis Anterior (TA)
		3.3 I.v. Administration
		3.4 S.c. or i.p. Administration
		3.5 Acute Toxicity Observation
		3.6 Removal of Tissue 7 Days or Longer After Treatment
		3.7 Cryosection
		3.8 Dystrophin and Laminin Double-Staining of mdx Muscles
		3.9 RT-PCR Protocol for Detection of Exon Skipping
	4 Notes
	References
Chapter 16: Near-Infrared In Vivo Whole-Body Fluorescence Imaging of PNA
	1 Introduction
	2 Materials
	3 Methods
		3.1 AF680-PNA Synthesis
		3.2 Preparation of Injection Solutions (See Note 3)
		3.3 Preparation of Injection Solution with Treatment Dose (See Note 4)
		3.4 In Vivo Epifluorescence Imaging (See Note 6)
		3.5 Post-scanning Data Processing
		3.6 In Vivo 3D Fluorescence Imaging Tomography (FLIT)
		3.7 Post-scanning Data Processing
	4 Notes
	References
Chapter 17: Poly(Lactic-co-Glycolic Acid) Nanoparticle Delivery of Peptide Nucleic Acids In Vivo
	1 Introduction
		1.1 Primer on PNA-Induced Gene Editing
		1.2 The Imperative of Nanoparticle-Mediated PNA/DNA Delivery for Gene Correction
		1.3 In Vivo Demonstrations of PNA/DNA-Induced Gene Editing Enabled by PLGA NPs
			1.3.1 Modification of CCR5
			1.3.2 Gene Correction in Cystic Fibrosis Models
			1.3.3 Gene Correction in β-Thalassemia Models
		1.4 In Vivo Applications of AntimiR PNAs Enabled by PLGA NPs
			1.4.1 Primer on AntimiR PNAs
			1.4.2 NP-Mediated Delivery of AntimiR-155 PNA
			1.4.3 NP-Mediated Delivery of PNA AntimiR-210
		1.5 PLGA NPs as Vehicles for Delivery of Bioactive PNA/DNA Reagents
	2 Materials
		2.1 Instruments and General Laboratory Equipment
		2.2 Chemicals
		2.3 Surfactant Solutions
		2.4 Nucleic Acids
	3 Methods
		3.1 Nanoparticle Formulation
			3.1.1 Polymer Preparation (Day 1)
			3.1.2 PNA and Donor DNA Encapsulation (Day 2)
			3.1.3 Nanoparticle Collection
		3.2 Nanoparticle Characterization
			3.2.1 Nanoparticle Diameter
			3.2.2 Nanoparticle Zeta Potential
			3.2.3 Nanoparticle Surface Morphology
			3.2.4 Nanoparticle Loading
		3.3 Representative Results
	4 Notes
	References
Chapter 18: Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting
	1 Introduction
	2 Materials
		2.1 Manual Solid-Phase Synthesis of the PNA-Based Hybridization Probes, HP1 and HP2
		2.2 Sortase A-Mediated Ligation
		2.3 Final RP-HPLC Purification of HP2 and ZHER2:342-SR-HP1
		2.4 Preparation of Solutions for Labelling
		2.5 Labelling of DOTA-PNA with 111In or 57Co
		2.6 Labelling of DOTA-PNA with 177Lu
		2.7 Labelling of DOTA-PNA with 68Ga
		2.8 Labelling of DOTA-Affibody-PNA Conjugate with 68Ga
		2.9 Quality Control Using HPLC
		2.10 Evaluation of Pretargeting In Vitro Specificity
	3 Methods
		3.1 Expression and Purification of Recombinant Proteins
		3.2 General Solid-Phase Synthesis of the PNA-Based Hybridization Probes, HP1 and HP2
		3.3 Synthesis of HP1 (GGG-PNA)
		3.4 Synthesis of HP2 (DOTA-PNA)
		3.5 Cleavage of the PNA-Based Hybridization Probes
		3.6 Sortase A-Mediated Affibody-PNA Ligation
		3.7 Purification of HP2
		3.8 Purification of ZHER2:342-SR-HP1
		3.9 Preparation of Buffers and Solution for Labelling
		3.10 Labelling of DOTA-PNA with 111In or 57Co
		3.11 Labelling of DOTA-PNA with 177Lu
		3.12 Labelling of DOTA-PNA with 68Ga
		3.13 Labelling of DOTA-Affibody-PNA with 68Ga
		3.14 Quality Control Using HPLC
		3.15 Evaluation of Pretargeting In Vitro Specificity
	4 Notes
	References
Index