Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Summary

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Abstract

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (γPNA) tiles. Each single-stranded tile (SST) is a 12-base γPNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these γPNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

Introduction

Successful construction of numerous complex nanostructures^{1,2,3,4,5,6,7,8,9,10,11,12} in aqueous or substantially hydrated media made using naturally occurring nucleic acids such as DNA^1,2,

Protocol

1. γPNA sequence design

1. Download the DNA Design Toolbox²² developed by the Winfree Lab at Caltech²³ into the folder containing programming scripts for designing sequences.
2. Within that sequence design folder, open a fourth-generation programming language compatible with the file extension “.m”, and then add the previously downloaded “DNAdesign” folder to the path using the following command:
   >> addpath DNAdesign
3. Subsequently run the following script named “PNA3nanofiber.m” (see Supplementary Figure 1) using the followi....

Results

The protocols discussed in the sections above describe the design of an adapted SST motif from DNA nanofibers for the robust generation of self-assembled nanofibers structures using multiple, distinct γPNA oligomers. This section describes the interpretation of data obtained from the successful recreation of the protocols described.

Following the protocol described in section 5 for TIRF imaging of samples of γPNA oligomers annealed in 75% DMSO: H₂O (v/v) most readily provides eviden.......

Discussion

This article focuses on adapting and improving existing nucleic acid nanotechnology protocols towards organic solvent mixtures. The methods described here focus on modifications and troubleshooting within a defined experimental space of select polar aprotic organic solvents. There is yet unexplored potential for other established nucleic acid nanotechnology protocols to be adapted within this space. This could improve potential applications through integration in other fields such as polymer and peptide synthesis which t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
γPNA strands/oligomers	Trucode Gene Repair Inc.		Section 2.1
UV-Vis Spectrophotometer	Agilent	Varian Cary 300	Section 3.1.2
Quartz cuvettes	Starna	29-Q-10	Section 3.1.1
Thermal cycler	Bio Rad	C1000 touch	Section 4.1
0.2 mL PCR tubes	VWR	53509-304	Section 4.5
Anhydrous DMF	VWR	EM-DX1727-6	Section 4.6
Anhydrous DMSO	VWR	EM-MX1457-6	Section 4.6
Anhydrous 1,4-Dioxane	Fisher Scientific	AC615121000	Section 4.6
10X Phosphate Buffered Saline (PBS)	VWR	75800-994	Section 3.1.1
Microscope slides	VWR	89085-399	Section 5.2
Glass cover slips	VWR	48382-126	Section 5.2
2% Collodion in Amyl Acetate	Sigma-Aldrich	9817	Section 5.2
Isoamyl Acetate	VWR	200001-180	Section 5.2
Biotinylated Bovine Serum Albumin (Biotin-BSA)	Sigma-Aldrich	A8549	Section 5.3
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A2153	Section 5.4
Streptavidin	Sigma-Aldrich	189730	Section 5.5
Trolox	Sigma-Aldrich	238813	Section 5.7
Total Internal Reflection Fluorescence microscope	Nikon	Nikon Ti2-E	Section 5.8
Transmission Electron Microscope	Joel	JEM 1011	Section 6.6
Tweezers	Dumont	0203-N5AC-PO	Section 6.3
Uranyl Acetate	Electron Microscopy Sciences	22400	Section 6.1
Formvar, 300 mesh, Copper grids	Ted Pella Inc.	1701-F	Section 6.2
Formvar-Silicon monoxide Type A, 300 mesh, Copper grids	Ted Pella Inc.	1829	Section 6.2
DNA oligomers/strands	IDT		Section 7.1
Sodium Dodecyl Sulphate (SDS)	VWR	97064-860	Section 8.1