Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

Summary

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

Abstract

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

Introduction

DNA molecules have been used to build many kinds of nanostructures over decades. Typical motifs include DAE (double crossover, antiparallel, even half-turns) and DAO tiles^1,2,3, star tiles^4,5,6,7, single stranded (ss) tiles^8,9,10, and DNA origami^11,12,13. These DNA....

Protocol

1. Preparation of Circular DNAs

1. Use all linear DNAs provided by commercial companies directly without further purification.
2. Centrifuge the DNA samples at 5,000 × g for 5 min to collect all DNA pellets at the bottom of the tubes. Add an appropriate volume of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to dissolve the DNA.
3. Measure the concentration of "a" ng/µL for each ss-DNA solution using a micro UV spectrometer at 260 nm. Convert "a" ng/µL to "b" µM following b = a × 10³ / (molecular weight of the DNA strand). Adjust the amount of TE solution to make a 10 µM DNA stock solu....

Results

The circular DNA moves slightly slower than its precursor linear DNA in denaturing PAGE (Figure 2) because the pore inside the circular DNA is penetrated and retarded by gel fibers^23,24,25. The correct ligation reaction efficiency for oligo-monomer cyclization depends on the substrate sequence and concentration, reaction temperature, time, etc. As the concen.......

Discussion

The protocols presented in this article focus on the synthesis of small circular DNA molecules and the assembly of DNA nanostructures. Most of randomly-sequenced DNA designs can be used in this protocol. The purity of circular DNAs is critical for the success of DNA assemblies. The production yield of cyclization can be improved by lowering the concentration of 5'-phosphorylated linear DNA; however, this will increase the workload to produce the same amounts of circular DNAs. The length of splint DNA strands also affects.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
T4 ligase	TaKaRa	2011A
T4 buffer	TaKaRa	2011A
TE buffer	Sangon	B548106
Thermo bottle	Thermos	SK-3000
Thermo cycler	Bio Gener	GE4852T
Exonuclease I	TaKaRa	2650A
Exonuclease I buffer	TaKaRa	2650A
30% (w/v) Acryl/Bis solution (19:1)	Sangon	B546016
TAE premix podwer	Sangon	B540023
Mg(Ac)2·4H2O	Nanjing Chemical Reagent	C0190550223
Urea	Sangon	A510907
TEMED	BBI	A100761
Ammonium Persulfate	Nanjing Chemical Reagent	13041920295
Power supply	Beijing Liuyi	DYY-8C
Water bath	Sumsung	DK-S12
Formamide	BBI	A100314
DNA Marker (25~500 bp)	Sangon	B600303
DNA Marker (100~3000 bp)	Sangon	B500347
Loading buffer	Sangon	B548313
PAGE electrophoresis systerm	Beijing Liuyi	24DN
Filter	ASD	5010-2225	0.22 µM
UV imaging System	Tanon	2500R
n-butanol	Sangon	A501800
Absolute Ethanol	SCR	10009257
NaOAc	Nanjing Chemical Reagent	12032610459
Centrifuge	eppendorf	Centrifuge 5424R
Vacuum concentrator	CHRIST	RVC 2-18
Ultraviolet spectrum	Allsheng	Nano-100
nucleic acid stain	Biotium	16G1010	GelRed
Agarose	Biowest	G-10
Agarose electrophoresis systerm	Beijing Liuyi	DYCP-31CN
Heating Plate	Jiangsu Jintan	DB-1
TBE premix podwer	Sangon	B540024
filter column	Bio-Rad	7326165	Freeze 'N Squeeze column
AFM	Bruker	Dimension FastScan
PEG8000	BBI	A100159
Mica	Ted Pella	BP50
triangular AFM probe in air	Bruker	FastScan-C
triangular AFM probe in fulid	Bruker	ScanAsyst-fluid+
DNA strands	Sangon