- Open Access
DNA-nanostructure-assembly by sequential spotting
© Breitenstein et al; licensee BioMed Central Ltd. 2011
- Received: 15 September 2011
- Accepted: 18 November 2011
- Published: 18 November 2011
The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions.
For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM) has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid) to incorporate advanced structures.
The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element.
- Atomic Force Microscope
- Peptide Nucleic Acid
- Persistence Length
- Adapter Oligonucleotide
- High Fluorescence Signal
The construction of nanostructures is a challenging and resource intensive task for biotechnology. The two classical ways are the top-down and bottom-up approaches. The best known top-down method was introduced in 1999 by Mirkin et al. [1–4] where an atomic force microscope tip was used for direct writing a chemically active ink on a gold-surface. The achieved method is now known as Dip-Pen-Nanolithography and is capable of generating feature sizes in the range of 50 nm [5, 6]. Further methods that aim on the preparation of nanostructures employ electrochemical deposition of metal salts , use of photomasks  or use of nanoscale stamps . But all these methods are cost intensive and very challenging in terms of applying more than one spotting component. On the other hand, in the bottom-up approach the self-assembly capabilities of biomolecules like DNA are exploited to the design of nanostructures. Up to now, many promising ways have been published for DNA [10–13] and even RNA-structures  to achieve molecular sized structures. But predominantly such complex DNA-structures are only randomly fixed on a surface without a defined position.
Here we present a simple method that combines top-down and bottom-up approaches to generate a DNA-based nanostructure. This toolbox uses the high flexibility of an atomic force microscope which is one of the most powerful mechanical tools in nanotechnology. The key elements of this device are the piezoelectric actuators, enabling nanoscopic small movements with high precision. In our approach the top-down tool is combined with the precision of bottom-up DNA base pairing. The simple building rules of DNA-base-pairing are being used for the creation of more complex nanostructures that easily skip the limitation of all mechanically based top-down techniques. The key element is to create sophisticated structures by hierarchical assembly. In addition we employ PNA (peptide nucleic acid [15, 16]) as a powerful tool to extend the DNA's building capabilities and to functionalize existing DNA strands with junctions.
The preparation of DNA nanostructures on a solid support is based on a recently developed method  in our group that provides fixed nanoscale anchors on a surface. Therefore we immobilized biomolecules like DNA-oligonucleotides on a functionalized glass support by using general AFM-techniques. This method facilitates the deposition of different biotinylated biomolecules by a single AFM-tip without the need to optimize spotting conditions for each substance. Only a single ink, namely neutravidin in glycerol, is used for spotting, which reduces technical requirements and can therefore be easily adapted to most AFMs. Neutravidin is used as the linking element because of its capability for binding up to four biotin molecules. Consequently this enables the immobilization of any biotinylated biomolecule on that specific neutravidin spot by AFM deposition.
Double-stranded DNA is quite a rigid macromolecule with a persistence length of about 50 nm which is, compared to single-stranded DNA with 1 - 4 nm persistence length, rather stiff . However, the use of dsDNA is advantageous not only because of its stability, but because of the possibility for protein induced and molecular recognition induced DNA-binding, as well as for PNA-interactions.
To have a universal tool, a DNA-oligonucleotide as adapter element can bind to the clasping PNA. A second DNA-oligonucleotide which is labelled by a fluorescent dye was used for visualisation of the nanoconstruct under fluorescence microscopy conditions. The adapter oligonucleotide is equipped with five repetitive sequence patterns to bind up to five identical, fluorescently labelled oligonucleotides for fluorescence enhancement. Additionally, it is important to take into consideration that the triplex invasion binding of PNA to the duplex DNA is significantly favoured at low ionic strength , which strongly disfavour DNA-DNA hybridization. Thus the PNA should be bound to the DNA-construct at low ionic strength before all the following steps. Once the triplex has formed the ionic strength has no impact on the triplex stability and facilitates to change to any desired buffer .
Furthermore, it shows that the orientation of the DNA-nanostructure in figure 5 is orientated according to the spotting pattern.
We have demonstrated a novel approach for using DNA as a building unit for surface-bound nanostructures. The structure does not depend on a fixed pattern and is immobilized at a defined position with well defined dimensions.
For generating such a structure only two key features are necessary: the first is a method that allows immobilization of different biomolecules, e.g. DNA oligonucleotides, on a surface with high spatial resolution. In this work we have shown that the sequential spotting method as illustrated in figure 1 fulfils this requirement. The second feature is a refined DNA-design that allows self-assembly of the nanostructure.
The prepared nanostructure also remains chemically accessible for subsequent biomolecular recognition such as by PNA. The PNA formed structure can be regarded as a versatile construction and linking element that facilitates the further building of complex superstructures. Binding PNA to dsDNA by triplex invasion has been tested and proved by microarray analysis and fluorescence microscopy.
The spotting-method itself is easy and does not require complex preparatory work. It has been designed with the aim to facilitate the employment of most atomic force microscopes. Therefore the presented method can be integrated readily into many nanotechnology applications and key questions. An upgrade of most AFMs is cogitable and thus is cost efficient because beside the common AFM-equipment or equivalent nanomanipulation tools only commonly available chemical compounds like biotinylated oligonucleotides and neutravidin in combination with DNA are required.
The outlook predominantly addresses the analysis of single molecule interactions. The investigation of RNA in single cells, for example, is limited by the faint concentration and might take advantage of structures on a molecular level. DNA based computing machines that are based on FRET (fluorescence resonance energy transfer), as described in , could benefit from the arrangement of the presented nanostructure and would result in a exciting combination of biology and electronic. We also have miniaturized array-technology together with micrufluidic point-of-care diagnostic approaches in our focus, which might lead us to lab-in-the-blood devices.
Silanization and biotinylation
Glass slides (Menzel Gläser, Menzel GmbH & Co. KG, 38116 Braunschweig, Germany) were cleaned with ultrasound in acetone for 15 minutes and again in ethanol (acetone and ethanol were obtained from Carl Roth GmbH & Co. KG, Karlsruhe, Germany). After rinsing with ultrapure water, the slides were put into NaOH (10 M) for 1 minute and washed thoroughly with water. Drying was carried out in a centrifuge (Varifuge 3.0R, Heraeus) for 1 minute. In the vapor phase at 120°C silanization with 3-Aminopropyltriethoxysilane (Fluka Chemie GmbH, 89552 Steinheim, Germany) was executed in a sealed beaker and finished after 60 minutes. For biotinylation, Sulfo-NHS-Biotin (20 mg) (Thermo Scientific, IL 61101 USA) was dissolved in DMSO (1 mL) (Carl Roth GmbH & Co. KG) because of its low stability and moisture-sensitivity. The DMSO solved Sulfo-NHS-Biotin can be stored at -20°C with desiccant.
Sulfo-NHS-Biotin (10 mL) solution was added to Na2HPO4 (100 mM, 21 mL), NaCl (150 mM) buffer at pH 7.4. Incubation of 5 silanized glass slides took place for 3 hours at room temperature. Slides were washed with PBS and rinsed with water. Blocking was carried out by incubating the glass slides in a freshly prepared, 0.1% (w/v) solution of blocking reagent CA from Applichem in 100 mM Tris-Cl. For cleaning, slides were washed three times for 5 minutes in Tris-Cl (100 mM Tris, 600 mM NaCl, pH 7.4) and finally rinsed with ultrapure water. NaOH, Na2HPO4, NaCl, PBS and blocking reagent CA were obtained from AppliChem GmbH, 64291 Dortmund, Germany.
Neutravidin (Thermo Scientific, IL 61101 USA) that had been spotted had to be addressed by biotinylated oligonucleotides (Biomers.net GmbH, 89077 Ulm, Germany). Sequences of the oligonucleotides: Side A LcF5: 5'-CTT ATC GCT TTA TGA CCG GAC C-3' (5': Biotin); Side B RcF6: 5'-CAA TGA AAC ACT AGG CGA GGA C-3' (5': Biotin). Staining of the outer frame was done with biotinylated DY-547 dye (Dyomics GmbH, 67745 Jena, Germany). All these three components were diluted in carbonate buffer pH 9.0 to a final concentration of 1 mM. Incubation time for binding was 5 minutes and was stopped by washing with 1× PBS-buffer and ultrapure water. The left DNA strand M13-L part and right DNA M13-R strand were diluted 1:50 and 5 μl of each solution were transferred onto the chip directly to the prepared array. After incubation in the dark for 60 minutes in TE-buffer at 37°C and 85% rel. humidity, the glass chip was washed by completely dipping it into PBS-Tween and rinsing it a second time in PBS.
An atomic force microscope CP-II from Veeco (Santa Barbara CA, 93117 USA) and AFM-tips from NanoSensors (NanoAndMore GmbH, 35578 Wetzlar, Germany) were used: DT-CONTR (force constant: 0.2 N/m; resonance frequency: 13 kHz). Movement of the AFM-tip and execution were controlled by the diNanolithography Software V.1.8. Approaching the biotinylated glass slide was achieved in contact mode with 3.4 mN contact force. The tip remained in contact for 4 seconds and changed to the next spotting positions by retraction. Ink was supplied to the tip by a hypodermic needle of Popper & Sons, Inc. (N.Y. 11040 USA).
The DNA-construct was generated by digesting 10 μg M13mp18 RF I DNA plasmid (New England - BioLabs GmbH, 65926 Frankfurt a. M., Germany) simultaneously with the restriction enzymes PstI, Acc65I and BamHI (New England - BioLabs GmbH, 65926 Frankfurt a. M., Germany) in NEBuffer-3 at 37°C for 2 h. Then the enzymes were inactivated by heating the batch to 80°C for 20 minutes and finally cooling down slowly (1 K/min.). Parallel to this, hybridization of the adapter segments in Tris-Cl buffer (100 mM Tris-Cl; 600 mM NaCl; pH 7.4) took place by heating the oligonucleotides up to 90°C for 5 minutes (see figure 4) and cooling down slowly (1 K/min.). The digested M13mp18 plasmid (120 μl) was then divided into a left and right batch. The left was incubated with 8 μl M13-L5 (10 μM) and 8 μl M13-M2 (10 μM). The right was incubated with 8 μl M13-R6 (10 μM) and 8 μl M13-M1 (10 μM). Prehybridization took place for 30 minutes at 40°C, then 30 minutes at 30°C followed by cooling down to 20°C. Both batches were then ligated separately with T4 DNA ligase (New England - BioLabs GmbH, 65926 Frankfurt a. M., Germany) over night at 4°C. To avoid rupture of the sensitive construct, ligation was not stopped by heating but by removing the enzyme by cleaning it with Sure Clean (Bioline GmbH, 14943 Luckenwalde, Germany) and dissolving it in 100 μl TE-buffer (50 mM Tris-Cl, 100 mM NaCl). The concentration of both, the left and the right batch, were equalized by adding TE-buffer to a final concentration of about 30 ng/μl. The product was then stored at -20°C.
The PNA 3927 was synthesized by conventional solid phase Boc chemistry as previously described [24, 25], and purified by reversed phase HPLC. The PNA was subsequently characterized by HPLC and MALDI-TOF mass spectrometry (see additional file 2). Furthermore, the thermal stability (T m ) of complexes with an oligonucleotide (5'-GAG GGA AGG-3') binding to the triplex domain and an ologonucleotide (5'-CAT CCA CAG GGG TAA-3') was determined as 87°C and 77°C, respectively (see additional file 3), showing that both domains are functional in terms of hybridization to a DNA target.
Glass slides (Menzel Gläser, Menzel GmbH & Co. KG, 38116 Braunschweig, Germany) were blocked 1 h with 0.1% blocking reagent CA (AppliChem GmbH, 06466 Gatersleben, Germany) after they were silanized and biotinylated as described above. The reactive glass slides were incubated over night with 25 ng/ml Avidin at room temperature. Microarrays were spotted contactless with the microarray spotter TopSpot (BioFluidiX GmbH, 79110 Freiburg, Germany) on the functionalized and blocked glass slides. The solutions that have been spotted were: 2.7 μl left DNA construct, 2.7 μl right DNA construct, 2.7 μl PNA-adapter and either 2.7 μl LcF5-Btn 1 μm or 2.7 μl RcF6-Btn 1 μm. For a negative sample one component was omitted (PNA or adapter oligonucleotide). Incubation took place at 25°C at 85% rel. humidity for 1 h. For detecting the PNA and the DNA's orientation a Cy5 labelled oligonucleotide (Cy5-cF6; 1 μl) and a Cy3 labeled oligonucleotide (Cy3-cF5; 1 μl) were hybridized at 35°C at 85% rel. humidity for 1 h and were finally detected by a fluorescence microarray scanner (Axon Instruments, GenePix 4200A).
Fluorescence microscopy was carried out with an upright epifluorescence microscope Olympus A BX51 (objective: UPlanFL N; 40 × 0.75). Fluorescence detection was accomplished with the following filter-cube combinations: DY-547 detection: excitation filter (Ex) BP 545/25, dichromatic mirror (Dm) 565, emission filter (Em) LP 605/70 and for SYBR-Green I detection: Ex BP 460 - 495, Dm 505, Em LP 510 - 550. For illumination a mercury arc lamp (100 W, OSRAM GmbH, 81543 München, Germany) in combination with a Uniblitz VCM-D1 shutter was used. Image acquisition was carried out with a CCD camera (FView II) with 12 bit dynamic range and 1376 × 1032 pixel resolution. Software aquisition was donw with cellˆR version 3.1 (build 1276). Image editing was realized with ImageJ V1.42q. Staining of DNA was performed with SYBR-Green I (1:10000 in DMSO).
We thank A. Christmann for technical assistance with the atomic force microscope and M. Schellhase for performing tests and her expertise in general spotting technology. We gratefully acknowledge critical commentary and reviewing the manuscript by Dr. habil. Axel Warsinke. We like to thank the European Commission for the support of this work (contract no. STRP13775, project Nucan).
- Piner RD, Zhu J, Xu F, Hong S, Mirkin CA: Dip-Pen Nanolithography. Science. 1999, 283: 661-663. 10.1126/science.283.5402.661.View ArticleGoogle Scholar
- Ginger DS, Zhang H, Mirkin CA: Zur Entwicklung der Dip-Pen-Nanolithographie. Angew Chem. 2004, 116: 30-46. 10.1002/ange.200300608.View ArticleGoogle Scholar
- Basnar B, Willner I: Dip-Pen-Nanolithographic Patterning of Metallic, Semiconductor, and Metal Oxide Nanostructures on Surfaces. Small. 2009, 5 (1): 28-44. 10.1002/smll.200800583.View ArticleGoogle Scholar
- Mirkin CA, Hong S, Demers L: Dip-Pen Nanolithography: Controlling Surface Architecture on the Sub-100 Nanometer Length Scale. ChemPhysChem. 2001, 283: 37-39.View ArticleGoogle Scholar
- Salaita K, Wang Y, Mirkin CA: Applications of dip-pen nanolithography. Nature Nanotechnology. 2007, 2:Google Scholar
- Salazar RB, Shovsky A, Sch¸cnherr H, Vancso GJ: Dip-Pen Nanolithography on (Bio)Reactive Monolayer and Block-Copolymer Platforms: Deposition of Lines of Single Macromolecules. Small. 2006, 2 (11): 1274-1282. 10.1002/smll.200600235.View ArticleGoogle Scholar
- Maynor BW, Filocamo SF, Grinstaff MW, Liu J: Direct-Writing of Polymer Nanostructures: Poly(thiophene) Nanowires on Semiconducting and Insulating Surfaces. J Am Chem Soc. 2002, 124 (4): 522-523. 10.1021/ja017365j.View ArticleGoogle Scholar
- Jang JW, Sanedrin RG, Senesi AJ, Zheng Z, Chen X, Hwang S, Huang L, Mirkin CA: Generation of Metal Photomasks by Dip-Pen Nanolithography. Small. 2009, 1-4. 10Google Scholar
- Thibault C, Berre VL, Casimirius S, Trévisiol E, François J, Vieu C: Direct microcontact printing of oligonucleotides for biochip applications. Journal of Nanobiotechnology. 2005, 3 (7):Google Scholar
- Lin C, Liu Y, Rinker S, Yan H: DNA Tile Based Self-Assembly: Building Complex Nanoarchitectures. ChemPhysChem. 2006, 1641-1647.Google Scholar
- Sharma J, Chhabra R, Liu Y, Ke Y, Yan H: DNA-Templated Self-Assembly of Two-Dimensional and Periodical Gold Nanoparticle Arrays. Angew Chem Int Ed. 2006, 730-735. 45Google Scholar
- Liu W, Zhong H, Wang R, Seeman NC: Crystalline Two-Dimensional DNA-Origami Arrays. Angew Chem. 2011, 278-281. 123Google Scholar
- Rothemund PWK: Folding DNA to create nanoscale shapes and patterns. Nature. 2006, 440: 297-302. 10.1038/nature04586.View ArticleGoogle Scholar
- Guo P: The emerging field of RNA nanotechnology. Nature Nanotechnology. 2010, 5: 833-842. 10.1038/nnano.2010.231.View ArticleGoogle Scholar
- Karkare S, Bhatnagar D: Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino. Appl Microbiol Biotechnol. 2006, 575-586. 71Google Scholar
- Nielsen PE, Egholm M: An Introduction to Peptide Nucleic Acid. Current Issues Molec Biol. 1999, 1 (2): 89-104.Google Scholar
- Breitenstein M, Hölzel R, Bier FF: Immobilization of different biomolecules by atomic force microscopy. Journal of Nanobiotechnology. 2010, 8 (10):Google Scholar
- Kim JY, Jeon JH, Sung W: A breathing wormlike chain model on DNA denaturation and bubble: Effects of stacking interactions. The Journal of Chemical Physics. 2008, 128-Google Scholar
- Nielsen PE: Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology. Current Opinion in Biotechnology. 2001, 12: 16-20. 10.1016/S0958-1669(00)00170-1.View ArticleGoogle Scholar
- Knauert MP, Glazer PM: Triplex forming oligonucleotides: sequence-specific tools for gene targeting. Human Molecular Genetics. 2001, 10 (20): 2243-2251. 10.1093/hmg/10.20.2243.View ArticleGoogle Scholar
- Kuhn H, Demidov VV, Frank-Kamenetskii MD, Nielsen PE: Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA. Nucleic Acids Research. 1998, 26 (2): 582-587. 10.1093/nar/26.2.582.View ArticleGoogle Scholar
- Haaima G, Lohse A, Buchardt O, Nielsen PE: Peptide Nucleic Acids (PNA) containing thymine monomers derived from chiral amino acids: Hybridization and solubility properties of D-lysine PNA. Angewandte Chemie. 1996, 35: 1939-1941. 10.1002/anie.199619391.View ArticleGoogle Scholar
- Dwyer C, Lebeck AR, Pistol C: Energy Transfer Logic on DNA Nanostructures: Enabling Molecular-Scale Amorphous Computing. 4th Workshop on Non-Silicon Computing. 2007: NSC-4(ISCA)Google Scholar
- Christensen L, Fitzpatrick R, Gildea B, Petersen KH, Hansen HF, Koch T, Egholm M, Buchardt O, Nielsen PE: Solid-Phase Synthesis of Peptide Nucleic Acids. Journal of Peptide Science. 1995, 1: 175-183. 10.1002/psc.310010304.View ArticleGoogle Scholar
- Egholm M, Christensen L, Dueholm KL, Buchardt O, Coull J, Nielsen PE: Efficient PH-Independent Sequence-Specific DNA Binding by Pseudoisocytosine-Containing Bis-PNA. Nucleic Acids Research. 1995, 23: 217-222. 10.1093/nar/23.2.217.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.