Enzyme-Directed Positioning of Nanoparticles on Large DNA Templates (original) (raw)

Control of Steric Hindrance on Restriction Enzyme Reactions with Surface-Bound DNA Nanostructures

Nano Letters, 2008

To understand better enzyme/DNA interactions and to design innovative detectors based on DNA nanoarrays, we need to study the effect of nanometric confinement on the biochemical activity of the DNA molecules. We focus on the study of the restriction enzyme reactions (Dpnll) within DNA nanostructures on flat gold films by atomic force microscopy (AFM). Typically we work with a few patches of DNA self assembled monolayers (SAMs) that are hundred nm in size and are lithographically fabricated within alkylthiol SAMs by AFM nanografting. We start by nanografting a few patches of a single-stranded DNA (ssDNA) molecule of 44 base pairs (bps) with a 4 bps recognition sequence (specific for Dpnll) in the middle. Afterwards, reaction-ready DNA nanopatches are obtained by hybridization with a complementary 44bps ssDNA sequence. The enzymatic reactions were carried out over nanopatches with different density. By carrying out AFM height measurements, we are able to show that the capability of the Dpnll enzyme to reach and react at the recognition site is easily varied by controlling the DNA packing in the nanostructures. We have found strong evidence that inside our ordered DNA nanostructures the enzyme (that works as a dimer) can operate down to the limit in which the space between adjacent DNA molecules is equal to the size of the DNA/enzyme complex. Similar experiments were carried out with a DNA sequence without the recognition site, clearly finding that in that case the enzymatic reaction did not lead to digestion of the molecules. These findings suggest that it is possible to tune the efficiency of an enzymatic reaction on a surface by controlling the steric hindrance inside the DNA nanopatches without vary any further physical or chemical variable. These findings are opening the door to novel applications in both the fields of biosensing and fundamental biophysics.

Synthesis and AFM visualization of DNA nanostructures

Thin Solid Films, 2004

We propose a novel bottom-up approach for the fabrication of various desired nanostructures, based on self-assembly of oligonucleotides governed by Watson-Crick base pairing. Using this approach, we designed Y-shaped, closed Y-shaped, H-shaped, and hexagonal structures with oligonucleotides. These structures were autonomously fabricated simply by mixing equimolar solutions of oligonucleotides and performing hybridization. After synthesis of the nanostructures, we confirmed their validity by agarose gel electrophoresis and atomic force microscope (AFM) visualization. We detected bands of the desired molecular sizes in the gel electrophoresis and observed the desired structures by AFM analysis. We concluded that the synthesized structures were consistent with our intended design and that AFM visualization is a very useful tool for the observation of nanostructures.

ChemInform Abstract: DNA Nanoarchitectures: Steps Towards Biological Applications

ChemInform, 2014

DNA's remarkable molecular recognition properties, flexibility and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During the past decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way, both generating nanostructures in one, two and three dimensions: the tile-based assembly process, which provides a useful tool to construct large and simple structures, and the DNA origami method, suitable for the production of smaller, more sophisticated and well defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures for molecular and cellular biology.

Controlled Confinement of DNA at the Nanoscale: Nanofabrication and Surface Bio-Functionalization

Methods in Molecular Biology, 2011

Nanopatterned arrays of biomolecules are a powerful tool to address fundamental issues in many areas of biology. DNA nanoarrays, in particular, are of interest in the study of DNA-protein interactions and for biodiagnostic investigations. In this context, achieving a highly specific nanoscale assembly of oligonucleotides at surfaces is critical. In this chapter, we describe a method to control the immobilization of DNA on nanopatterned surfaces; the nanofabrication and the bio-functionalization involved in the process will be discussed.

Selective DNA-Mediated Assembly of Gold Nanoparticles on Electroded Substrates

Langmuir, 2008

Motivated by the technological possibilities of electronics and sensors based on gold nanoparticles (Au NPs), we investigate the selective assembly of such NPs on electrodes via DNA hybridization. Protocols are demonstrated for maximizing selectivity and coverage using 15mers as the active binding agents. Detailed studies of the dependences on time, ionic strength, and temperature are used to understand the underlying mechanisms and their limits. Under optimized conditions, coverage of Au NPs on Au electrodes patterned on silicon dioxide (SiO 2 ) substrates was found to be ∼25-35%. In all cases, Au NPs functionalized with non-complementary DNA show no attachment and essentially no nonspecific adsorption is observed by any Au NPs on the SiO 2 surfaces of the patterned substrates. DNA-guided assembly of multilayers of NPs was also demonstrated and, as expected, found to further increase the coverage, with three deposition cycles resulting in a surface coverage of approximately 60%.

Multimodal Characterization of a Linear DNA-Based Nanostructure

ACS Nano, 2012

9 10 D eveloping methods for patterning 11 discrete particles and molecules at 12 the nanoscale has become an im-13 portant avenue for nanotechnological re-14 search as the limitations and inefficiencies 15 of conventional top-down patterning be-16 come ever more apparent. For such work, 17 investigators are increasingly turning to 18 self-assembly methodologies. While there 19 are many systems that allow for self-assem-20 bly of simple molecular structures, DNA-21 based technologies provide inherent ad-22 vantages due to the precise nature of 23 WatsonÀCrick base pairing and the molec-24 ular-level control one has over base se-25 quence. In the simplest examples, linear 26 DNA constructs have been assembled with 27 a wide variety of particle and molecular 28 attachments including fluorescent dyes, 29 semiconductor quantum dots (QDs), gold 30 nanoparticles (AuNPs), and proteins, clearly 31 demonstrating the potential of DNA functio-32 nalization chemistry for nanoscale control. 1À4 33 Greatly expanding the potential application 34 space, Seeman pioneered methods that use 35 DNA as a self-assembled structural material. 5 36 As he elegantly demonstrated, the use of 37 crossovers, tiles, and junctions permits a wide 38 range of structures to be realized. 6À10 Rothe-39 mund extended this methodology further 40 with DNA origami, 11 an approach for creating 41 arbitrary two-dimensional DNA structures 42 using a long scaffold strand and many smaller 43 staple strands. 12À16 In all implementations, 44 since each base or set of bases within the 45 DNA structure is uniquely addressable, there 46 is potential for a self-assembly approach that 47 allows for arbitrary particle or molecular pla-48 cement with a resolution approaching the 49 base-to-base separation distance of ∼3 Å. 50 In order to reach a full understanding of 51 the accuracy with which such DNA struc-52 tures form and the consequent precision 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 that can be realized in DNA-based pattern-77 ing, it is necessary to examine a variety of 78 assembled structures in intimate detail 79 using a diverse set of techniques. To date, 80 such a study has not been performed, nor is 81 it clear what exactly would constitute a "full" 82 characterization. In molecular biology, X-ray 83 crystallography is the gold standard in di-84 ABSTRACT Designer DNA structures have garnered much interest as a way of assembling novel nanoscale architectures with exquisite control over the positioning of discrete molecules or nanoparticles.