Nucleic Acid Nanotechnology Research Articles

A New Approach to Model and Directly Control Co-Transcriptional RNA Folding

RNAs are synthesized exclusively in a 5' to 3' direction and it is well established that many (particularly long noncoding, but also riboswitch) RNAs begin to fold before the transcript is fully synthesized. Indeed, in many cases, directional folding is thought to limit the formation of nonproductive, nonnative structures that might arise from a more random condensation of secondary and tertiary structures or to facilitate formation of necessary (and transient on pathway) folding intermediates. Site-specifically programmed pauses in transcription are often necessary to direct these interactions.Using tools from synthetic biology and nucleic acid nanotechnology, we are developing an alternative approach to direct and control 5' to 3' sequential RNA folding. The approach allows the introduction of pauses into the sequential release and allows the experimenter to halt and then resume RNA release/folding. Initial studies use the malachite green binding RNA aptamer, with a simple fluorescence readout of formation of the final structure. Subsequent studies will footprint intermediates with a variety of probes to follow the progress of folding and detect intermediates.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Isothermal Self-Assembly of Complex DNA Structures under Diverse and Biocompatible Conditions

Nucleic acid nanotechnology has enabled researchers to construct a wide range of multidimensional structures in vitro. Until recently, most DNA-based structures were assembled by thermal annealing using high magnesium concentrations and nonphysiological environments. Here, we describe a DNA self-assembly system that can be tuned to form a complex target structure isothermally at any prescribed temperature or homogeneous condition within a wide range. We were able to achieve isothermal assembly between 15 and 69 °C in a predictable fashion by altering the strength of strand-strand interactions in several different ways, for example, domain length, GC content, and linker regions between domains. We also observed the assembly of certain structures under biocompatible conditions, that is, at physiological pH, temperature, and salinity in the presence of the molecular crowding agent polyethylene glycol (PEG) mimicking the cellular environment. This represents an important step toward the self-assembly of geometrically precise DNA or RNA structures in vivo.

BiotecVisions 2012, February

BiotecVisions 2012, February

Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology.

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA 'staples'. This revolutionary approach has led to the creation of a multitude of two-dimensional and three-dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy.

Abstracts7th Annual Meeting of the Oligonucleotide Therapeutics SocietyCopenhagen, DenmarkSeptember 8–10, 2011

The use of Antisense Oligonucleotides (ASOs) for re-directing splicing has shown promising results as a therapeutic approach for several diseases. A showcase for this technology comes from work done on the Survival of Motor Neuron 2 (SMN2) gene. SMN2 only generates a small amount of full-length mRNA due to a point mutation in exon 7 that causes it to be predominantly skipped. We have previously identified an 18mer 2′-O-methoxyethyl (MOE) modified ASO that promotes almost complete exon 7 inclusion by targeting ISS-N1, an hnRNP A1/A2-dependent intronic splicing silencer in intron 7. We have extended these findings by evaluating how various chemical modifications of the nucleotide sugar moiety of the 18mer ASO affect SMN2 exon 7 splicing. Unexpectedly, we found that incorporating fluorine at the 2′ position (2′-F) of the ribose sugar caused almost complete exon 7 skipping. This skipping activity is dependent on hybridization to the target pre-mRNA and is still robust when the ASO binding is shifted 3′. Furthermore, the 2′-F ASO causes skipping of exon 7 on chromatin associated RNA. We have explored the mechanisms by which the MOE ASO causes SMN2 exon 7 inclusion and the 2′-F ASO causes exon 7 skipping, by doing pull-down experiments with SMN2 RNA immobilized on magnetic beads. We found that the MOE ASO displaces hnRNPA1/A2 from ISS-N1. The 2′-F ASO is still able to displace hnRNPA1/A2, but the resulting 2′-F ASO-RNA duplex is recognized by Interleukin Enhancer Binding Factor 2 and 3 (ILF2 and ILF3). To determine if ILF2 and ILF3 are responsible for the skipping of exon 7, we reduced their expression with siRNAs. Indeed, reduction of ILF2 and ILF3 expression reversed the 2′-F ASO-mediated exon skipping. When administered to SMN2 transgenic mice, the 2′-F ASO caused SMN2 exon 7 skipping in a variety of tissues. These results demonstrate that ASOs with different chemical modifications can be used to promote exon inclusion or exon skipping by either displacing proteins from the pre-mRNA or by recruiting specific proteins to the heteroduplex.

The Resurgence of Acyclic Nucleic Acids

This review examines acyclic nucleoside analogs as therapeutic agents, potential progenitor candidates to RNA, and novel building blocks for nucleic-acid nanotechnology. Together, these areas of research provide new insights into the structural and functional properties of nucleic acids and suggest new paradigms for nucleic acid self-assembly.

Nucleic acid nanotechnology-towards Angstrom-scale engineering.

Nucleic acids and analogues are suitable building blocks for reliable self-assembly of nanometer-sized two- or three-dimensional materials. In order to mimic or approach nature with respect to size and function, Angstrom-scale chemical engineering is emerging as pivotal for future developments. Efforts within nucleic acid nanotechnology will be focussed on generating rigid and stable low nanometer-sized structures carrying functionalities with predictable spatial positioning allowing, by encoded self-assembly, functional nucleic acid architectures to be built towards applications within the biological and material sciences.

Intercourse after Syphilis

Nucleic acid interactions under crowded environments are of great importance for biological processes and nanotechnology. However, the kinetics and thermodynamics of nucleic acid interactions in a crowded environment remain poorly understood. We use a coarse-grained model of DNA to study the kinetics and thermodynamics of DNA duplex and hairpin formation in crowded environments. We find that crowders can increase the melting temperature of both an 8-mer DNA duplex and a hairpin with a stem of 6-nt depending on the excluded volume fraction of crowders in solution and the crowder size. The crowding induced stability originates from the entropic effect caused by the crowding particles in the system. Additionally, we study the hybridization kinetics of DNA duplex formation and the formation of hairpin stems, finding that the reaction rate <i>k</i>_on is increased by the crowding effect, while <i>k</i>_off is changed only moderately. The increase in <i>k</i>_on mostly comes from increasing the probability of reaching a transition state with one base pair formed. A DNA strand displacement reaction in a crowded environment is also studied with the model and we find that rate of toehold association is increased, with possible applications to speeding up strand displacement cascades in nucleic acid nanotechnology.