Molecular self-assembly is the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates joined by noncovalent bonds. Molecular self-assembly is ubiquitous in biological systems and underlies the formation of a wide variety of complex biological structures. Understanding self-assembly and the associated noncovalent interactions that connect complementary interacting molecular surfaces in biological aggregates is a central concern in structural biochemistry. Self-assembly is also emerging as a new strategy in chemical synthesis, with the potential of generating nonbiological structures with dimensions of 1 to 10(2) nanometers (with molecular weights of 10(4) to 10(10) daltons). Structures in the upper part of this range of sizes are presently inaccessible through chemical synthesis, and the ability to prepare them would open a route to structures comparable in size (and perhaps complementary in function) to those that can be prepared by microlithography and other techniques of microfabrication.

Molecular self-assembly and nanochemistry: A chemical strategy for the synthesis of nanostructures

Nucleic acid aptamers, often termed 'chemical antibodies', are functionally comparable to traditional antibodies, but offer several advantages, including their relatively small physical size, flexible structure, quick chemical production, versatile chemical modification, high stability and lack of immunogenicity. In addition, many aptamers are internalized upon binding to cellular receptors, making them useful targeted delivery agents for small interfering RNAs (siRNAs), microRNAs and conventional drugs. However, several crucial factors have delayed the clinical translation of therapeutic aptamers, such as their inherent physicochemical characteristics and lack of safety data. This Review discusses these challenges, highlighting recent clinical developments and technological advances that have revived the impetus for this promising class of therapeutics.

Aptamers as targeted therapeutics: current potential and challenges

Aptamers are single-stranded nucleic acid molecules that possess properties comparable to those of protein monoclonal antibodies, and thus are clear alternatives to long established antibody-based diagnostic or biotechnological products for research, diagnostics, and therapy. 1-4 This class of functional nucleic acids can fold into complex three-dimensional shapes, 5-7 forming binding pockets and clefts for the specific recognition and tight binding of any given molecular target, 8-10 from metal ions and small chemicals to large proteins and higher order protein complexes, whole cells, viruses, or parasites. Aptamers can be isolated in Vitro from vast combinatorial libraries that comprise trillions of different sequences, by a process called “ in Vitro selection”, or “SELEX”, an acronym for “systematic evolution of ligands by exponential enrichment”. An in Vitro selection experiment comprises a number of sequential steps, the first of which is the generation of a nucleic acid library of random sequences. This starting pool of mainly nonfunctional RNA or DNA sequences is generated using a standard DNA-oligonucleotide synthesizer. 11 The design of such libraries involves the synthesis of a short defined sequence, followed by a random region of variable length and another defined sequence at the 5 ′-end. This pool of synthetic single-stranded DNA (ssDNA) is amplified in the polymerase chain reaction (PCR), generating several copies of each DNA in its double-stranded form. By in Vitro transcription, a corresponding library of RNA molecules can be generated which can then be used for the in Vitro selection. If the transcription reaction contains nucleoside triphosphate derivatives that are chemically modified but still are substrates for RNA polymerases, libraries of modified RNAs can be generated in which sequences are equipped with a broad variety of additional chemical functionalities, normally not present in natural nucleic acids. 12-14 Aptamers selected from chemically modified libraries can, in some cases, be completely resistant toward degradation by nucleases. The additional functional groups can lead to ligands with novel * To whom correspondence should be addressed: telephone, +49-228735661; fax,+49-228-735388; e-mail, m.famulok@uni-bonn.de. † LIMES Institute. ‡ University of Konstanz. Volume 107, Number 9

Functional Aptamers and Aptazymes in Biotechnology, Diagnostics, and Therapy

Recent developments show encouraging results for the use of DNA as a construction material for nanometer-sized objects. Today, however, DNA-based molecular nanoarchitectures are constructed with mainly unmodified or at best end-modified oligonucleotides, thus shifting the development of functionalized DNA structures into the limelight. One of most recent developments in this direction is the substitution of the canonical Watson-Crick base pairs by metal complexes. In this way "metal-base pairs" are created, which could potentially impart magnetic or conductive properties to DNA-based nanostructures. This review summarizes research which started almost 45 years ago with the investigation of how metal ions interact with unmodified DNA and which recently culminated in the development of artificial ligand-like nucleobases so far able to coordinate up to ten metal ions inside a single DNA duplex in a programmable fashion.

DNA--metal base pairs.

Aptamers are small single-stranded nucleic acids that fold into a well-defined three-dimensional structure. They show a high affinity and specificity for their target molecules and inhibit their biological functions. Aptamers belong to the nucleic acids family and can be synthesized by chemical or enzymatic procedures, or a combination of the two. They can, therefore, be considered as both chemical and biological substances. This Review summarizes the most convenient approaches to their preparation and new developments in the field of aptamers. The application of aptamers in chemical biology is also discussed.

The chemical biology of aptamers.

To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on “demanding” templates containing consecuti...

A Versatile Toolbox for Variable DNA Functionalization at High Density

The programmable self-association of molecular units into higher ordered structures plays a key role in the bottom-up construction of nanomaterials. Crucial for the successful supramolecular assembly of nanoobjects, however, is the choice of the functional molecular units themselves. Nucleic acids have emerged as a convenient target for these purposes because of their unique properties in molecular recognition and the ease by which oligonucleotides can be accessed synthetically. The assembly of DNA nanoobjects with defined twoor three-dimensional geometries requires either rigid subunits, such as in double crossover (DX) or paranemic crossover (PX) elements, or inherently rigid triangular shapes such as tetrahedra or bipyramids. From a structural point of view, DNA minicircles are probably the simplest rigid objects with a nanometer size. Small DNA circles were first prepared by designing two 21mer DNA precursor sequences which, upon hybridization and ligation, resulted in a statistical distribution of DNA minicircles containing 105, 126, 147, and 168 base pairs (bp). Atomic force microscopy (AFM) analysis of 168-bp minicircles confirmed their smooth circular structure without any ring deformation or supercoiling. These features predestine them as building blocks for the assembly of objects on the nanometer scale. However, a major drawback of DNA minicircles in the construction of higher ordered DNA architectures is their unbranched, continuously doublestranded (ds) nature that prevents the guided aggregation of multiple rings. Furthermore, the statistical assembly of the short oligonucleotides that were applied in the known strategies for the synthesis of DNA minicircles prevents the controlled introduction of “customized” sequences into the circle that can serve as defined handles for the selfassembly of multiple rings. We recently reported the assembly of two DNA minicircles, guided by a “strut” of Dervan-type polyamides that specifically held two rings together by binding to different 9mer double-stranded “custom” sequences that were present in each 168-mer ring exactly once. Here we report on the construction of minicircles that contain a customized singlestranded gap sequence at a defined position. The gap can serve as a versatile site for the modification of DNA minicircles, thereby allowing, for example, their guided modification with functional groups through hybridization with synthetic oligonucleotides. The key step in the preparation of DNA minicircles with sequence-specific functionalization focuses on a preformed incomplete minicircle MCgap containing a 21-nucleotide single-stranded (ss) gap region (Figure 1). Hybridizing and

DNA Minicircles with Gaps for Versatile Functionalization

Owing to its well-established synthetic access, its well-known structural properties, and its size, DNA is a very interesting material for building nanoarchitectures in a bottom-up approach. This has already been proven in a number of studies. For example, cubes, octahedra, Borromean rings, as well as tubes (nanowires) have been built with DNA. However, DNA can not only be used as a structural material on a nanometer scale but also as a functional material, for example, to measure temperature force. However, as DNA is a linear polymer, it is difficult to build stable, branched three-dimensional architectures. Branched DNA duplexes offer a possible solution, but complex “tiles” have to be used for stable structures. Other approaches, for example, rely on the use of synthetic DNA derivatives to build trisoligonucleotidyl junctions. Polyamides derived from nonproteinogenic amino acids containing N-methlypyrrole, N-methylimidazole, or N-methylhydroxypyrrole groups can bind in a hairpin motif to the minor groove of double-stranded (ds) DNA. The base sequence to which they can bind selectively and with high affinity can be programmed by the sequence of the respective residues in the two antiparallel strands of the hairpin polyamide. A complete set of pairing rules is available for the straight-forward recognition of the majority of possible DNA sequences. DNA-binding polyamides of this type can be used for a variety of applications, like, for example, gene regulation or as sequence-selective nuclear stains. As most of the names for this class of compound are either lengthy or imprecise, we propose the term “Dervan-type polyamide” in honor of P. B. Dervan who made the major contributions in this field. Herein, we report on the use of Dervan-type polyamides as a possible second orthogonal structural element—besides Watson–Crick base pairing—for DNA-based architectures. Our rationale is that by connecting two hairpin polyamides with a long and flexible linker, we generate “DNA struts” that target their matching binding sites on different DNA duplex strands and can thus function as “sequence-selective glue”, which will make it easier to build up complex stable architectures. Only once before has a Dervan polyamide been shown to bind across twoDNAduplexes, but in this case, the two duplexes were preorganized next to each other in the nucleosome core particle. To test whether Dervan polyamides alone can be used to hold DNA objects together, we synthesized the heterobifunctional strut S-AB shown in Figure 1. It consists of the two

Polyamide Struts for DNA Architectures

https://www.limes-institut-bonn.de/fileadmin/user_upload/Group-Famulok/pdf/Publikatonen/Ackermann2010b.pdf

Goran Rasched

Papers

A Versatile Toolbox for Variable DNA Functionalization at High Density

DNA Minicircles with Gaps for Versatile Functionalization

Polyamide Struts for DNA Architectures

Assembly of dsDNA nanocircles into dimeric and oligomeric aggregates

DNA‐Ringe mit Einzelstrangdomänen zur vielseitigen Funktionalisierung