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We have developed modular assays based on aptamer displacement or protein-dependent reporter ribozymes for the screening of small molecule inhibitors. Thereby, chemical space can be explored in a rapid, focused, and modular manner, by indirectly taking advantage of the highest molecular diversity currently amenable to screening, namely that of 10 16 different nucleic acid sequences. In this account, we discuss the application of these approaches to find new inhibitors for target proteins. Examples showing that these modulators can be used as tools for gaining novel biological insight are provided. We also discuss some recent data on interlocked DNA architectures such as entirely double-stranded DNA rotaxanes.

Mechanically interlocked molecules such as rotaxanes and catenanes have potential as components of molecular machinery. Rotaxanes consist of a dumb-bell-shaped molecule encircled by a macrocycle that can move unhindered along the axle, trapped by bulky stoppers. Previously, rotaxanes have been made from a variety of molecules, but not from DNA. Here, we report the design, assembly and characterization of rotaxanes in which both the dumb-bell-shaped molecule and the macrocycle are made of double-stranded DNA, and in which the axle of the dumb-bell is threaded through the macrocycle by base pairing. The assembly involves the formation of pseudorotaxanes, in which the macrocycle and the axle are locked together by hybridization. Ligation of stopper modules to the axle leads to the characteristic dumb-bell topology. When an oligonucleotide is added to release the macrocycle from the axle, the pseudorotaxanes are either converted to mechanically stable rotaxanes, or they disassemble by means of a slippage mechanism to yield a dumb-bell and a free macrocycle. Our DNA rotaxanes allow the fields of mechanically interlocked molecules and DNA nanotechnology to be combined, thus opening new possibilities for research into molecular machines and synthetic biology.

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Phenanthrene-1,2-dimethanol was incorporated into oligodeoxynucleotides via formation of phosphodiester bonds (cf. Scheme 1). If placed at internal positions in a DNA duplex, a strong reduction of duplex stability is observed (Table 1). Terminal attachment of stretches of phenanthrene residues, however, leads to a substantial increase in stability. The stabilization is attributed to a cooperative interaction of the phenanthrene residues of the two strands rather than to dangling end effects. Chimeric oligomers containing a stretch of six phenanthrene residues show two separate transitions (Table 2): one arising from the denaturation of the DNA stem (observable by a hyperchromic effect at 260 nm) and a second one from the denaturation of the phenanthrene part (observable by temperature-dependant gel mobility assays). Based on these findings, a model of the chimeric hybrids is proposed, in which the phenanthrene residues stack in a zipper-like manner on top of the DNA base pairs without disrupting the B-form of the DNA stem (see Fig. 7).

The synthesis of a trifluoromethylphenyl diazirine photoaffinity probe of the cytohesin inhibitor SecinH3 is described. The probe exhibits improved labelling efficiency over a benzophenone-based probe and thus is more suitable for photoaffinity labelling in complex biological samples.

Two sets of RNA phosphoramidites, carrying the (fluoride-labile) 2'-O-[(triisopropylsilyl)oxy]methyl (tom) group and the (photolabile) [(R)-1-(2-nitrophenyl)ethoxy]methyl ((R)-npeom) group, were prepd. The two protecting groups were completely orthogonal to each other. Three ribozyme-substrate constructs, protected each by a (R)-npeom group, were synthesized; on photolysis, efficient cleavage of this remaining protecting group occurred. It could be demonstrated that the presence of one (R)-npeom group within a RNA strand has only a minor influence on the pairing properties of corresponding duplexes. [on SciFinder (R)]

The structural reorganization of nanoscale DNA architectures is a fundamental aspect in dynamic DNA nanotechnology. Commonly, DNA nanoarchitectures are reorganized by means of toehold-expanded DNA sequences in a strand exchange process. Here we describe an unprecedented, toehold-free switching process that relies on pseudo-complementary peptide nucleic acid (pcPNA) by using a mechanism that involves double-strand invasion. The usefulness of this approach is demonstrated by application of these peptide nucleic acids (PNAs) as switches in a DNA rotaxane architecture. The monomers required for generating the pcPNA were obtained by an improved synthesis strategy and were incorporated into a PNA actuator sequence as well as into a short DNA strand that subsequently was integrated into the rotaxane architecture. Alternate addition of a DNA and PNA actuator sequence allowed the multiple reversible switching between a mobile rotaxane macrocycle and a stationary pseudorotaxane state. The switching occurs in an isothermal process at room temperature and is nearly quantitative in each switching step. pcPNAs can potentially be combined with light- and toehold-based switches, thus broadening the toolbox of orthogonal switching approaches for DNA architectures that open up new avenues in dynamic DNA nanotechnology.

A recent trend in DNA nanotechnology consists of the assembly of architectures with dynamic properties that can be regulated by employing external stimuli. Reversible processes are important for implementing molecular motion into DNA architectures as they allow for the regeneration of the original state. Here we describe two different approaches for the reversible switching of a double-stranded DNA rotaxane architecture from a stationary pseudorotaxane mode into a state with movable components. Both states only marginally differ in their respective topologies but their mechanical properties are fundamentally different. In the two approaches, the switching operation is based on strand-displacement reactions. One of them employs toehold-extended oligodeoxynucleotides whereas in the other one the switching is achieved by light-irradiation. In both cases, multiple back and forth switching between the stationary and the mobile states was achieved in nearly quantitative fashion. The ability to reversibly operate mechanical motion in an interlocked DNA nanostructure opens exciting new avenues in DNA nanotechnology.

The synthesis of a trifluoromethylphenyl diazirine photoaffinity probe of the cytohesin inhibitor SecinH3 is described. The probe exhibits improved labelling efficiency over a benzophenone-based probe and thus is more suitable for photoaffinity labelling in complex biological samples.

Kissing complexes formed on the basis of highly specific noncanonical interactions of RNA hairpins can be harnessed for the controlled assembly of DNA nanoobjects. Two DNA minicircles, each equipped with a different RNA hairpin motif (see picture), mediate a tight and specific binding of the two circular DNA nanoobjects. These interactions may offer the possibility to construct DNA nanoobjects with increased complexity.

Our group is working on the development of reliable access to biologically relevant, long, and modified RNA sequences. Here, we briefly present the chemical synthesis of such sequences with 2?-O-triisopropylsilyloxymethyl (= 2?-O-tom) protected ribonucleoside phosphoramidites, their template-directed enzymatic ligation, and some examples of modified nucleotides, designed to promote structural and biological studies.

The assembly of double-stranded (ds) DNA nanocircles both by hybridization with branched oligodeoxynucleotides (ODNs) and by intercalation was analyzed by atomic force microscopy (AFM). Branched ODNs ligated to single-stranded (ss) gap regions of dsDNA nanocircles led to defined, dumbbell-shaped architectures. ODNs containing an aromatic intercalator yielded oligomeric aggregates.

The prepn. and the pairing properties of the new 3'-deoxyribopyranose (4'->2')-oligonucleotide (= p-DNA) pairing system, based on 3'-deoxy-b-D-ribo-pyranose nucleosides is presented. D-Xylose was efficiently converted to the prefunctionalized 3-deoxyribopyranose deriv. 4-O-[(tert-butyl)dimethylsilyl]-3-deoxy-D-ribo-pyranose 1,2-diacetate (obtained as a 4:1 mixt. of a- and b-D-anomers). From this sugar building block, the corresponding, appropriately protected thymine, guanine, 5-methylcytosine, and purine-2,6-diamine nucleoside phosphoramidites were prepd. in a minimal no. of steps. These building blocks were assembled on a DNA synthesizer, and the corresponding p-DNA oligonucleotides were obtained in good yields after a one-step deprotection under std. conditions, followed by HPLC purifn. Qual., p-DNA shows the same pairing behavior as p-RNA, forming antiparallel, exclusively Watson-Crick-paired duplexes that are much stronger than corresponding DNA duplexes. Duplex stabilities within the three related (i.e., based on ribo-pyranose nucleosides) oligonucleotide systems p-RNA, p-DNA, and 3'-O-Me-p-RNA were compared with each other. Intrinsically, p-RNA forms the strongest duplexes, followed by p-DNA, and 3'-O-Me-p-RNA. However, by introducing the nucleobases purine-2,6-diamine and 5-methylcytosine instead of adenine and cytosine, a substantial increase in stability of corresponding p-DNA duplexes was obsd. [on SciFinder (R)]

By automated synthesis, we prepd. hybrid oligonucleotides consisting of covalently linked RNA and p-DNA sequences (p-DNA = 3'-deoxyribopyranose (4'->2')-oligonucleotides). The pairing properties of corresponding hybrid duplexes formed from fully complementary single strands were investigated. An uninterrupted p-p-stacking at the p-DNA/RNA interface and cooperative pairing between the two systems was achieved by connecting them via a 4'-p-DNA-2'->5'-RNA-3' and 5'-RNA-2'->4'-p-DNA-2' phosphodiester linkage, resp. The RNA 2'-phosphoramidites required for the formation of the RNA-2'->4'-p-DNA phosphodiester linkage were synthesized from the corresponding, 3'-O-tom-protected ribonucleosides (tom = [(triisopropylsilyl)oxy]methyl). Analogs of the FMN binding aptamer and the hammerhead ribozyme were prepd. Each of these analogs consisted of two p-DNA/RNA hybrid single strands with complementary p-DNA sequences, designed to substitute stem/loop and stem motifs within the parent compds. By comparative binding and cleavage studies, it was found that mixing of the two complementary p-DNA/RNA hybrid sequences resulted in the formation of the fully functional analogs of the FMN-binding aptamer and of the hammerhead ribozyme, resp. [on SciFinder (R)]

The determination of the solution structure of small non-natural oligopeptides and oligonucleotides by NMR, which is one of the main research topics of our group, is illustrated on the example of an 8-mer p-DNA duplex. p-DNA, the 3'-desoxy analogue of p-RNA, forms an highly selective pairing system but its pairing strength is less than that of analogous p-RNA sequences. The NMR study reveals that the backbone of p-DNA corresponds more closely to the conformation predicted for pentapyranose nucleic acids by qualitative conformational analysis than p-RNA.