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Interlocked molecular architectures are well known in supramolecular chemistry and are widely used for various applications like sensors, molecular machines and logic gates. The use of DNA for constructing these interlocked structures has increased significantly within the current decade. Because of Watson-Crick base pairing rules, DNA is an excellent material for the self-assembly of well-defined interlocked nanoarchitectures. These DNA nanostructures exhibit sufficient stability, good solubility in aqueous media, biocompatibility, and can be easily combined with other biomolecules in bio-hybrid nano-assemblies. Therefore, the study of novel DNA-based interlocked systems is of interest for nanotechnology, synthetic biology, supramolecular chemistry, biotechnology, and for sensing purposes. Here we summarize recent developments and applications of interlocked supramolecular architectures made of DNA. Examples illustrating that these systems can be precisely controlled by switching on and off the molecular motion of its mechanically trapped components are discussed. Introducing different triggers into such systems creates molecular assemblies capable of performing logic gate operations and/or catalytic activity control. Interlocked DNA-based nanostructures thus represent promising frameworks for building increasingly complex and dynamic nanomachines with highly controllable functionality., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nucleic acid aptamers, or simply aptamers, are oligonucleotides that bind specific ligands that vary from small molecules to proteins. An aptamer for a specific ligand is routinely identified through the process of systematic evolution of ligands by exponential enrichment, although some aptamers are found in nature as ligand-binding sites of special RNA structures called riboswitches. Aptamers have significant value in biotechnology and for the development of aptamer-based therapeutics. This perspective briefly highlights the tight connection between the journal Chemistry & Biology and in vitro selection technologies over the past two decades. We then focus our discussion on the summary of the current state of the art of aptamer technologies and provide our view of the future challenges and opportunities for the field., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lymphatic filariasis and onchocerciasis are severe diseases caused by filarial worms and affect more than 150 million people worldwide. Endosymbiotic α-proteobacteria Wolbachia are essential for these parasites throughout their life cycle. Using a high-throughput chemical screen, we identified a benzimidazole compound, wALADin1, that selectively targets the δ-aminolevulinic acid dehydratase (ALAD) of Wolbachia (wALAD) and exhibits macrofilaricidal effects on Wolbachia-containing filarial worms in vitro. wALADin1 is a mixed competitive/noncompetitive inhibitor that interferes with the Mg(2+)-induced activation of wALAD. This mechanism inherently excludes activity against the Zn(2+)-dependent human ortholog and might be translatable to Mg(2+)-responsive orthologs of other bacterial or protozoan pathogens. The specificity profile of wALADin1 derivatives reveals chemical features responsible for inhibitory potency and species selectivity. Our findings validate wALADins as a basis for developing potent leads that meet current requirements for antifilarial drugs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report on the direct electrochemical detection of aptamer-protein interactions, namely between a DNA aptamer and lysozyme (LYS) based on electrochemical impedance spectroscopy (EIS) technique. First, the affinity of the aptamer to LYS and control proteins was presented by using filter retention assay. An amino-modified version of the DNA aptamer-recognizing lysozyme was covalently immobilized on the surface of multiwalled carbon nanotube-modified screen-printed electrodes (MWCNT-SPEs), which were employed for measurements and have improved properties compared with bare SPEs. This carbon nanotube setup enabled the reliable monitoring of the interaction of lysozyme with its cognate aptamer by EIS transduction of the resistance to charge transfer (R(ct)) in the presence of 2.5 mM [Fe(CN)₆]³⁻/⁴⁻. This assay system provides a means for the label-free, concentration-dependent, and selective detection of lysozyme with an observed detection limit of 12.09 μg/ml (equal to 862 nM)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aptamers targeting reverse transcriptase (RT) from HIV-1 inhibit viral replication in vitro, presumably by competing with binding of the primer/template complex. This site is not targeted by the currently available small-molecule anti-HIV-1 RT inhibitors. We have identified SY-3E4, a small-molecule inhibitor of HIV-1 RT, by applying a screening assay that utilizes a reporter-ribozyme regulated by the anti-HIV-1 RT aptamer. SY-3E4 displaces the aptamer from the protein, selectively inhibits DNA-dependent, but not RNA-dependent, polymerase activity, and inhibits the replication of both the wild-type virus and a multidrug-resistant strain. Analysis of available structural data of HIV-1 and HIV-2 RTs rationalizes many of the observed characteristics of the inhibitory profiles of SY-3E4 and the aptamer and suggests a previously not considered region in these RTs as a target for antiviral therapy. Our study reveals unexplored ways for rapidly identifying alternative small-molecule target sites in proteins and illustrates strategies for overcoming resistance-conferring mutations with small molecules.

Directed in vitro evolution can create RNA catalysts for a variety of organic reactions, supporting the "RNA world" hypothesis, which proposes that metabolic transformations in early life were catalyzed by RNA molecules rather than proteins. Among the most fundamental carbon-carbon bond-forming reactions in nature is the aldol reaction, mainly catalyzed by aldolases that utilize either an enamine mechanism (class I) or a Zn(2+) cofactor (class II). We report on isolation of a Zn(2+)-dependent ribozyme that catalyzes an aldol reaction at its own modified 5' end with a 4300-fold rate enhancement over the uncatalyzed background reaction. The ribozyme can also act as an intermolecular catalyst that transfers a biotinylated benzaldehyde derivative to the aldol donor substrate, coupled to an external hexameric RNA oligonucleotide, supporting the existence of RNA-originated biosynthetic pathways for metabolic sugar precursors and other biomolecules.

Here we report the in vitro selection of novel small peptide motifs that bind to human alpha-thrombin. We have applied mRNA display to select for thrombin binding peptides from an unbiased library of 1.2 x 10(11) different 35-mer peptides, each containing a random sequence of 15 amino acids. Two clones showed binding affinities ranging from 166 to 520 nM. A conserved motif of four amino acids, DPGR, was identified. Clot formation of human plasma is inhibited by the selected clones, and they downregulate the thrombin-mediated activation of protein C. The identified peptide motifs do not share primary sequence similarities to any of the known natural thrombin binding motifs. As new inhibitors for human thrombin open interesting possibilities in thrombosis research, our newly identified peptides may provide further insights into this field of investigation and may be possible candidates for the development of new anti-thrombotic agents.

In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. We describe an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to pefloxacin and other quinolone derivatives. Within 16 selection cycles, highly sensitive clones responding to drug levels in the sub-micromolar range were obtained. The morpholine moiety of the quinolone derivatives was required for inhibition of the self-cleavage of the selected ribozymes: modifications of the aromatic system were tolerated better than modifications of the morpholine ring. We also present a theoretical model that analyzes the predicted fraction of ribozymes with a given binding constant and cleavage rate recovered after each selection cycle. This model precisely predicts the actual experimental values obtained with the selection procedure. It can thus be used to determine the optimal conditions for an in vitro selection of an allosteric ribozyme with a desired dissociation constant and cleavage rate for a given application., (Copyright 2001 Academic Press.)

Aptamers are valuable tools for studying numerous aspects of biological processes, opening up new experimental opportunities to analyse the function of a wide range of cellular molecules. Functional RNA molecules can be rapidly selected in vitro from complex combinatorial mixtures of different sequences. Recently, it was shown that in vitro selection processes can be automated: the first generation selection robots will soon mean aptamers for several targets can be isolated in parallel within days rather than weeks. Aptamers not only exhibit highly specific molecular recognition properties but are also able to modulate the function of their cognate targets in a highly specific manner by agonistic or antagonistic mechanisms. These properties prompted the development of novel technologies to exploit the use of aptamers to modulate distinct functions of biological targets. Recent controlled expression of aptamers inside cells demonstrated their impressive potential as rapidly generated intracellular inhibitors of biomolecules. Intracellularly applied aptamers are also called 'intramers'. Here we discuss recent developments and strategies for intramer-based technologies that have the potential to greatly facilitate characterisation of unknown protein functions in the context of their natural expression status in vivo. Thus, intramer-based technologies offer many promising applications in functional genomics, proteomics and drug discovery.

Background: In vitro selected ribozymes with nucleotide synthase, peptide and carbon-carbon bond forming activity provide insight into possible scenarios on how chemical transformations may have been catalyzed before protein enzymes had evolved. Metabolic pathways based on ribozymes may have existed at an early stage of evolution., Results: We have isolated a novel ribozyme that mediates Michael-adduct formation at a Michael-acceptor substrate, similar to the rate-limiting step of the mechanistic sequence of thymidylate synthase. The kinetic characterization of this catalyst revealed a rate enhancement by a factor of approximately 10(5). The ribozyme shows substrate specificity and can act as an intermolecular catalyst which transfers the Michael-donor substrate onto an external 20-mer RNA oligonucleotide containing the Michael-acceptor system., Conclusion: The ribozyme described here is the first example of a catalytic RNA with Michael-adduct forming activity which represents a key mechanistic step in metabolic pathways and other biochemical reactions. Therefore, previously unforeseen RNA-evolution pathways can be considered, for example the formation of dTMP from dUMP. The substrate specificity of this ribozyme may also render it useful in organic syntheses.

In vitro selection of combinatorial nucleic acid libraries leads to specific target-binding molecules--RNA, single stranded DNA, modified RNA or modified DNA, commonly designated as aptamers--and to novel catalytic nucleic acids. The current state of aptamer and ribozyme technology is such that it establishes itself as a means of obtaining useful tools for molecular biology, diagnostics, molecular medicine and bio-organic chemistry.

Background: The 'RNA world' hypothesis proposes that the early history of life on earth consisted of a period in which chemical transformations were catalyzed exclusively by ribozymes. Ribozymes that act as acyl transferases, or catalyze the formation of amide or peptide bonds seem particularly attractive candidates to link the RNA world to the modern protein-nucleic acid world. The central role played by aminoacylated RNAs in today's processes of translating RNA into protein suggests that aminoacyl transfer reactions catalyzed by RNA might have facilitated the development and optimization of the translation apparatus during early evolution., Results: We describe the isolation and characterization of a novel ribozyme that catalyzes the transfer of an amino-acid ester from an aminoacyl donor substrate onto the ribozyme itself. The site of aminoacylation was determined to be at an internal 2'-OH group of a cytosine residue. The aminoacylation depends on the presence of Mg2+ and can be competitively inhibited by the AMP moiety of the aminoacyl donor substrate, suggesting that there is a specific binding pocket for this substrate. The originally selected ribozyme was engineered to act as intermolecular catalyst that transfers the amino acid onto an external 28-residue oligonucleotide. The aminoacylated oligonucleotide was further used to quantify the reverse reaction catalyzed by the ribozyme., Conclusions: The ribozyme we have isolated is an example of a catalytic RNA with ester transferase activity which uses a substrate that is not templated by Watson-Crick-pairing hydrogen bonds. The reaction catalyzed by the ribozyme expands the scope of RNA catalysis to include acyl transferase activity from an RNA 3' end to an internal 2' position and the reverse. Ribozymes with such activity have been postulated to be evolutionary precursors of ribosomal RNA.

By in vitro selection, a variety of catalytic DNA oligonucleotides were obtained which cleave chimeric oligonucleotides at a single ribonucleotide position embedded within a deoxyribonucleotide context in the presence or absence of divalent metal ions. After several cycles of selection/amplification in the absence and in the presence of low amounts of Mg2+ two different types of catalysts emerged: one type depended strongly on Mg2+ or other divalent metal ions, the other type performed cleavage reactions independently of Mg2+ in the presence of spermine. Experimental analysis of the secondary structure of some of the selected deoxyribozymes was carried out by chemical probing. The ribonucleotide in the selected catalysts is unpaired and presents the cleavage site to the attacking nucleophile. Our results suggest that the main selection criterion under metal-free conditions was a favourable arrangement of the attacking nucleophile and the phosphate leaving group. The cleavage rates of the selected divalent metal independent catalysts are within the same order of magnitude as the rate of metal independent substrate hydrolysis in the hammerhead ribozyme. One of the metal dependent catalysts showed an unexpected preference for Ca2+ instead of Mg2+. In this deoxyribozyme binding of Ca2+ occurred co-operatively whereas binding of Mg2+ did not. Comparison of the secondary structure and reactivity of this catalyst with Mg2+ and Ca2+ suggests that here a special binding pocket for Ca2+ was selected. This deoxyribozyme achieved a rate acceleration of substrate cleavage in the order of at least 10(4) compared to the uncatalysed reaction performing a cleavage mechanism similar to that of the hammerhead or hairpin ribozyme.

Background: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection., Results: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb2+-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot., Conclusions: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.

Background: Antibiotics can interfere with RNA activity. Translation of RNA by the prokaryotic ribosome, self-splicing of group I introns, HIV replication and hammerhead ribozyme cleavage are inhibited by the aminoglycoside neomycin B. To explore the molecular basis by which small molecules such as antibiotics inhibit RNA function, we undertook an in vitro selection to obtain a variety of RNA molecules with the capacity to recognize neomycin., Results: The majority of the RNA molecules selected to specifically bind neomycin share a region of nucleotide sequence homology. From chemical probing and covariations among different clones we show that in all sequences this region folds into a hairpin structure, which from footprinting and partial alkaline hydrolysis experiments is shown to be the neomycin-binding site. Neomycin is recognized with high affinity (Kd approximately equal to 100 nM) and high specificity (> 100-fold higher affinity for neomycin than for paromomycin)., Conclusions: The fact that RNAs containing the consensus sequence, as well as sequences that display variations within this region, specifically recognize neomycin suggests that a structural motif rather than a particular nucleotide sequence is required for neomycin recognition. We propose that a hairpin stem-loop structural motif, which might feature a widened major groove, may be a prerequisite for neomycin recognition. This structural pattern can be extrapolated to other natural neomycin-responsive RNAs.