ATP-Triggered, Allosteric Self-Assembly of DNA Nanostructures.
Li Qian,Liu Longfei,Mao Dake,Yu Yuyan,Li Weili,Zhao Xinfeng,Mao Chengde
Journal of the American Chemical Society
Responsive self-assembly is a general process in biological systems and is highly desired in engineered systems. DNA nanostructures provide a versatile molecular platform for studying such responsive self-assembly. Various triggers have been explored for DNA nanostructures. However, each trigger requires a unique mechanism for its response. This situation brings a great challenge to engineer the responsiveness. Herein, we propose an aptamer-based, allosteric mechanism for responsive DNA self-assembly. The aptamer-ligand binding causes the DNA motif to change its conformation and thus influences the motif assembly. With a model of an ATP aptamer, we have demonstrated the responsive assembly. Such responsive behavior, we believe, will be an important element for molecular machines, bioimaging/biosensing, and drug delivery.
Identification and characterization of nucleobase-modified aptamers by click-SELEX.
Pfeiffer Franziska,Tolle Fabian,Rosenthal Malte,Brändle Gerhard Markus,Ewers Jörg,Mayer Günter
Aptamers are single-stranded oligonucleotides that are in vitro-selected to recognize their target molecule with high affinity and specificity. As they consist of the four canonical nucleobases, their chemical diversity is limited, which in turn limits the addressable target spectrum. Introducing chemical modifications into nucleic acid libraries increases the interaction capabilities of the DNA and thereby the target spectrum. Here, we describe a protocol to select nucleobase-modified aptamers by using click chemistry (CuAAC) to introduce the preferred chemical modification. The use of click chemistry to modify the DNA library enables the introduction of a wide range of possible functionalities, which can be customized to the requirements of the target molecule and the desired application. This protocol yields modified DNA aptamers with extended interaction properties that are not accessible with the canonical set of nucleotides. After synthesis of the starting library containing a commercially available, alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU)) instead of thymidine, the library is functionalized with the modification of choice by CuAAC. The thus-modified DNA is incubated with the target molecule and the best binding sequences are recovered. The chemical modification is removed during the amplification process. Therefore, this protocol is compatible with conventional amplification procedures and avoids enzymatic incompatibility problems associated with more extensive nucleobase modifications. After single-strand generation, the modification is reintroduced into the enriched library, which can then be subjected to the subsequent selection cycle. The duration of each selection cycle as outlined in the protocol is ∼1 d.
Structural distinctions between NAD+ riboswitch domains 1 and 2 determine differential folding and ligand binding.
Chen Hao,Egger Michaela,Xu Xiaochen,Flemmich Laurin,Krasheninina Olga,Sun Aiai,Micura Ronald,Ren Aiming
Nucleic acids research
Riboswitches are important gene regulatory elements frequently encountered in bacterial mRNAs. The recently discovered nadA riboswitch contains two similar, tandemly arrayed aptamer domains, with the first domain possessing high affinity for nicotinamide adenine dinucleotide (NAD+). The second domain which comprises the ribosomal binding site in a putative regulatory helix, however, has withdrawn from detection of ligand-induced structural modulation thus far, and therefore, the identity of the cognate ligand and the regulation mechanism have remained unclear. Here, we report crystal structures of both riboswitch domains, each bound to NAD+. Furthermore, we demonstrate that ligand binding to domain 2 requires significantly higher concentrations of NAD+ (or ADP retaining analogs) compared to domain 1. Using a fluorescence spectroscopic approach, we further shed light on the structural features which are responsible for the different ligand affinities, and describe the Mg2+-dependent, distinct folding and pre-organization of their binding pockets. Finally, we speculate about possible scenarios for nadA RNA gene regulation as a putative two-concentration sensor module for a time-controlled signal that is primed and stalled by the gene regulation machinery at low ligand concentrations (domain 1), and finally triggers repression of translation as soon as high ligand concentrations are reached in the cell (domain 2).
Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures.
Kappel Kalli,Zhang Kaiming,Su Zhaoming,Watkins Andrew M,Kladwang Wipapat,Li Shanshan,Pintilie Grigore,Topkar Ved V,Rangan Ramya,Zheludev Ivan N,Yesselman Joseph D,Chiu Wah,Das Rhiju
The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
Programming Biomimetically Confined Aptamers with DNA Frameworks.
Mao Xiuhai,Liu Mengmeng,Yan Lei,Deng Mengying,Li Fan,Li Min,Wang Fei,Li Jiang,Wang Lihua,Tian Yang,Fan Chunhai,Zuo Xiaolei
Active sites of proteins are generally encapsulated within three-dimensional peptide scaffolds that provide the molecular-scale confinement microenvironment. Nevertheless, the ability to tune thermodynamic stability in biomimetic molecular confinement relies on the macromolecular crowding effect of lack of stoichiometry and reconfigurability. Here, we report a framework nucleic acid (FNA)-based strategy to increase thermodynamic stability of aptamers. We demonstrate that the molecular-scale confinement increases the thermodynamic stability of aptamers facilitated folding kinetics, which is confirmed by the single-molecule FRET (smFRET). Unfavorable conformations of aptamers are restricted as revealed by the Monte Carlo simulation. The binding affinity of the DNA framework-confined aptamer is improved by ∼3-fold. With a similar strategy we improve the catalytic activity of hemin-binding aptamer. Our approach thus shows high potential for designing protein-mimicking DNA nanostructures with enhanced binding affinity and catalytic activity for biosensing and biomedical engineering.