Multistep small-molecule synthesis programmed by DNA templates

The translation of DNA sequences into synthetic products is a key requirement of our approach to evolving synthetic molecules through iterated cycles of translation, selection, and amplification. Here we report general linker and purification strategies for sequence-specific DNA-templated synthesis that collectively enable the product of a DNA-templated reaction to be isolated and to undergo subsequent DNA-templated reactions. Using these strategies, we have achieved the first multistep nucleic acid-templated small-molecule syntheses to generate two different molecules. In addition to representing a method for translating DNA templates sequence-specifically into corresponding multistep synthetic products, our findings also provide experimental support for previously proposed models invoking multistep nucleic acid-templated synthesis as mediating the prebiotic translation of replicable information into the earliest functional molecules.     

Translation of DNA into synthetic N-acyloxazolidines

The translation of DNA into synthetic molecules enables their manipulation by powerful evolution-based methods previously available only to proteins and nucleic acids. The development of increasingly sophisticated DNA-templated small-molecule syntheses is crucial to broadening the scope of this approach. Here, we report the translation of DNA templates into monocyclic and bicyclic N-acyloxazolidines using multistep DNA-templated organic synthesis. Second-generation template architectures, used for the first time in a multistep DNA-templated synthesis, together with reactions and linker cleavage strategies not previously described in a DNA-templated format, were crucial to the successful translation. The products generated in this work represent the most complex small molecules to date synthesized in a DNA sequence-programmed manner and provide the basis for DNA-templated synthetic heterocycle libraries.     

Effects of template sequence and secondary structure on DNA-templated reactivity

DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. As the size of DNA-templated libraries increases, the possibility of forming intramolecularly base-paired structures within templates that impede templated reactions increases as well. To achieve uniform reactivity across many template sequences and to computationally predict and remove any problematic sequences from DNA-templated libraries, we have systematically examined the effects of template sequence and secondary structure on DNA-templated reactivity. By testing a series of template sequences computationally designed to contain different degrees of internal secondary structure, we observed that high levels of predicted secondary structure involving the reagent binding site within a DNA template interfere with reagent hybridization and impair reactivity, as expected. Unexpectedly, we also discovered that templates containing virtually no predicted internal secondary structure also exhibit poor reaction efficiencies. Further studies revealed that a modest degree of internal secondary structure is required to maximize effective molarities between reactants, possibly by compacting intervening template nucleotides that separate the hybridized reactants. Therefore, ideal sequences for DNA-templated synthesis lie between two undesirable extremes of too much or too little internal secondary structure. The relationship between effective molarity and intervening nucleic acid secondary structure described in this work may also apply to nucleic acid sequences in living systems that separate interacting biological molecules.     

DNA-templated nickel nanostructures and protein assemblies

We report a straightforward method for the fabrication of DNA-templated nickel nanostructures on surfaces. These nickel nanomaterials have potential to be applied as nanowires, as templated catalyst lines, as nanoscale magnetic domains, or in directed protein localization. Indeed, we show here that histidine-tagged phosducin-like protein (His-PhLP) binds with high selectivity to both Ni2+-treated surface DNA and DNA-templated nickel metal to create linear protein assemblies on surfaces. The association of His-PhLP with DNA-templated nickel ions or metal is reversible under appropriate rinsing conditions. Nanoscale DNA-templated protein assemblies might be useful in the construction of high-density protein lines for proteomic analysis, for example. Importantly, these nanofabrication procedures are not limited to linear DNA and can be applied readily to other self-assembled DNA topologies.     

From Interaction to Function in DNA-Templated Supramolecular Self-Assemblies

DNA-templated self-assembly represents a rich and growing subset of supramolecular chemistry where functional self-assemblies are programmed in a versatile manner using nucleic acids as readily-available and readily-tunable templates. In this review, we summarize the different DNA recognition modes and the basic supramolecular interactions at play in this context. We discuss the recent results that report the DNA-templated self-assembly of small molecules into complex yet precise nanoarrays, going from 1D to 3D architectures. Finally, we show their emerging functions as photonic/electronic nanowires, sensors, gene delivery vectors, and supramolecular catalysts, and their growing applications in a wide range of area from materials to biological sciences.     

A new solid-phase nanoparticle FRET assays based on GO/CS/ZnS nanocomposite film and developed in sensing for bromonium ion

Composite thin films consisting of nano-sized ZnS particles dispersed in chitosan/GO films have been prepared by in-situ method. The films obtained were characterized by FTIR and UV–Vis spectroscopy. The ZnS nanoparticles with 90 nm in diameter were dispersed uniformly in the film matrix. Optical absorption peak due to the size of ZnS particles was observed around 350 nm. The fluorescence emission at 430 nm of the GO/CS/ZnS nanocomposite films is very sensitive to the presence of bromonium ion from aqueous solutions. New solid-phase nanoparticles FRET assays are firstly immobilized on the substrate and then interacted with functionalized acceptor molecules in the solution to trigger the FRET effect to detect Br^–.     

Deep-UV surface-enhanced resonance Raman scattering of adenine on aluminum nanoparticle arrays

We report the ultrasensitive detection of adenine using deep-UV surface-enhanced resonance Raman scattering on aluminum nanostructures. Well-defined Al nanoparticle arrays fabricated over large areas using extreme-UV interference lithography exhibited sharp and tunable plasmon resonances in the UV and deep-UV wavelength ranges. Theoretical modeling based on the finite-difference time-domain method was used to understand the near-field and far-field optical properties of the nanoparticle arrays. Raman measurements were performed on adenine molecules coated uniformly on the Al nanoparticle arrays at a laser excitation wavelength of 257.2 nm. With this technique, less than 10 amol of label-free adenine molecules could be detected reproducibly in real time. Zeptomole (~30,000 molecules) detection sensitivity was readily achieved proving that deep-UV surface-enhanced resonance Raman scattering is an extremely sensitive tool for the detection of biomolecules.     

Translation of DNA into a library of 13,000 synthetic small-molecule macrocycles suitable for in vitro selection

DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in vitro selection of a pilot library of 65 macrocycles. In this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. Integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.     

Amplified Self-Immolative Release of Small Molecules by Spatial Isolation of Reactive Groups on DNA-Minimal Architectures

Triggering the release of small molecules in response to unique biomarkers is important for applications in drug delivery and biodetection. Due to low quantities of biomarker, amplifying release is necessary to gain appreciable responses. Nucleic acids have been used for both their biomarker-recognition properties and as stimuli, notably in amplified small-molecule release by nucleic-acid-templated catalysis (NATC). The multiple components and reversibility of NATC, however, make it difficult to apply in vivo. Herein, we report the use of the hybridization chain reaction (HCR) for the amplified, conditional release of small molecules from standalone nanodevices. We couple HCR with a DNA-templated reaction resulting in the amplified, immolative release of small molecules. We integrate the HCR components into single nanodevices as DNA tracks and spherical nucleic acids, spatially isolating reactive groups until triggering. This could be applied to biosensing, imaging, and drug delivery.     

Highly sensitive in vitro selections for DNA-linked synthetic small molecules with protein binding affinity and specificity

We have developed in vitro selections for DNA-linked synthetic small molecules with protein binding affinity and specificity. These selections require only generally accessible equipment, offer high degrees of enrichment of active molecules from mixtures of predominantly inactive species, can be applied to a variety of unrelated proteins, and require approximately 108-fold less material than existing synthetic molecule screening methods. Iterating these selections multiplies the net enrichment of active molecules, enabling enormous overall enrichment factors exceeding 106 to be achieved. Further, the selections can be adapted to select for binding specificity in addition to binding affinity. The application of methods described in this work may play a key role in the discovery of desired molecules from DNA-templated synthetic libraries.     

Template-directed synthesis in 3'- and 5'-direction with reversible termination

Extending both ways: A method for DNA-templated synthesis on solid support is described. Controlled, stepwise chain extension was demonstrated both in the direction favored by nature (3'-extension; see scheme) and in the direction typical for conventional DNA synthesizers (5'-extension).     

Oriented structure of octadecyl acridine orange intercalated in the monolayer and Langmuir–Blodgett film of octadecyl adenine‐thymine base pairs

The oriented structure of acridine orange (AO) in both monolayer and Langmuir–Blodgett (LB) film has been studied by optical waveguide (OWG) spectroscopy using polarized incident light. Mixed monolayer and LB films, consisting of octadecyl acridine orange (C₁₈‐AO) incorporated in stacked base pairs of octadecyl adenine (C₁₈‐Ade) and octadecyl thymine (C₁₈‐Thy), were prepared on a quartz waveguide. Absorption of transverse electric field (TE) polarized light was about twice that of transverse magnetic field (TM) polarized light. Both OWG spectra have λ_max at 500 nm, which is characteristic of monomeric AO molecules. This result strongly suggests that C₁₈‐AO molecules were dispersed uniformly in the mixed monolayer and were excited more effectively by the TE polarized light. Since the absorption moment of AO molecules is related to their long axis, it is proposed that C₁₈‐AO molecules are incorporated in C₁₈‐Ade/C₁₈‐Thy pairs with the long axis parallel to the layer surface. The absorbance at 500 nm was proportional to the number of layers on the waveguide. The dichroic ratio of the absorbance at 500 nm for TE polarized light to that for TM polarized light was constant regardless of the number of layers. The C₁₈‐AO molecules were uniformly incorporated in each layer with the long axis relatively parallel to the layer surface. Copyright © 2004 John Wiley & Sons, Ltd.     

Preparation of Ni nanoparticles between montmorillonite layers utilizing dimethylaminoborane as reducing agent

Preparation of Ni nanoparticles between montmorillonite layers using dimethylaminoborane (DMAB) as a reducing agent is reported. The DMAB molecules are first intercalated into the interlayer space of Ni-montmorillonite (Ni-mont). Then, as a result of a heating process, the DMAB is decomposed to release electrons for the reduction of the Ni ions. From high-resolution TEM images, it is demonstrated that the deposited Ni nanoparticles with about 1-2 nm in size are formed uniformly over the entire area of the Ni-mont matrix. Considering the gallery height calculated by subtracting the silicate sheet thickness from the basal spacing (1.30 nm), the morphology of the formed Ni nanoparticles in the interlayer space is thought to be disc-like in shape with a thickness of 0.3-0.4 nm and an average lateral size of 1.2 nm.     

DNA-Templated Gold Nanoclusters for Fluorescence Resonance Energy Transfer-Based Human Serum Albumin Detection

Journal of Analytical Chemistry - A new method for the determination of human serum albumin (HSA) was developed using gold nanoclusters (AuNCs) as a probe. DNA-templated gold nanoclusters with...     

Poly(adenine) DNA-Templated Gold Nanocluster-Based Fluorescent Strategy for the Determination of Thiol-Containing Pharmaceuticals

The fluorescence intensity of poly(adenine) DNA-templated gold nanoclusters was shown to be significantly quenched by N-acetylcysteine through the formation of the Au-S metal-ligand bonds. On the basis of the fluorescence quenching phenomenon, a sensitive, turn-off, and label-free fluorescence method has been designed for the determination of thiol-containing pharmaceuticals using poly(adenine) DNA-templated gold nanoclusters as fluorescent probes. The assay exhibited sensitive determination of N-acetylcysteine with a linear dynamic range from 10?nM to 10?μM and a limit of detection of 3?nM. Furthermore, the proposed strategy was successfully applied for the determination of N-acetylcysteine levels in acetylcysteine granule samples. Thus, the method could provide a sensitive, simple, and rapid fluorescent sensing platform for the determination of thiol-containing pharmaceuticals.     

DNA模板指导的有机合成

DNA模板指导的有机合成反应具有序列特异性特点.本文综述了模板DNA指导的多种类型的有机合成反应,包括还原胺化、亲核取代、Henry、Wittig烯化、光化学连接以及多步小分子的合成等反应;介绍了DNA指导的组合库的合成反应;总结了DNA模板结构对反应的影响以及反应中立体选择性的问题.     

DNA-templated dimerization of hairpin polyamides

Double-helical DNA accelerates the rate of ligation of two six-ring hairpin polyamides which bind adjacent sites in the minor groove via a 1,3-dipolar cycloaddition to form a tandem dimer. The rate of the templated reaction is dependent on DNA sequence as well as on the distance between the hairpin-binding sites. The tandem dimer product of the DNA-templated reaction has improved binding properties with respect to the smaller hairpin fragments. Since cell and nuclear uptake of DNA-binding polyamides will likely be dependent on size, this is a minimum first step toward the design of self-assembling small gene-regulating fragments to produce molecules of increasing complexity with more specific genomic targeting capabilities.     

基于氧化石墨烯和DNA量子点组装体的DNA二元逻辑检测及循环可逆设计

制备了水溶性的氧化石墨烯(GO)和以DNA为模板的Cd Te量子点(P1),通过GO与P1的π-π堆积作用组装构建了纳米生物传感器,将其用于双目标DNA分子的逻辑检测,实现了较高的选择性;通过改进DNA序列,实现了该传感器对双目标分子的可逆循环检测及重复利用.利用原子力显微镜(AFM)、透射电子显微镜、电泳和荧光光谱等方法对传感器的构建和检测过程进行了表征.该P1-GO纳米生物传感器在核酸检测等领域具有较大的应用前景.     

二氧化钛表面包覆氧化硅纳米膜的热力学研究

热力学计算结果表明,氧化硅的临界成核半径为2.8nm.由起伏引起的核胚如果小于2.8nm则不会形成晶核而继续生长.上述热力学分析虽然是半理想化的,但是非常有效.可以找到这样一个体系,其溶液条件不发生均匀成核,而是异相表面成核.这在理论上为氧化硅包覆在二氧化钛表面形成均匀膜而不生成单独的氧化硅相提供了可能.     

Structural and optical investigations of SiO2-CdS core-shell particles

Cadmium sulfide nanoparticles (approximately 5 nm), chemically capped using thioglycerol molecules, have been anchored onto silica particles (approximately 80 nm) functionalized with 3-aminopropyltrimethoxysilane. Transmission electron microscopy clearly showed that at a low concentration of cadmium sulfide, nanoparticles were discretely and more or less uniformly attached onto the silica particles. At a high concentration of cadmium sulfide nanoparticles, an approximately 6-nm-thick compact shell of cadmium sulfide was formed on the silica particles. In both cases the nanocrystalline nature of cadmium sulfide particles was preserved, as is evident from X-ray diffraction and optical absorption spectra.