Reprogramming DNA-directed reactions on the basis of a DNA conformational change

A strategy has been developed for switching chemical reactions between two identical reagents on the basis of DNA duplex-triplex transition. In response to the change of solution pH, a DNA complex changes its conformation and repositions chemical reagents that are conjugated with DNA strands. As a result, chemical reactions are reprogrammed. This strategy is expected to be applicable to sophisticated chemical syntheses.     

Deciphering nucleic acid modifications by chemical derivatization-mass spectrometry analysis

Chemical derivatization in combination with mass spectrometry (MS) analysis is a promising strategy for the sensitive and effective analysis of nucleic acid modifications. In this review, we summarize the recent advances for deciphering modifications in DNA and RNA by chemical derivatization-MS analysis.     

4-硫脱氧胸苷及类似物在UVA光辅助治疗抗癌药物方面的研究

本文叙述了含硫碱基DNA的化学和医疗应用。硫碱基（Thio-base）有其独特的性质,比如,易烷基化,易氧化和强的紫外长波(400-315 nm,UVA)吸收。这些特定化学和物理性质存于含硫碱基、含硫核苷和含硫碱基DNA中,它们对制备修饰的DNA和对硫碱基的DNA与其它生物大分子之间进行光诱导的交联反应是非常有用的。功能化的含有硫碱基的DNA及其类似物可用于对DNA的修复的研究。本文也介绍和评述了利用紫外光/含硫脱氧胸（腺嘧啶脱氧核）苷（UVA/Thiothymidine）的抗癌疗法,同时,探讨了含硫核苷类似物和紫外线光之间的协同作用,为癌症和其它疾病治疗提供一种新的方法。     

Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates

DNA-directed chemical synthesis has matured into a useful tool with applications such as fabrication of defined (nano)molecular architectures, evolution of amplifiable small-molecule libraries, and nucleic acid detection. Most commonly, chemical methods were used to join oligonucleotides under the control of a DNA or RNA template. The full potential of chemical ligation reactions can be uncovered when nonnatural oligonucleotide analogues that can provide new opportunities such as increased stability, DNA affinity, hybridization selectivity, and/or ease and accuracy of detection are employed. It is shown that peptide nucleic acid (PNA) conjugates, nonionic biostable DNA analogues, allowed the fashioning of highly chemoselective and sequence-selective peptide ligation methods. In particular, PNA-mediated native chemical ligations proceed with sequence selectivities and ligation rates that reach those of ligase-catalyzed oligodeoxynucleotide reactions. Usually, sequence-specific ligations can only be achieved by employing short-length probes, which show DNA affinities that are too low to allow stable binding to target segments in large, double-stranded DNA. It is demonstrated that the PNA-based ligation chemistry allowed the development of a homogeneous system in which rapid single-base mutation analyses can be performed even on double-stranded PCR DNA templates.     

Discovery of Small‐Molecule Interleukin‐2 Inhibitors from a DNA‐Encoded Chemical Library

Libraries of chemical compounds individually coupled to encoding DNA tags (DNA‐encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high‐quality DNA‐encoded chemical library comprising 30 000 drug‐like compounds; this was screened in 170 different affinity capture experiments. High‐throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor‐associated antigen carbonic anhydrase IX (CA IX) and the pro‐inflammatory cytokine interleukin‐2 (IL‐2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL‐2 was confirmed by molecular docking. Our findings suggest that DNA‐encoded chemical libraries allow the facile identification of drug‐like ligands principally to any protein of choice, including molecules capable of disrupting high‐affinity protein–protein interactions.     

A Photo‐responsive Small‐Molecule Approach for the Opto‐epigenetic Modulation of DNA Methylation

Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small‐molecule approach to modulate DNA methylation with light. The strategy uses a photo‐tuneable version of a clinically used drug (5‐aza‐2′‐deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo‐regulated molecule can be light‐controlled to reduce genome‐wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine.     

HPLC技术对细菌基因组DNA碱基组成的精确测定

高效液相色谱技术(HPLC)在精确分析细菌基因组DNA碱基组成时存在着重复性差和杂质干扰的问题。本研究结合反相HPLC技术对化学水解(高氯酸法和甲酸法)和生物酶水解(磷酸二酯酶Ⅰ和牛肠粘膜碱性磷酸酶)DNA的水解条件和产物进行分析,建立了一套系统的分析方法,改善了分析的重复性问题,排除了主要的误差来源,达到了对细菌DNA碱基组成精确分析测定的目的。     

Structure and function of noncanonical nucleobases

DNA and RNA contain, next to the four canonical nucleobases, a number of modified nucleosides that extend their chemical information content. RNA is particularly rich in modifications, which is obviously an adaptation to their highly complex and variable functions. In fact, the modified nucleosides and their chemical structures establish a second layer of information which is of central importance to the function of the RNA molecules. Also the chemical diversity of DNA is greater than originally thought. Next to the four canonical bases, the DNA of higher organisms contains a total of four epigenetic bases: m(5) dC, hm(5) dC, f(5) dC und ca(5) dC. While all cells of an organism contain the same genetic material, their vastly different function and properties inside complex higher organisms require the controlled silencing and activation of cell-type specific genes. The regulation of the underlying silencing and activation process requires an additional layer of epigenetic information, which is clearly linked to increased chemical diversity. This diversity is provided by the modified non-canonical nucleosides in both DNA and RNA.     

Chemical ligation as a method for the assembly of double-stranded nucleic acids: Modifications and local structure studies

A new procedure was developed as an alternative to the enzymatic assembly of natural and modified double-stranded DNAs using chemical reagent (chemical ligation). BrCN was suggested as an efficient coupling reagent, which induces superfast reactions in DNA duplexes. The physicochemical properties and the structure of new types of DNA duplexes, which are the substrates for chemical ligation, with breaks in phosphodiester chains, including concatemers, were studied. Chemical ligation was applied to prepare biologically active 17–200 base-pair double-stranded DNAs and DNA-RNA block-copolymers, to incorporate various modifications into DNA duplexes including pyrophosphate and phosphoramidate unnatural internucleotide bonds. The unique possibilities of this approach were demonstrated in the development of methods for circularization of oligodeoxy ribonucleotides and assembly of branched DNAs. The structural-kinetic concept of chemicalligation was created and the relationship between the reactivity of interacting groups and sequence-dependent local conformation of the ligation site in B-DNA was established. The lesser efficiency of chemical ligation of RNA fragments in comparison to that of DNA analogs was demonstrated and rationalized. This approach was used as a sensitive monitor of a stable double helix formation and third-strand binding to a DNA duplex.Translated from Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1889–1911, August, 1996.     

Discovery and synthesis of new UV-induced intrastrand C(4-8)G and G(8-4)C photolesions

UV irradiation of cellular DNA leads to the formation of a number of defined mutagenic DNA lesions. Here we report the discovery of new intrastrand C(4-8)G and G(8-4)C cross-link lesions in which the C(4) amino group of the cytosine base is covalently linked to the C(8) position of an adjacent dG base. The structure of the novel lesions was clarified by HPLC-MS/MS data for UV-irradiated DNA in combination with chemical synthesis and direct comparison of the synthetic material with irradiated DNA. We also report the ability to generate the lesions directly in DNA with the help of a photoactive precursor that was site-specifically incorporated into DNA. This should enable detailed chemical and biochemical investigations of these lesions.     

Site-specific assembly of DNA and appended cargo on arrayed carbon nanotubes

We describe a strategy that permits discrete regions of arrayed carbon nanotubes (CNTs) to be functionalized simultaneously and specifically with DNA oligonucleotides. The different chemical properties of two regions on single CNTs and orthogonal chemical coupling strategies have been exploited to derivatize CNTs within highly ordered arrays with multiple DNA sequences. Through duplex hybridization, we then targeted different DNA sequences with appended metal nanoparticles to distinct sites on the CNT architecture with precise spatial control. The materials generated from these studies represent the first CNTs with bipartite functionalization. The approach described provides a high level of precision in parallel and directed assembly of DNA sequences and appended cargo and is useful for the preparation of novel hybrid bionanomaterials.     

Functionality‐Independent DNA Encoding of Complex Natural Products

DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late‐stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end‐products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL (nDEL).     

Deoxyribonucleotides: the unusual chemistry and biochemistry of DNA precursors

Deoxyribonucleotides, monomers of macromolecular DNA and the chemical matter of genes, have received surprisingly little attention among chemists and molecular biologists alike, although their origin, properties, and mechanism of enzyme-catalyzed formation bear unique chemical traits which are the basis of DNA replication. Apart from providing insights in bioorganic free radical chemistry, present interest in deoxyribonucleotides stems from the expected demand of hundreds of kilograms per year for DNA chips and antisense constructs used in gene therapy, difficult to produce by conventional methods. A novel approach towards deoxyribonucleotide, and hence DNA formation in a putative primordial 'RNA world' has also recently emerged.     

Chemical properties of fatty acid derivatives as inhibitors of DNA polymerases

In this study, the chemical properties of organic acids as DNA polymerase inhibitors were examined. In total, we assayed the inhibitory activities of 23 compounds. We found that the DNA synthesis activity of DNA polymerase was usually reduced to less than 50% in the presence of 100 microM monoprotic acids, which have a Clog P value greater than 7.0 and a pK(a) value less than 5.4. With a minor modification these chemical properties applied to several organic fatty acids previously reported as DNA polymerase inhibitors. Moreover, we also examined the inhibitory activities of perfluorooctadecanoic acid (PFOdA) and perfluorooctanesulfonic acid (PFOS) against DNA polymerase beta in detail. These compounds inhibited the polymerase activity of pol beta competitively with template-primer DNA, and non-competitively with dNTPs. In addition, the 8 kDa domain-defective pol beta was also sensitive to these compounds. Our results suggest that the inhibitory mode of action of PFOdA and PFOS is different from that mediated by the classic fatty acid inhibitors against DNA polymerase beta.     

An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling

DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.     

DNA‐Encoded Dynamic Combinatorial Chemical Libraries

Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA‐encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal‐to‐noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA‐encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high‐affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA‐templated chemical reactions.     

Solid-Phase Synthesis of Oligodeoxynucleotides containing phosphoramidate internucleotide linkages and their specific chemical cleavage

Oligonuclecotides bearing phosphoramidate internucleotide linkages can be prepared chemically by standard solid-phase DNA synthesis. Thus, phosphoramidate internucleotide bonds can be placed at will into specific positions within a given DNA fragment. The backbone-modified DNA fragments prepared in this way are susceptible to a specific chemical cleavage.     

Formation, characterization, and chemistry of undecanoic acid-terminated silicon surfaces: patterning and immobilization of DNA

This paper describes a simple strategy for DNA immobilization on chemically modified and patterned silicon surfaces. The photochemical modification of hydrogen-terminated Si(111) with undecylenic acid leads to the formation of an organic monolayer covalently attached to the surface through Si-C bonds without detectable reaction of the carboxylic acid group, providing indirect support of a free radical mechanism. Chemical activation of the acid function was achieved by a simple chemical route using N-hydroxysuccinimide (NHS) in the presence of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride. Single strand DNA with a 5'-dodecylamine group was then coupled to the NHS-activated surface by amide bond formation. Using a previously reported chemical patterning approach, we have shown that DNA can be immobilized on silicon surfaces in spatially well-resolved domains. Methoxytetraethyleneglycolamine was used to inhibit nonspecific adsorption. The resulting DNA-modified surfaces have shown good specificity and chemical and thermal stability under hybridization conditions. The sequential reactions on the surface were monitored by ATR-FTIR, X-ray Photoelectron Spectroscopy, and fluorescence spectroscopy.     

Programmable protein-protein conjugation via DNA-based self-assembly

Protein molecules were precisely arrayed on a designable DNA scaffold close to each other using a DNA aptamer. By adding a chemical cross-linker, the neighboring protein molecules were effectively and covalently cross-linked to each other without losing their activities.