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Artificial DNA: PNA & XNA最新文献

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Effect of 2′-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells 2 ' - o -甲基/硫代膦酸酯修饰的反义寡核苷酸对患者源性细胞中亨廷顿蛋白表达的影响
Pub Date : 2014-12-15 DOI: 10.1080/1949095X.2016.1146391
Masayuki Matsui, Richard Threlfall, M. Caruthers, D. Corey
ABSTRACT Optimizing oligonucleotides as therapeutics will require exploring how chemistry can be used to enhance their effects inside cells. To achieve this goal it will be necessary to fully explore chemical space around the native DNA/RNA framework to define the potential of diverse chemical modifications. In this report we examine the potential of thiophosphonoacetate (thioPACE)-modified 2′-O-methyl oligoribonucleotides as inhibitors of human huntingtin (HTT) expression. Inhibition occurred, but was less than with analogous locked nucleic acid (LNA)-substituted oligomers lacking the thioPACE modification. These data suggest that thioPACE oligonucleotides have the potential to control gene expression inside cells. However, advantages relative to other modifications were not demonstrated. Additional modifications are likely to be necessary to fully explore any potential advantages of thioPACE substitutions.
优化寡核苷酸作为治疗药物将需要探索如何利用化学来增强其在细胞内的作用。为了实现这一目标,有必要充分探索天然DNA/RNA框架周围的化学空间,以确定各种化学修饰的潜力。在本报告中,我们研究了硫代膦乙酸酯(thiiopace)修饰的2 ' - o -甲基寡核苷酸作为人类亨廷顿蛋白(HTT)表达抑制剂的潜力。抑制作用发生了,但比缺乏硫代ace修饰的类似锁定核酸(LNA)取代低聚物的抑制作用要小。这些数据表明,硫代ace寡核苷酸具有控制细胞内基因表达的潜力。然而,相对于其他修改的优势没有被证明。为了充分挖掘硫代ace替代的任何潜在优势,可能需要进行额外的修改。
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引用次数: 3
Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage. 纯化可恒温 Cy5 标记的 γ-PNA 并将其组装到三维 DNA 纳米笼中。
Pub Date : 2014-12-15
Justin D Flory, Trey Johnson, Chad D Simmons, Su Lin, Giovanna Ghirlanda, Petra Fromme

PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled NA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.

PNA 是一种混合分子,是连接多肽功能与 DNA 纳米结构多样性的理想分子。然而,由于 PNA 具有疏水性,因此在水性溶剂中使用 PNA 会更具挑战性。作为构建可在三维 DNA 纳米支架中排列的环状 PNA 多肽的第一步,我们开发了一种使用应变促进、无铜点击化学的溶液相方法,将荧光染料 Cy5 与 2 条双功能 PNA 链共轭。我们设计了一种三维 DNA 纳米支架,其上的 2 条荧光标记 PNA 链的结合位点非常接近模拟蛋白质的活性位点。变性聚丙烯酰胺凝胶电泳(PAGE)是一种从大量过量的未反应染料和未反应的中性 PNA 中纯化带电的染料标记 NA 结合物的有效方法。通过荧光监测凝胶在水中的洗脱情况,发现可溶性较高的 PNA 链的洗脱效率更高。原生 PAGE 显示,两条 PNA 链都杂交到了 DNA 纳米笼内的预定结合位点。利用与 Cy3 标记的 DNA 纳米笼的佛斯特共振能量转移(FRET),确定一种 PNA-Cy5 共轭物的解离温度接近 50℃。稳态和时间分辨荧光用于研究各种复合物中的染料取向和相互作用。双功能、可恒温的 PNA 分子是控制肽在小型 DNA 纳米笼内组装和定向的有趣候选分子,可用于模拟蛋白质催化位点。
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引用次数: 0
Anomeric DNA quadruplexes. 异头DNA四联体。
Pub Date : 2014-01-01 DOI: 10.4161/adna.28422
Natalia A Kolganova, Anna M Varizhuk, Roman A Novikov, Vladimir L Florentiev, Galina E Pozmogova, Olga F Borisova, Anna K Shchyolkina, Igor P Smirnov, Dmitry N Kaluzhny, Edward N Timofeev

Thrombin-binding aptamer (TBA) is a 15-nt DNA oligomer that efficiently inhibits thrombin. It has been shown that TBA folds into an anti-parallel unimolecular G-quadruplex. Its three-dimensional chair-like structure consists of two G-tetrads connected by TT and TGT loops. TBA undergoes fast degradation by nucleases in vivo. To improve the nuclease resistance of TBA, a number of modified analogs have been proposed. Here, we describe anomeric modifications of TBA. Non-natural α anomers were used to replace selected nucleotides in the loops and core. Significant stabilization of the quadruplex was observed for the anomeric modification of TT loops at T4 and T13. Replacement of the core guanines either prevents quadruplex assembly or induces rearrangement in G-tetrads. It was found that the anticoagulant properties of chimeric aptamers could be retained only with intact TT loops. On the contrary, modification of the TGT loop was shown to substantially increase nuclease resistance of the chimeric aptamer without a notable disturbance of its anticoagulant activity.

凝血酶结合适体(TBA)是一种有效抑制凝血酶的15-nt DNA低聚体。结果表明,TBA可折叠成反平行的单分子g -四联体。它的三维椅子状结构由两个由TT和TGT环连接的g四分体组成。TBA在体内被核酸酶快速降解。为了提高TBA对核酸酶的抗性,人们提出了许多修饰的类似物。在这里,我们描述了TBA的端粒修饰。用非天然α异头取代环和核中的选定核苷酸。在T4和T13处对TT环进行了明显的四联体稳定化处理。核心鸟嘌呤的替换可以阻止四重体的组装或诱导g -四分体的重排。发现嵌合适体的抗凝血特性只有在TT环完整的情况下才能保留。相反,TGT环的修饰可以显著提高嵌合适体的核酸酶抗性,而不会显著影响其抗凝血活性。
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引用次数: 13
Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage. 高温Cy5标记γ-PNAs的纯化和组装成三维DNA纳米笼。
Pub Date : 2014-01-01 DOI: 10.4161/1949095X.2014.992181
Justin D Flory, Trey Johnson, Chad R Simmons, Su Lin, Giovanna Ghirlanda, Petra Fromme

PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled PNA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50°C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.

PNA是一种杂交分子,非常适合于连接多肽的功能景观和结构多样性,可以用DNA纳米结构进行工程设计。然而,由于PNA的疏水性,在水溶液中处理PNA更具挑战性。一种使用菌株促进的溶液相方法,开发了铜自由点击化学将荧光染料Cy5偶联到2双功能PNA链,作为构建环状PNA多肽的第一步,环状PNA多肽可以排列在3D DNA纳米支架内。设计了一个3D DNA纳米笼,将2条荧光标记的PNA链的结合位点靠近模拟蛋白质活性位点。变性聚丙烯酰胺凝胶电泳(PAGE)是一种从大量未反应的染料和未反应的中性PNA中纯化带电的、染料标记的PNA偶联物的有效方法。通过荧光监测凝胶在水中的洗脱,发现对更可溶性的PNA链更有效。原生页面显示,两个PNA链杂交到DNA纳米笼内的预期结合位点。Förster共振能量转移(FRET)与Cy3标记的DNA纳米笼被用来确定一个PNA-Cy5缀合物的解离温度接近50℃。稳态和时间分辨荧光被用来研究染料取向和各种配合物之间的相互作用。双功能、耐热的PNA分子是控制小DNA纳米笼内肽的组装和取向以模拟蛋白质催化位点的有趣候选者。
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引用次数: 3
Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags. 利用寡核苷酸标签的化学连接对小分子文库进行dna编码的通用策略。
Pub Date : 2014-01-01 DOI: 10.4161/adna.27896
Alexander Litovchick, Matthew A Clark, Anthony D Keefe

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method "relay primer-dependent bypass" utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3'-terminus prior to ligation to adjacent oligonucleotides. The second reading method "repeat-dependent bypass" utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3'-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries.

亲和介导的选择dna编码小分子的大文库越来越多地被用于启动药物发现程序。我们提出了使用寡核苷酸的化学连接来编码这些文库的通用方法。这些方法可用于记录组合合成过程中单个库成员的化学历史。我们展示了三种不同的化学连接方法作为这些文库的信息记录过程(写入)的例子,以及两种不同的dna生成方法作为这些文库的信息检索过程(读取)的例子。示例写入方法包括非催化和Cu(I)催化的炔-叠氮化物环加成和新型光化学胸腺嘧啶-补骨脂素环加成。第一种读取方法“中继引物依赖旁路”利用一种中继引物,该引物在嵌入固定序列的化学连接结上杂交,并在连接相邻寡核苷酸之前在其3'端延伸。第二种读取方法“重复依赖旁路”利用化学连接连接,其两侧是重复序列。上游重复序列在重排事件之前被复制,在重排事件中,cDNA的3'端与下游重复序列杂交,聚合继续进行。原则上,这些读取方法可用于任何连接化学,并为dna编码化学文库的编码(写入)和解释(读取)提供通用策略。
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引用次数: 16
The genetic code. Rewritten, revised, repurposed. 遗传密码。重写,修改,改变用途。
Pub Date : 2014-01-01 DOI: 10.4161/adna.29408
Roy D Sleator

Despite remaining apparently frozen through the millennia, the genetic code is far more flexible than previously believed and can be extended and repurposed with relative ease.

尽管在数千年的时间里,遗传密码显然是冻结的,但它比以前认为的要灵活得多,可以相对容易地扩展和改变用途。
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引用次数: 4
Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases. 含有修饰鸟嘌呤碱基的富g三聚体寡核苷酸生物活性的提高。
Pub Date : 2014-01-01 DOI: 10.4161/adna.27792
Faye A Rogers, Janice A Lloyd, Meetu Kaushik Tiwari

Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis.

由序列特异性三联体形成寡核苷酸(TFOs)产生的三联体结构已被证明是基因靶向策略的有前途的工具。此外,三重体技术已被广泛应用于DNA修复、重组和诱变的分子机制研究。然而,利用富含鸟嘌呤的寡核苷酸作为第三链的三联体形成可以通过钾诱导的自结合导致g -四联体形成而被抑制。我们在这里报道,与含有天然鸟嘌呤的tfo相比,部分取代8-氮杂-7-二氮杂-鸟嘌呤(PPG)的富鸟嘌呤tfo改善了钾的靶位结合。我们设计了ppg取代的TFOs与supFG1报告基因中的多嘌呤序列结合。采用电泳迁移率凝胶位移法分析ppg取代的TFOs与目标序列的结合效率。我们已经确定,在钾的存在下,非取代的TFO, AG30不能与目标序列结合,但在高达140 mM KCl的条件下,可以观察到与ppg取代的AG30结合。通过基因靶向诱变测定,ppg - tfo能够保持其诱导基因组修饰的能力。此外,这些化合物能够诱导DNA双链断裂,从而导致细胞凋亡的激活。
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引用次数: 8
Synthesis and spectral characterization of environmentally responsive fluorescent deoxycytidine analogs. 环境响应型荧光脱氧胞苷类似物的合成与光谱表征。
Pub Date : 2014-01-01 DOI: 10.4161/adna.29174
Adam A H Elmehriki, Mojmír Suchý, Kirby J Chicas, Filip Wojciechowski, Robert H E Hudson

Herein, we describe the synthesis and spectroscopic properties of five novel pyrrolodeoxycytidine analogs, and the related 5-(1-pyrenylethynyl)-2'-deoxycytidine analog; as well as fluorescence characterization of 5-(p-methoxyphenylethynyl)-2'-deoxyuridine. Within this series of compounds, rigidification of the structure from 6-phenylpyrrolodeoxycytidine to 5,6-benzopyrroldeoxycytidine made remarkable improvement of the fluorescence quantum yield (Φ ~1, EtOH) and substantially increased the Stokes shift. Exchange of the phenyl group of 6-phenylpyrrolodeoxycytidine for other heterocycles (benzofuryl or indolyl) produced an increase in the extinction coefficient at the excitation wavelength while preserving high quantum yields. The steady-state fluorescence response to the environment was determined by sensitivity of Stokes shift to solvent polarity. The effect of solvent polarity on fluorescence emission intensity was concurrently examined and showed that 5,6-benzopyrrolodeoxycytidine is highly sensitive to the presence of water. On the other hand, the previously synthesized 5-(p-methoxyphenylethynyl)-2'-deoxyuridine was found to be sensitive to solvent viscosity indicating molecular rotor behavior.

本文描述了五种新型吡咯脱氧胞苷类似物的合成和光谱性质,以及相关的5-(1-芘乙炔基)-2'-脱氧胞苷类似物;以及5-(对甲氧基苯乙基)-2'-脱氧尿苷的荧光表征。在这一系列化合物中,从6-苯基吡咯烷脱氧胞苷结构固化为5,6-苯并吡咯烷脱氧胞苷,荧光量子产率显著提高(Φ ~1, EtOH), Stokes位移显著增加。6-苯基吡咯脱氧胞苷的苯基与其他杂环(苯并呋喃基或吲哚基)交换,在保持高量子产率的同时,增加了激发波长处的消光系数。稳态荧光对环境的响应由Stokes位移对溶剂极性的敏感性决定。同时考察了溶剂极性对荧光发射强度的影响,结果表明,5,6-苯并吡咯脱氧胞苷对水的存在高度敏感。另一方面,先前合成的5-(对甲氧基苯乙基)-2'-脱氧尿嘧啶对溶剂粘度敏感,表明分子转子行为。
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引用次数: 7
Investigation of B-Z transitions with DNA oligonucleotides containing 8-methylguanine. 含8-甲基鸟嘌呤DNA寡核苷酸的B-Z跃迁研究。
Pub Date : 2014-01-01 DOI: 10.4161/adna.28226
Frederic Y-H Chen, Soyoung Park, Haruka Otomo, Sohei Sakashita, Hiroshi Sugiyama

Among various Z-form DNA inducers, such as transition metal complexes, polyamines and high ionic concentrations, 8-methylguanine have received attention as efficient chemical modifications. Although it is clear that m8-modified guanine base markedly stabilizes the Z conformation of short oligonucleotides under physiological salt conditions, how sequence composition affects the preference of Z-DNA is still not well established. In this study, various oligomers of d(CG)n or d(GC)n containing either 8-methylguanine in a different position were synthesized and their capacity of stabilizing Z-DNA were evaluated by CD spectra and then compared with each other. It is was found out that the Z-DNA stabilizing effect depend on the order of arrangement of m(8)G and m(8)rG in DNA strands and the center position is the most effective to stabilize the Z-DNA and promote the B to Z transition.

在各种z型DNA诱导剂中,如过渡金属配合物、多胺和高离子浓度,8-甲基鸟嘌呤作为一种高效的化学修饰受到了人们的关注。虽然m8修饰的鸟嘌呤碱基在生理盐条件下明显稳定短寡核苷酸的Z构象,但序列组成如何影响Z- dna的偏好仍然没有很好的确定。本研究合成了不同位置含有8-甲基鸟嘌呤的d(CG)n或d(GC)n低聚物,用CD谱评价了它们稳定Z-DNA的能力,并进行了比较。结果表明,稳定Z-DNA的效果与m(8)G和m(8)rG在DNA链中的排列顺序有关,中心位置对稳定Z-DNA和促进B向Z过渡最有效。
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引用次数: 15
Improvement of sequence selectivity in triple helical recognition of RNA by phenylalanine-derived PNA. 苯丙氨酸衍生PNA对RNA三螺旋识别序列选择性的提高。
Pub Date : 2013-07-01 Epub Date: 2013-10-08 DOI: 10.4161/adna.26599
Thomas Zengeya, Artem Gindin, Eriks Rozners

Modified peptide nucleic acids (PNA) containing one or two thymine PNA monomers derived from phenylalanine were synthesized. Triple helix formation by these modified PNAs with RNA and DNA hairpins having a variable base pair in the middle of the helix were studied using isothermal titration calorimetry and compared with triple helix formation by non-modified PNAs. While unmodified PNA had low sequence selectivity against mismatched hairpins, introduction of one or two phenylalanine-derived monomers significantly increased the mismatch discrimination and sequence selectivity of the modified PNA. Consistent with our previous observations, PNA formed more stable triple helices with RNA than with DNA. Interestingly, the phenylalanine modification further improved the preference of PNA for RNA over DNA hairpin.

合成了由苯丙氨酸衍生的含有一个或两个胸腺嘧啶PNA单体的修饰肽核酸(PNA)。用等温滴定量热法研究了这些修饰的PNAs形成的三螺旋结构,其中RNA和DNA发夹在螺旋中间具有可变碱基对,并与未修饰的PNAs形成的三螺旋结构进行了比较。未修饰的PNA对错配发卡的序列选择性较低,但引入一两个苯丙氨酸衍生单体可显著提高修饰后的PNA对错配的辨别能力和序列选择性。与我们之前的观察一致,PNA与RNA形成比与DNA更稳定的三螺旋。有趣的是,苯丙氨酸修饰进一步提高了PNA对RNA的偏好,而不是DNA发夹。
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引用次数: 7
期刊
Artificial DNA: PNA & XNA
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