这项工作得到了新加坡教育部 (教育部) 1 级 (RGT3/13 和 RG42/15 至 gc) 和教育部2级 (MOE2013-T2-2-024 和 MOE2015-T2-1-028 至 gc) 的支持。

基于此处所报告的工作的专利申请 (PAT/179/14/15/PCT) 已提交。

RNA 双结合肽寡聚物 (例如, 10-市场汇率) 是中型分子, 因此可以显示电泳移动性转移后, 与 rna 具有可比或稍大的大小 (如, 50-或更小)。如果 rna 的数量明显大于肽核酸, 那么由于凝胶迁移率的变化, 肽核酸在 rna 中的滴定可能不起作用。因此, 大的 RNA 可能被截断的变性页的化验。一个大的 RNA 的滴定到一个荧光标记的巴肽允许对三重形成的监视由变性琼脂糖凝胶与样品装载在中间的胶凝体40。
对于一个恒定的总 rna 浓度的变性页的滴定实验, 我们通常使用1µM 的无标记 rna 浓度的有效后染色的自由 rna 和三重带溴溴。rna 浓度低至0.2 µM 也可能是足够的取决于 rna 构造31。未标记 RNA (0.2 µM) 的浓度决定了可以精确测量的Kd值应该是大约0.2 µM 或更大。其他染色染料可用于提高染色效率。另外, 我们未公布的数据表明, Cy3 染料标记 rna 可用于变性页实验, 以测量紧密结合事件。
由于 2-嘌呤是仅适度萤光的事实, 2-嘌呤荧光滴定也被限制到绑定的测量与Kd值接近或高于0.2 µM31。RNA 或肽核酸可以用相对鲜艳的染料标记, 通过荧光滴定来量化溶液中相对紧密的结合, 如果该结合导致荧光信号的变化53,54,55。
针对 rna 结构的 dsRNA 结合 PNAs 的策略已经测试了有限数量的 rna。具有不同序列和基对组合的 dsRNAs 的绑定属性可能会有所不同。一个人可能总是选择 TFPNAs 的设计的双相嘌呤丰富的股。了解连续 Q·三重可能会影响三倍的稳定性。显然需要更广泛的序列依赖性研究来理解 TFPNAs 的序列依赖性结合特性。
通过增加长度和/或进一步修改基和主干5657的 TFPNAs, 可以进一步增强 TFPNAs 的绑定关联性。但是, 连续的双工区域可能不经常包含超过10连续的基对, 而不受非沃森-克里克结构的干扰。一个可以共轭 TFPNAs 与小分子, 以识别非沃森-克里克结构毗邻 dsRNA 地区。原则上, 一个 TFPNA-小分子共轭有望增强结合亲和力和特异性相比, 单 TFPNA 或小分子。但是, 链接器的化学和物理属性为共轭58,59,60,61,62,63,64必须优化。
事实上, TFPNAs 可以有选择地绑定到 dsRNAs 超过 ssRNAs 和 dsDNAs 表明, 它可以发展 TFPNAs 作为非常有用的化学探针和潜在的治疗配体通过调节 RNA 结构动力学和相互作用与蛋白质和代谢物。细胞摄取 TFPNAs 可通过与细胞穿透基, 如小分子, 多肽, 和纳米颗粒, 或与超分子结构的络合物, 如脂质体5,6 进行共轭. ,12,17,25,31,4165,66。进一步功能化的 TFPNAs 与 bioimaging 标签, 如荧光和放射性同位素可能有助于检测, 成像, 并在活生物体的 RNA 结构的目标。

rna 是重要的生物标记物和治疗靶点, 由于在不同的生物过程的调控和催化作用上的 rna 的发现的最近进展1,2,3。传统上, 反义股已被用来绑定到 ssRNAs 通过沃森-克里克复式编队 3,4,5,6,7,89,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 27. 最近, 三重形成的肽核酸 (TFPNAs) 被设计通过 Hoogsteen 氢键结合 (图 1)3,28的 dsRNAs。dsRNA 地区是存在于大多数传统反义的 rna, 包括 pre-mRNAs 和基因, 或 pri rna3, 和许多其他编码 rna1,26,27。以 TFPNAs 为目标的 dsRNAs 通过三重螺旋形成可能是有利的, 由于其结构的特殊性, 并具有巨大的潜力, 用于恢复正常功能的 rna, 这是失调在疾病, 例如。
最近发布的工作由 Rozners et al.和 us3、28、29、30、3132、33 34,35,36,373839,4041, 报告了改善改良 TFPNAs 对 dsRNAs 的选择性结合增强亲和性。我们已经开发了合理设计的肽单体 (图 2) 的合成方法, 包括硫代 pseudoisocytosine (L) 单体的30和胍改性的5甲基胞嘧啶 (Q) 单体31。通过各种生物化学和生物物理特性的方法, 我们已经证明, 相对短的 PNAs (6-10 残留) 纳入 L 和 Q 残留显示, 改善了沃森-克里克 g-c 和 c-g 基对, 分别在 dsRNAs。此外, 与未修饰的 PNAs 相比, PNAs 含 L 和 Q 残留物对 dsRNA ssRNA 和 dsDNA 具有更具选择性的约束力。在 Q 基地的胍功能42使 PNAs 进入 HeLa 细胞31。
在我们的实验室, 我们合成 PNAs 由手工中银化学 (boc 或t-boc 代表叔 butyloxycarbony (参见图 2) 固相多肽合成方法4。以 boc 为胺保护基团合成的肽基单体, 与 fluorenylmethyloxycarbonyl (芴甲氧羰基) 胺保护基团相比, 具有更大的阻, 在肽类单体偶联中可能有益坚实的支持。中银集团是酸不稳定的, 可以很容易地去除固体支持的20-50% 乙酸酸 (TFA) 在二氯甲烷 (DCM) 在肽的合成。一种可用于合成肽类寡聚物的自动缩氨酸合成物;然而, 在自动多肽合成器中, 需要3-5 倍的蛋白质肽单体。手动合成需要大大减少肽单体 (2-3 倍多余), 每个耦合容易监测的凯泽测试43。此外, 许多自动合成器不兼容的 boc 战略综合, 由于使用腐蚀性 TFA 在中行的去除步骤。
采用反相高效液相色谱 (RP-HPLC), 通过 MALDI 的分子量表征 (图 3和 4)30,可以纯化肽寡聚物。31. 我们使用变性页来监视三重形成, 由于自由 RNA 双工构造和肽结合 triplexes 经常显示不同的迁移率 (图 5)30,31.如果能实现两个 RNA 双工和 PNA·的高效 post-staining, 则不需要标记RNA2三重带。变性页实验需要一个相对较小的样本量。但是, 加载 (孵化) 缓冲器和运行缓冲 (ph 8.3) 可能不一样, 导致测量被限制在动力学稳定的 triplexes, 因为相对较高的 ph 值8.3 可能会极大地动摇一个三重。
2-嘌呤是腺嘌呤的异构体 (6-嘌呤);2-嘌呤荧光强度 (发射峰在大约 370 nm) 是敏感的局部结构环境的变化, 并适用于监测的三重形成与 2-嘌呤残留物附近的肽结合位点 (图 6)31. 与其他许多在可见范围内显示荧光发射的染料不同, 2-嘌呤标记的 RNA 可以暴露在室温下, 而不需要光漂白。不像页面实验, 其中一个运行的缓冲 ph 8.3 是经常需要的, 2 嘌呤的荧光滴定法允许测量的约束力在一个解决方案在指定的 ph 值, 从而可以允许测量和检测相对薄弱和动力学不稳定的束缚在平衡。
紫外吸收-检测热熔实验允许测量复式 (图 7) 的热稳定性31和三丛30,32,44,45。根据长度和序列组成, 熔化的 triplexes 可能或可能不会显示一个明确的过渡。如果加热和冷却曲线重叠, 就可以得到热力学参数。等温滴定量热法 (ITC)32可获得准确的热力学参数;但是, 贸易中心通常需要大量的样品。

<table style="border-collapse:collapse;width:491pt" width="654">
	<colgroup>
		<col style="width:176pt" width="235" />
		<col style="width:116pt" width="155" />
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			<td class="xl25" height="60" style="width:176pt;height:45.0pt" width="235">Molecular sieves, 4A, 1-2 mm diameter pellets&#160;</td>
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			<td class="xl74" height="60" style="width:132pt;height:45.0pt" width="176">87956</td>
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			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">4-Methylbenzhydrylamine hydrochloride (MBHA&#376;HCl)</td>
			<td class="xl30">Sigma-Aldrich</td>
			<td class="xl68" height="60" style="width:132pt;height:45.0pt" width="176">532444</td>
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			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">A11801</td>
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			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">(Benzotriazol-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)</td>
			<td class="xl30">Alfa Aesar</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">B25251</td>
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			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">Acetic anhyride</td>
			<td class="xl30">Sigma-Aldrich</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">320102</td>
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			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">Kaiser Test Kit&#160;</td>
			<td class="xl30">Sigma-Aldrich</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">60017</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">Trifluoroacetic acid (TFA)</td>
			<td class="xl30">Alfa Aesar</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">L06374</td>
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			<td class="xl36" style="width:116pt" width="155">ASM Research Chemicals GmbH</td>
			<td class="xl70" height="60" style="width:132pt;height:45.0pt" width="176">5004007, 5004008, 5004009, 5004010</td>
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			<td class="xl36" style="width:116pt" width="155">Sigma-Aldrich</td>
			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">B8389 / 47624</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Thioanisole</td>
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			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">A14846</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">1,2-Ethanedithiol</td>
			<td class="xl36" style="width:116pt" width="155">Alfa Aesar</td>
			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">L12865</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Trifluoromethanesulfonic acid</td>
			<td class="xl36" style="width:116pt" width="155">Alfa Aesar</td>
			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">A10173</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">LiChrosper&#174; 100 RP-18 endcapped (5 &#181;m) LiChroCART&#174; 250-4</td>
			<td class="xl41">Merck Millipore</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">150838</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">RNA Oligos&#160;</td>
			<td class="xl41">Sigma-Aldrich</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">Customized&#160;</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">&#945;-cyano-4-hydroxycinnamic acid (CHCA)</td>
			<td class="xl41">Sigma-Aldrich</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">39468</td>
			<td></td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td class="xl41">Alfa Aesar</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">J15694</td>
			<td></td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">HEPES&#160;</td>
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			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">17-737E</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Acrylamide</td>
			<td class="xl41">Sigma-Aldrich</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">A8887</td>
			<td></td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">N.N&#39;-methylenebisacrylamide</td>
			<td class="xl41">Sigma-Aldrich&#160;</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">146072</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Ammonium persulfate (APS)</td>
			<td class="xl41">Bio-rad</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">161-0700</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Tetramethylethylenediamine (TEMED)</td>
			<td class="xl41">Bio-rad</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">161-0800</td>
			<td></td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">10X Tris-Borate-EDTA (TBE) Buffer, pH 8.3</td>
			<td class="xl41">1st Base</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">BUF-3010-10X1L</td>
			<td></td>
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			<td class="xl44" height="60" style="width:176pt;height:45.0pt" width="235">Glycerol&#160;</td>
			<td class="xl45">Promega&#160;</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">H5433</td>
			<td></td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl44" height="60" style="width:176pt;height:45.0pt" width="235">Ethidium bromide (10 mg/mL)</td>
			<td class="xl45">Bio-rad&#160;</td>
			<td class="xl64" height="60" style="width:132pt;height:45.0pt" width="176">161-0433</td>
			<td></td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl48" height="60" style="width:176pt;height:45.0pt" width="235">High Precision Cell (Quartz Suprasil, 200-2500 nm)</td>
			<td class="xl49">Hellma Analytics&#160;</td>
			<td class="xl61" height="60" style="width:132pt;height:45.0pt" width="176">105.250-QS</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequence-specific and selective recognition of double-stranded rnas over single-stranded rnas by chemically modified peptide nucleic acids

rna 正逐渐成为重要的生物标志物和治疗靶点。因此, 在 RNA 序列和结构的识别方面, 开发化学探针和治疗配体具有很大的潜力。化学修饰的核酸 (肽) 寡聚物最近被开发, 可以识别 RNA 复式在序列特定的方式。PNAs 的化学稳定性与中性肽样骨干。PNAs 可通过人工合成的 Boc-化学固相肽综合法相对容易合成。采用反相高效液相色谱法纯化 PNAs, 然后通过基质辅助激光解吸/电离-飞行时间 (MALDI) 对分子量进行表征。非变性聚丙烯酰胺凝胶电泳 (PAGE) 技术促进了三重形成的成像, 因为精心设计的自由 RNA 复式结构和肽结合 triplexes 经常显示不同的迁移率。非变性页与溴溴后染色往往是一个简单的和信息技术, 以表征结合亲和力和特异性肽寡聚物。通常, 多个 RNA 发夹或复式单碱基对突变可用于表征肽结合性质, 如结合的亲和力和特殊性。2-嘌呤是腺嘌呤的异构体 (6-嘌呤);2-嘌呤荧光强度对局部结构环境变化敏感, 适用于在肽结合位点附近的 2-嘌呤残留物的三重形成监测。2-嘌呤荧光滴定法还可用于确认修饰 PNAs 对靶双 rna (dsRNAs) 的结合选择性 (单 rna)。紫外吸收-检测热熔实验允许测定肽-RNA 复式和 PNA·的热稳定性RNA2 triplexes。在这里, 我们描述了合成和纯化的肽寡聚体包含修饰残留物, 并描述了生物化学和生物物理方法的表征, RNA 复式的识别的修改 PNAs。

反相 HPLC 法可以纯化肽核酸寡聚物。我们可以得到纯肽寡聚物与两个回合的 HPLC 纯化 (图 3)。PNAs 的身份可以通过 MALDI-飞行时分析 (图 4) 进行确认。
非变性页是一种简单的信息技术, 用于表征肽寡聚物的结合亲和力和特异性。我们通常使用多个 RNA 发夹或复式单基对突变来表征绑定属性 (图 5)。图 5中显示的变性页数据清楚地表明, Q 和 L 修饰的肽可以识别一个具有 C-G 对的 dsRNA 区域 (图 5B, 底部面板) 但没有一个没有 C-G 对 (图 5B, 顶部面板)。这种特定的和增强的识别是通过 T·L·Q·PNA·RNA2基三 (图 1A, C, D) 形成。不同的 PNAs 有单个或多个突变, 也可以用来证明改良的肽的结合性能。我们已经表明, 在孵化缓冲中添加 2 mM Mg2 +不会影响绑定显着31。
我们已经证明了 2-嘌呤荧光滴定, 一个 Q 和 L 修饰的肽结合到一个目标的 dsRNA 区域 (图 6a, 6C, 6D), 但不是 ssRNA (图 6B, 6E, 6F)。肽 P3 绑定到2嘌呤标记的 dsRNA, 其Kd值为0.8 ±0.1 µM。370 nm 的 2-嘌呤标记 ssRNA 的荧光强度保持相对恒定, P3 浓度不同, 表明肽 P3 对 ssRNA 缺乏约束力。
含有 Q 残滓的 PNAs (P2 和 P3) 显示没有热熔转变 (图 7), 建议不绑定到 ssRNA。这是由于在沃森-克里克像 Q G 对的空间空间冲突。与未修饰的肽 P1、PNAs P4 和 P5 含有修正的 l 残留物, 但没有 Q 残基, 显示了相应的 RNA 肽复式的熔融温度下降, 由于在沃森-克里克像 l-G 对的空间位碰撞存在。紫外吸光度检测的热熔数据与 2-嘌呤荧光滴定数据一致, 这也表明含有 Q 和 L 残留物的肽基不明显地绑定到 ssRNA (图 6B, 6E, 6F)。合并 Q 基地比 L 基地更不稳定作为一个 q 基有一个更重要的空间空间冲突形成一个沃森-克里克样的 Q G 对 (图 1F) 相比, 沃森-克里克样的 l-g 对 (图 1E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56221/56221fig1v2.jpg"> 
图 1: 稳定基三和不稳定基对结构的化学结构.(A-D)主槽 PNA·RNA2基 T·的三元组U (a), C+·G-C (B), L·G-c (c) 和 Q·C-G (D)。(E, F)不稳定的沃森-克里克喜欢肽-RNA 基对的 l-g (E) 和 Q-g (F)。字母 R 代表 RNA 的糖磷酸盐中坚。氢键由黑色虚线表示。该图从引用31中复制。<a href="//ecsource.jove.com/files/ftp_upload/56221/56221fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56221/56221fig2v2.jpg"> 
图 2: 肽单体的化学结构.显示四肽单体 (T、C、L 和 Q)。<a href="//ecsource.jove.com/files/ftp_upload/56221/56221fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56221/56221fig3.jpg"> 
图 3: 肽寡聚物的化学结构和高效液相色谱法提纯.(A) 肽核酸序列 P3 的化学结构。(B, C)粗肽 P3 (B) 和 re-purified 肽 P3 (C) 的反相 HPLC 数据。<a href="//ecsource.jove.com/files/ftp_upload/56221/56221fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56221/56221fig4.jpg"> 
图 4:MALDI P3 的纯化肽谱.<a href="//ecsource.jove.com/files/ftp_upload/56221/56221fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56221/56221fig5.jpg"> 
图 5:RNA 发夹和肽核酸序列和绑定特性的变性页。(A) RNA 发夹 (rHP1 和 rHP2), 肽 P3, 和 PNA·RNA2三重形成于肽 P3 和 rHP2 之间。(B) 非变性页 (12%) 结果为 rHP1 和 rHP2 绑定到肽 P3。孵化缓冲液为200毫米氯化钠, 0.5 毫米 EDTA, 20 毫米 HEPES, pH 7.5。装载的 RNA 发夹 (rHP1 和 rHP2) 在1µM 在20µL。在车道从左到右的肽浓度是 0, 0.2, 0.4, 1, 1.6, 2, 4, 10, 16, 20, 28, 和50µM. 肽 P3 不绑定到 rHP1 (顶部面板), 但绑定到 rHP2 (底部面板)。(C) Kd确定 P3 绑定到 rHP2。三层组成 (Y) 的分数是针对肽的浓度而绘制的。该图是从引用31改编的。<a href="//ecsource.jove.com/files/ftp_upload/56221/56221fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56221/56221fig6.jpg"> 
图 6: 肽 P3 结合的荧光滴定研究2-嘌呤标记的 rna。在 RNA 序列中, 2-嘌呤残留物被指定为 "2"。孵化缓冲液为200毫米氯化钠, 0.5 毫米 EDTA, 20 毫米 HEPES, pH 7.5。(a) PNA·RNA2三重形成于 P3 和一个2嘌呤标记的 dsRNA (dsRNA2-2AP) 之间。(B) 在 P3 和2嘌呤标记的 ssRNA (ssRNA2-2AP) 之间形成的一种假想的肽-RNA 双工。(C, E)2-嘌呤标记的 RNA 双工 (1 µM) 和 ssRNA (1 µM) 的荧光发射谱, 分别为不同的 P3 浓度在 pH 7.5。峰值在大约 475 nm 是由于弱荧光发射的 L 基地在巴肽。(D, F)Kd基于 RNA 双工和 ssRNA 的 2-嘌呤荧光强度 (370 nm) 的地块, 分别与肽 P3 浓度的测定。该图是从引用31改编的。<a href="//ecsource.jove.com/files/ftp_upload/56221/56221fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56221/56221fig7.jpg"> 
图 7: RNA 肽复式的热熔化结果.孵化缓冲液为200毫米氯化钠, 0.5 毫米 EDTA, 20 mm NaH2PO4, pH 7.5。所有样本包含5µM 的单 RNA (ssRNA1) 和巴肽在130µL. (a) 单绞 rna (ssRNA1)、PNAs (P1、P2、P3、P4 和 P5) 以及在 P3 和 ssRNA1 之间以平行方向形成的假想的肽-rna 双工。对像 Q g 和 l-g 对的沃森-克里克的空间位场冲突进行了说明。(B) 为不同的 PNAs 绑定到 ssRNA1 的熔融曲线。熔化的温度在熔化的转折的曲线显示。该图是从引用31改编的。

Watch this Scientific Journal Video about Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids at JoVE.com

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

我们报告了合成和纯化多肽核酸 (巴氨酸) 齐聚物的协议。本文介绍了复式修饰 PNAs 识别 RNA 的生物化学和生物物理方法。

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

sequence-specific-selective-recognition-double-stranded-rnas-over

Research

JoVE Journal

Genetics

9.4K 次观看.  Nanyang Technological University. 我们报告了合成和纯化多肽核酸 (巴氨酸) 齐聚物的协议。本文介绍了复式修饰 PNAs 识别 RNA 的生物化学和生物物理方法。

视频: Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

科研

遗传学

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

原子力显微镜检查DNA损伤识别核苷酸切除修复

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

基于细胞的 sineup 非编码 rna 检测, 可专门增强 mrna 的翻译能力

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

使用基因激活 crispr 过度表达长非编码 rna

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

高密度DNA和RNA微阵列.光刻合成、杂交和制备大型核酸库

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...