Name: sequence-specific and selective recognition of double-stranded rnas over single-stranded rnas by chemically modified peptide nucleic acids
Uploaded: 2017-09-21T14:00:00.000+00:00
Duration: 9 min 4 s
Description: 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

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><tbody><tr><td>Molecular sieves, 4A, 1-2 mm diameter pellets&amp;nbsp;</td><td>Alfa Aesar</td><td>87956</td><td></td></tr><tr><td>4-Methylbenzhydrylamine hydrochloride (MBHA&lt;span class=&quot;font6&quot;&gt;&amp;Yuml;&lt;/span&gt;&lt;span class=&quot;font0&quot;&gt;HCl)&lt;/span&gt;</td><td>Sigma-Aldrich</td><td>532444</td><td></td></tr><tr><td>N,N-diisopropylethylamine (DIPEA)</td><td>Alfa Aesar</td><td>A11801</td><td></td></tr><tr><td>(Benzotriazol-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)</td><td>Alfa Aesar</td><td>B25251</td><td></td></tr><tr><td>Acetic anhyride</td><td>Sigma-Aldrich</td><td>320102</td><td></td></tr><tr><td>Kaiser Test Kit&amp;nbsp;</td><td>Sigma-Aldrich</td><td>60017</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Alfa Aesar</td><td>L06374</td><td></td></tr><tr><td>Unmodified PNA monomers</td><td>ASM Research Chemicals GmbH</td><td>5004007, 5004008, 5004009, 5004010</td><td></td></tr><tr><td>Boc-Lys(Z)-OH / Fmoc-Lys(Boc)-OH</td><td>Sigma-Aldrich</td><td>B8389 / 47624</td><td></td></tr><tr><td>Thioanisole</td><td>Alfa Aesar</td><td>A14846</td><td></td></tr><tr><td>1,2-Ethanedithiol</td><td>Alfa Aesar</td><td>L12865</td><td></td></tr><tr><td>Trifluoromethanesulfonic acid</td><td>Alfa Aesar</td><td>A10173</td><td></td></tr><tr><td>LiChrosper&amp;reg; 100 RP-18 endcapped (5 &amp;micro;m) LiChroCART&amp;reg; 250-4</td><td>Merck Millipore</td><td>150838</td><td></td></tr><tr><td>RNA Oligos&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Customized&amp;nbsp;</td><td></td></tr><tr><td>&amp;alpha;-cyano-4-hydroxycinnamic acid (CHCA)</td><td>Sigma-Aldrich</td><td>39468</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Alfa Aesar</td><td>J15694</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Lonza</td><td>17-737E</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A8887</td><td></td></tr><tr><td>N.N&#039;-methylenebisacrylamide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>146072</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Bio-rad</td><td>161-0700</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Bio-rad</td><td>161-0800</td><td></td></tr><tr><td>10X Tris-Borate-EDTA (TBE) Buffer, pH 8.3</td><td>1st Base</td><td>BUF-3010-10X1L</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>H5433</td><td></td></tr><tr><td>Ethidium bromide (10 mg/mL)</td><td>Bio-rad&amp;nbsp;</td><td>161-0433</td><td></td></tr><tr><td>High Precision Cell (Quartz Suprasil, 200-2500 nm)</td><td>Hellma Analytics&amp;nbsp;</td><td>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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

遗传学

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

核苷三磷酸 - 从合成到生化特征

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

化学

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

探索序列空间,识别调节RNA结合蛋白的结合位点

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

免疫与感染

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5&#39;-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

使用MALDI-TOF质谱法鉴定酶降解产生的RNA片段

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

<meta charset="utf8"></head><body>用于鉴别性 RNA 和 DNA 分析的精密 DNAzyme 纳米机器

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

<meta charset="utf8"></head><body>作者聚焦:DNA 纳米传感器的进步 – 解决分子检测中的灵敏度和选择性挑战

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

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

摘要

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此视频中的章节

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

的快速方法新颖的RNA结合蛋白隔离

核苷三磷酸 - 从合成到生化特征

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

探索序列空间,识别调节RNA结合蛋白的结合位点

肽为基础的识别功能图案和具有约束力的合作伙伴

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

使用MALDI-TOF质谱法鉴定酶降解产生的RNA片段

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme 10-23 - 用于核酸识别的基于纳米机器