Fluorescence correlation spectroscopy measurements of DNA unwinding/bending fluctuations with DNA backbone-incorporated dyes
Fluorescence correlation spectroscopy measurements of DNA unwinding/bending fluctuations with DNA backbone-incorporated dyes
复制标题
使用 DNA 主链掺入染料进行 DNA 解旋/弯曲波动的荧光相关光谱测量
DOI：
10.1016/j.bpj.2023.11.559
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发表时间：
2024
影响因子：
3.4
通讯作者：
Ansari, Anjum
中科院分区：
文献类型：
--
作者：
Ten, Timour;Zvoda, Viktoriya;Baral, Saroj;Ansari, Anjum
星期日，2024年2月11日81a和动态与FRET标签连接到DNA与接头。然而，归因于FRET的变化，DNA构象的变化可能是复杂的，因为在分离DNA动力学连接器/染料动力学的困难。最近，我们成功地测量了用位于3个碱基对错配位点任一侧的FRET对ATTO 550-ATTO 647 N标记的DNA寡聚体中的解旋/弯曲波动（Ten et al.（2022），J.Biol.Phys.48：253 - 272）。平衡FRET测量显示，较高的FRET在错配与匹配的DNA相比，表明在错配的存在下，更弯曲/解绕构象。荧光相关光谱（FCS）揭示错配DNA上的$100 - 300 ms动态，而匹配DNA没有检测到动态。然而，对这些构建体的荧光寿命测量显示出多个FRET状态，即使对于匹配的DNA，我们将其归因于这些带正电荷的ATTO染料对DNA堆叠/解堆叠的可能性。我们接下来尝试用带负电荷的ATTO 532代替ATTO 550作为供体，以促进更有利于所连接的染料自由旋转的条件。令人惊讶的是，使用这种染料对，我们失去了检测匹配和错配DNA之间任何构象差异的能力，并且使用这种染料对的FCS在两种构建体上都没有显示出动力学。在这里，我们探索FRET/FCS测量使用Cy3-Cy5对纳入DNA骨架。这些刚性堆叠的染料显示出降低FRET中的不确定性，因为DNA分子内的固定取向（Ranjit et al.（2009），J. Phys. Chem. B. 113：7861 - 7866）。因此，我们预计他们使用FCS.423-Pos报告DNA构象动力学的能力具有更高的灵敏度固定离心力显微镜上的桶角Jai Dreher、Audrey Orlowski、阿什利卡特。美国马萨诸塞州阿默斯特市阿默斯特学院物理系。鱼精蛋白质在精子细胞内显著折叠DNA。我们想用离心力显微镜（CFM）观察鱼精蛋白对DNA的体外折叠和去折叠。CFM利用离心机的离心力展开DNA。力的大小取决于离心机中铲斗的角度。在这里，我们开始设计和制造一个机加工部件，以恒定的45度角将铲斗固定在CFM中。考虑到旋转力，以及同心对称和平衡，我们设计并建造了一个环，以保持水桶在所需的角度。最终，我们能够在Solidworks中设计模型，用塑料3D打印原型，测试原型，并开始加工零件。以恒定的角度保持桶，将在CFM中提供恒定的力，并将允许我们测量由鱼精蛋白浓缩的DNA的解折叠。
Sunday, February 11, 2024 81a and dynamics with FRET labels attached to the DNA with linkers. However, attributing changes in FRET to changes in DNA conformations can be complicated because of the difficulty in separating DNA dynamics from linker/dye dynamics. Recently, we successfully measured unwinding/bending fluctuations in DNA oligomers labeled with the FRET pair ATTO550-ATTO647N placed on either side of a 3 base-pair mismatched site (Ten et al.(2022), J. Biol. Phys. 48: 253-272). Equilibrium FRET measurements showed higher FRET in mismatched compared with matched DNA, indicating a more bent/unwound conformation in the presence of the mismatch. Fluorescence correlation spectroscopy (FCS) revealed $100-300 ms dynamics on mismatched DNA with no dynamics detected for matched DNA. However, fluorescence lifetime measurements on these constructs showed multiple FRET states even for the matched DNA, which we attributed to the likelihood of these positively charged ATTO dyes stacking/unstacking against the DNA. We next tried replacing ATTO550 by the negatively charged ATTO532 as the donor to promote conditions more conducive to free rotation of the attached dye. Surprisingly, with this dye pair we lost the ability to detect any conformational differences between matched and mismatched DNA, and FCS with this pair showed no dynamics on either construct. Here, we explore FRET/FCS measurements using Cy3-Cy5 pair incorporated within the DNA backbone. These rigidly stacked dyes were shown to reduce uncertainty in FRET because of fixed orientation within the DNA molecule (Ranjit et al.(2009), J. Phys. Chem. B. 113: 7861–7866). Accordingly, we anticipate higher sensitivity in their ability to report on DNA conformational dynamics with FCS.423-Pos Fixing the bucket angle on a centrifugal force microscope Jai Dreher, Audrey Orlowski, Ashley Carter. Department of Physics, Amherst College, Amherst, MA, USA. Protamine proteins dramatically fold DNA within sperm cells. We want to use a centrifugal force microscope (CFM) to view in vitro folding and unfolding of DNA by protamine. A CFM uses the centrifugal force from a centrifuge to unfold the DNA. The amount of force depends on the angle of the bucket in the centrifuge. Here, we set out to design and build a machined part that holds the buckets in the CFM at a constant 45-degree angle. Taking into account rotational forces, as well as concentric symmetry and balance, we designed and built a ring to hold the buckets at the desired angle. Ultimately, we were able to design a model in Solidworks, 3D print a prototype in plastic, test the prototype, and begin work on the machined part. Holding the buckets at a constant angle, will provide a constant force in the CFM and will allow us to measure the unfolding of DNA condensed by protamine.