Suitability of double-stranded DNA as a molecular standard for the validation of analytical ultracentrifugation instruments
Suitability of double-stranded DNA as a molecular standard for the validation of analytical ultracentrifugation instruments
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双链 DNA 作为分子标准品用于验证分析超速离心仪器的适用性
DOI：
10.1007/s00249-023-01671-y
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Demeler, Borries
中科院分区：
文献类型：
--
作者：
Ranasinghe, Maduni;Fogg, Jonathan M.;Catanese, Daniel J.;Zechiedrich, Lynn;Demeler, Borries
为了解决目前缺乏经验证的分析超离心（AUC）分子标准品的问题，我们研究了双链DNA分子的适用性。我们比较了线性和环状DNA的流体动力学性质作为温度的函数。研究了负超螺旋、切口和线性化的333和339 bp微环。我们量化了这些DNA在5个不同温度下的流体动力学性质，范围从4 ° C到37 °C。为了提高我们测量的精度，每个样品在三个重复和五个转子速度上进行全局拟合。DNA的特殊稳定性允许每个样品在几个月的时间内重复沉淀，而不会发生聚集或降解，并且具有优异的重现性。线性化和切口的小环DNA的沉降和扩散系数表现出高度均匀的样品，并随温度增加，表明摩擦减少。线性DNA的沉降速度最慢，超螺旋DNA的沉降速度最快。随着温度的升高，超螺旋样品转移到较慢的沉降，但沉降速度比刻痕微环。这些结果表明，负超螺旋DNA在较高温度下变得不那么紧凑。从细菌中纯化的超螺旋微环显示出异质性。因此，从细菌中分离的超螺旋DNA不适合作为分子标准。线性和切口样品非常适合作为AUC的分子标准品，并且在AUC池中具有出色的胶体稳定性。即使在4个月的时间里，在不同速度和温度下进行了60次实验，DNA的所有拓扑状态仍然保持胶体状态，它们的浓度基本保持不变。
To address the current lack of validated molecular standards for analytical ultracentrifugation (AUC), we investigated the suitability of double-stranded DNA molecules. We compared the hydrodynamic properties of linear and circular DNA as a function of temperature. Negatively supercoiled, nicked, and linearized 333 and 339 bp minicircles were studied. We quantified the hydrodynamic properties of these DNAs at five different temperatures, ranging from 4 to 37 °C. To enhance the precision of our measurements, each sample was globally fitted over triplicates and five rotor speeds. The exceptional stability of DNA allowed each sample to be sedimented repeatedly over the course of several months without aggregation or degradation, and with excellent reproducibility. The sedimentation and diffusion coefficients of linearized and nicked minicircle DNA demonstrated a highly homogeneous sample, and increased with temperature, indicating a decrease in friction. The sedimentation of linearized DNA was the slowest; supercoiled DNA sedimented the fastest. With increasing temperature, the supercoiled samples shifted to slower sedimentation, but sedimented faster than nicked minicircles. These results suggest that negatively supercoiled DNA becomes less compact at higher temperatures. The supercoiled minicircles, as purified from bacteria, displayed heterogeneity. Therefore, supercoiled DNA isolated from bacteria is unsuitable as a molecular standard. Linear and nicked samples are well suited as a molecular standard for AUC and have exceptional colloidal stability in an AUC cell. Even after sixty experiments at different speeds and temperatures, measured over the course of 4 months, all topological states of DNA remained colloidal, and their concentrations remained essentially unchanged.
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影响因子：
16.6
作者：
Irobalieva RN;Fogg JM;Catanese DJ Jr;Sutthibutpong T;Chen M;Barker AK;Ludtke SJ;Harris SA;Schmid MF;Chiu W;Zechiedrich L
通讯作者：
Zechiedrich L
影响因子：
2.4
作者：
Demeler, Borries;Brookes, Emre
通讯作者：
Brookes, Emre
凝胶电泳中超螺旋 DNA 的特性。
影响因子：
5.6
作者：
Y. Zivanovic;I. Goulet;A. Prunell
通讯作者：
A. Prunell
影响因子：
2.9
作者：
HANSEN, JC;LEBOWITZ, J;DEMELER, B
通讯作者：
DEMELER, B
影响因子：
14.9
作者：
Brunet A;Salomé L;Rousseau P;Destainville N;Manghi M;Tardin C
通讯作者：
Tardin C