Determination of Charge-Structure Relationships for DNA

Laue9807550电泳法是分子生物学中应用最广泛的技术之一，然而，对电泳法的彻底和正确的理论处理一直是难以捉摸的。这项研究与佐治亚州立大学的斯图尔特·艾利森博士合作，将实验测量与最近开发的边界元素建模方法相结合，旨在开发适用于水溶液中小多离子的理论。确定大小、电荷和电荷密度的DNA寡核苷酸将在一系列溶剂中进行检查。电泳迁移率和有效电荷将由膜受限分析电泳法测定。分析性超速离心法将用于测定溶液质量，以及沉降值、摩擦系数和病毒系数。在不同离子强度、盐类型和盐价的溶剂中，有效电荷将在广泛的DNA长度范围内测量。结构-电荷关系也将使用具有形式电荷密度的寡核苷酸来探索，这种电荷密度可以通过甲基膦取代选择性地改变。这使得可以检查具有相同形式电荷但不同电荷密度分布的DNA的有效电荷和流体动力学。最后，将检查确定结构的核糖体RNA片段，以表征推测的二价金属离子结合位点。为了补充核酸工作，牛核糖核酸酶的流动性和有效电荷将在一定的pH和盐浓度范围内测定。结构数据表明，核糖核酸酶可能具有组氨酸依赖的阴离子结合部位。P.I.S的初步迁移率数据的模拟表明，阴离子是松散结合的。实验将确定离子的离解常数，确定组氨酸在阴离子结合中的作用，并确定可逆离子结合对电泳迁移率的影响。综上所述，实验数据和理论分析的结合将使广泛适用于电泳的理论得以发展。此外，这项研究的数据将确定用稳态电泳法测量的有效电荷与其他分子电荷测量之间的实验关系。总之，这项工作将为生物化学中有效电荷的常规测定和操作奠定基础。电泳法是生物化学、分子生物学和分子遗传学中应用最广泛的方法之一。例如，这项技术是人类基因组计划DNA测序方法的基础。尽管它得到了广泛的应用和基本的重要性，但在过去的一个世纪里，科学家们一直没有找到一个全面的电泳学理论。这项研究是为了对合作实验室中开发的模型进行实验分析，以完善对电泳法的理解。这将为合理地操纵生物分子上的电荷，从而开发出更好的医药、农业和聚合物制剂奠定基础。

Laue9807550Electrophoresis is one of the most widely-used techniques in molecular biology, however a thorough and correct theoretical treatment of electrophoresis has proven elusive. This study, in collaboration with Dr. Stuart Allison at Georgia State University, combines experimental measurements with recently developed boundary-element modeling methods aiming at the development of a theory applicable to small polyions in aqueous solvents. DNA oligonucleotides of defined size, charge and charge density will be examined in a range of solvents. Electrophoretic mobility and effective charge will be determined by membrane-confined analytical electrophoresis. Analytical ultracentrifugation will be used to determine solution mass, as well as sedimentation, frictional and viral coefficients. The effective charge will be measured over a wide range of DNA lengths in solvents with varying ionic strengths, salt types and salt valences. Structure-charge relationships also will be explored using oligonucleotides that have formal charge densities that are selectively altered by methylphosphonate substitutions. This allows the examination of the effective charge and hydrodynamics of DNA having the same formal charge but different distributions of charge density. Finally, a ribosomal RNA fragment of defined structure will be examined in order to characterize putative divalent metal ion binding sites. To complement the nucleic acid work, the mobility and effective charge of bovine ribonuclease will be determined over a range of pH and salt concentrations. Structural data indicate that ribonuclease may have a histidine-dependent anion binding site. The modeling of P.I.'s preliminary mobility data indicate that the anion is loosely bound. Experiments will determine the dissociation constant for the ion, define the role of the histidine in anion binding and determine the effects of reversible ion binding on electrophoretic mobility. Taken together, the combination of experimental data and theoretical analysis will allow the development of a broadly applicable theory for electrophoresis. Furthermore, data from this study will define the experimental relationship between the effective charge measured by steady state electrophoresis and other measures of molecular charge. Overall, this work will lay the foundation for the routine determination and manipulation of the effective charge in biochemistry.Electrophoresis is amongst the most widely used methods in biochemistry, molecular biology and molecular genetics. For example, the technique is at the foundation of the methods used in sequencing DNA for the human genome project. Despite its wide-spread use and fundamental importance, a comprehensive theory for electrophoresis has eluded scientists for the last century. This study is to experimentally analyze models developed in a collaborating laboratory in order to refine the understanding of electrophoresis. It will lay the foundation for the rational manipulation of charge on biomolecules so that better pharmaceutical, agricultural and polymer agents can be developed.