Determination of Charge-Structure Relationships for DNA

电泳是分子生物学中最广泛使用的技术之一，然而，对电泳进行彻底和正确的理论处理已被证明是困难的。这项研究与格鲁吉亚州立大学的Stuart Allison博士合作，将实验测量与最近开发的边界元建模方法相结合，旨在开发适用于水溶剂中小聚离子的理论。将在一系列溶剂中检查规定大小、电荷和电荷密度的DNA寡核苷酸。电泳迁移率和有效电荷将通过膜限制分析电泳来确定。将使用分析超离心法测定溶液质量以及沉降、摩擦和病毒系数。有效电荷将在具有不同离子强度、盐类型和盐价态的溶剂中在宽范围的DNA长度上测量。结构-电荷关系也将使用具有通过甲基膦酸酯取代选择性改变的形式电荷密度的寡核苷酸来探索。这允许检查具有相同形式电荷但不同电荷密度分布的DNA的有效电荷和流体力学。最后，将检查具有确定结构的核糖体RNA片段，以表征推定的二价金属离子结合位点。为了补充核酸工作，牛核糖核酸酶的迁移率和有效电荷将在一定范围的pH值和盐浓度下测定。结构数据表明，核糖核酸酶可能有一个组氨酸依赖的阴离子结合位点。P.I.的初步迁移率数据表明阴离子是松散结合的。实验将确定离子的解离常数，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。两者合计，实验数据和理论分析的结合，将允许一个广泛适用的电泳理论的发展。此外，本研究的数据将定义通过稳态电泳测量的有效电荷与分子电荷的其他测量之间的实验关系。电泳是生物化学、分子生物学和分子遗传学中应用最广泛的方法之一。例如，该技术是人类基因组计划DNA测序方法的基础。尽管电泳的广泛使用和根本的重要性，在上个世纪，科学家们一直没有找到一个全面的电泳理论。本研究旨在通过实验分析合作实验室开发的模型，以完善对电泳的理解。它将为合理操纵生物分子上的电荷奠定基础，从而开发出更好的药物，农业和聚合物试剂。

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.