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Hydration of r(UGGGGU) Quadruplexes


For about 50 years it has been known that guanine-rich sequence can, under appropriate

conditions, adopt a distinctive, four-stranded, helical fold known as a G-quadruplex.

Interest in quadruplex folds has grown in recent years as evidence of their biological

relevance has accumulated from both sequence analysis and function-specific assays.

The folds are unusually stable and their formation appears to require close management

to maintain cell health; regulatory failure correlates with genomic instability and a

number of cancer phenotypes.

This thesis examines, by x-ray crystallography, the solvent structure of a pre-

viously reported tetramolecular RNA quadruplex, UGGGGU stabilized by Sr 2+ ions.

Crystal forms of the octameric assembly formed by this sequence exhibit unusually

strong diffraction and anomalous signal enabling the construction of reliable models to

a resolution of 0.85A. The solvent structure confirms hydration patterns reported for

other nucleic acid helical conformations and provides support for the greater stability

of RNA quadruplexes relative to DNA. Novel features detected in the octameric RNA

assembly include a new crystal form and evidence of multiple conformations, among

which one leading to the formation of a well-hydrated internal cavity.

Though solvent is generally acknowledged to play a fundamental role in nucleic

acid structure, its characterization from diffraction data remains challenging. To assist

with this task, the thesis investigates two novel additions to the crystallographic meth-

ods arsenal. The first is segmentation of electron density maps into Morse-Smale basins

characterized by uniform gradient flow. The second is the use of level set Fast Marching

methods to compute the full distance field defined by the molecular surface. Both tech-

niques show promise, though additional work will be required to yield effective tools.

Gradient-flow segmentation provides an unambiguous way to gather all map density as-

sociated with a modeled atom and enables the calculation of novel volumetric properties

including total basin density. Distance field calculation provides a unified framework for

combining molecular surface calculation with surface-related queries including pocket

and cavity detection and solvent travel depth.

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