Login

Publications  •  Project Statistics

Glossary  •  Schools  •  Disciplines
People Search: 
   
Title/Abstract Search: 

Dissertation Information for Vanvimon Saksmerprome

NAME:
- Vanvimon Saksmerprome

DEGREE:
- Ph.D.

DISCIPLINE:
- Chemistry

SCHOOL:
- Indiana University (USA) (2005)

ADVISORS:
- Donald H Burke

COMMITTEE MEMBERS:
- James T. Drummond
- John Richardson
- Theodore S Widlanski

MPACT Status: Incomplete - Not_Inspected

Title: Kinetic analysis of natural and engineered hammerhead ribozymes

Abstract: The hammerhead ribozyme (hRz) is a small catalytic RNA motif that cleaves a specific phosphodiester bond, generating a terminal 5 ' hydroxyl and a 2 ' 3 ' -cyclic phosphate. Natural hRz's are found in satellite RNAs of some plant viroid pathogens and in the repetitive DNA of newts, cave crickets, and flukes. Since the active core, termed "minimal hRz," can be readily engineered into a trans -acting catalyst with programmable specificity, the ribozyme has become a popular tool in biology and medicine. In the TRAP design (t[barbelow]argeted r[barbelow]eversibly-a[barbelow]ttenuated p[barbelow]robes), two sequences are appended onto the hRz's catalytic core, generating an oligonucleotide-responsive ribozyme. Because cleavage activity of TRAP ribozyme results from formation of an active structure, monitoring cleavage kinetics sheds light on the parameters that affect RNA folding. Resulting kinetic and thermodynamic parameters favors a model in which regulation arises from equilibration between the active and inactive conformations of the TRAP, and that cleavage kinetics are primarily governed by the relative stabilities of the activating and attenuating helices. Probing conformational equilibrium of TRAPS as a function of pH and urea concentrations suggests the possibility that deprotonation of the 2 ' hydroxyl group serves a dual role of favoring productive folding and enhancing the nucleophilicity of this oxygen.

The hRz in the biological context contains tertiary stabilized motif (TSM) that enhance considerably the cleavage efficiency at sub-millimolar Mg 2+ concentrations, whereas the minimal hRz requires several millimolar MgCl 2 for optimal activity. Stabilizing hRz by in vitro selected TSM is observed to function nearly independent of the substrate sequences flanking TSM. This sequence flexibility enables the use of highly active hRz targeting substrate derived from the U5 genomic region of HIV-1 at physiological conditions. The hRz's can catalyze the ligation reaction through reversible transesterification. Investigations of the internal equilibrium of the ribozyme help to understand how a conformational switch between the cleavage and ligation activities is regulated in natural hRz.

MPACT Scores for Vanvimon Saksmerprome

A = 0
C = 0
A+C = 0
T = 0
G = 0
W = 0
TD = 0
TA = 0
calculated 2010-12-13 17:37:56

Advisors and Advisees Graph