Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target
Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2002.
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | eng |
| Published: |
Massachusetts Institute of Technology
2008
|
| Subjects: | |
| Online Access: | http://hdl.handle.net/1721.1/43615 |
| _version_ | 1826210643225608192 |
|---|---|
| author | Walton, S. Patrick (Stephen Patrick), 1973- |
| author2 | Martin Y. Yarmush and Gregory N. Stephanopoulos. |
| author_facet | Martin Y. Yarmush and Gregory N. Stephanopoulos. Walton, S. Patrick (Stephen Patrick), 1973- |
| author_sort | Walton, S. Patrick (Stephen Patrick), 1973- |
| collection | MIT |
| description | Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2002. |
| first_indexed | 2024-09-23T14:52:54Z |
| format | Thesis |
| id | mit-1721.1/43615 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T14:52:54Z |
| publishDate | 2008 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/436152019-04-10T23:27:01Z Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target Walton, S. Patrick (Stephen Patrick), 1973- Martin Y. Yarmush and Gregory N. Stephanopoulos. Massachusetts Institute of Technology. Dept. of Chemical Engineering. Massachusetts Institute of Technology. Dept. of Chemical Engineering. Chemical Engineering. Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2002. Includes bibliographical references (p. 165-178). Antisense oligonucleotides have the potential to selectively inhibit the expression of any gene with a known sequence. Antisense-based therapies are under development for the treatment of infectious diseases as well as complex genetic disorders. Although there have been some remarkable successes, realizing this potential is proving difficult because of problems with oligonucleotide stability, specificity, affinity, and delivery. Each of these limitations has been addressed experimentally through the use of chemically-modified oligonucleotides and oligonucleotide conjugates, with much success in enhancing oligonucleotide efficacy. These early studies have shown that selection of target site, once considered a trivial problem, is critical to the success of antisense strategies. It has become clear that the efficacy of antisense oligonucleotides is a strong function of the structure of the target mRNA. Though single-stranded, RNA molecules are typically folded into complex three-dimensional structures, formed primarily by intramolecular Watson-Crick base-pairing. If an oligonucleotide is complementary to a sequence embedded in the three dimensional structure, the oligonucleotide may not be able to bind to its target site and exert its therapeutic effect. Because the majority of the structure of RNA molecules is due to Watson-Crick base-pairing, relatively accurate predictions of these folding interactions can be made from algorithms that locate the structure with the most favorable free energy of folding. (cont.) Taking advantage of the predictability of RNA structures, this thesis addresses the problem of antisense target site selection, first from a theoretical and subsequently an experimental standpoint. A thermodynamic model to predict the binding affinity of oligonucleotides for their target mRNA is described and validated using multiple in vitro and cell-culture based experimental data sets. Subsequently, direct experimental comparisons with theoretical predictions are made on the well-characterized rabbit-[beta]-globin (RBG) mRNA, using a novel, centrifugal, binding affinity assay. The importance of the hybridization kinetics is also explored, as is the role of association kinetics in defining the rate of cleavage by the enzyme ribonuclease H (RNase H). Finally, the applicability of the model in identifying biologically active oligonucleotides is demonstrated. by S. Patrick Walton. Sc.D. 2008-11-07T20:18:15Z 2008-11-07T20:18:15Z 2001 2002 Thesis http://hdl.handle.net/1721.1/43615 50406833 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 178 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemical Engineering. Walton, S. Patrick (Stephen Patrick), 1973- Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target |
| title | Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target |
| title_full | Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target |
| title_fullStr | Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target |
| title_full_unstemmed | Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target |
| title_short | Thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mRNA target |
| title_sort | thermodynamics and kinetics of antisense oligonucleotide hybridization to a structured mrna target |
| topic | Chemical Engineering. |
| url | http://hdl.handle.net/1721.1/43615 |
| work_keys_str_mv | AT waltonspatrickstephenpatrick1973 thermodynamicsandkineticsofantisenseoligonucleotidehybridizationtoastructuredmrnatarget |