. Synthetic RNAs discovered via in vitro selection experiments have wide-ranging applications in biomedical sciences and biotechnology, including therapeutic aptamers that inhibit protein function and ribozymes that control gene expression. The RNA in vitro selection process, however, lacks systematic computational analysis to potentially increase the probability of discovering complex synthetic RNAs. To fill this significant gap, we have developed computational approaches for designing structured RNA pools and optimizing RNA functions to improve the productivity of in vitro selection and directed evolution experiments. In vitro selection experiments and our computational analysis suggest that designed RNA pools possessing diverse structural motifs can enhance discovery of complex RNA motifs which are rarely found in random pools. This is the main hypothesis we aim to demonstrate and apply in this proposal. To address current limitations and develop computational in vitro selection, we aim to develop:
(Aim A) computational approaches to structured RNA pool design;(B) methods for screening and testing designed RNA pools;
and (Aim C) a computational approach for simulating directed evolution for optimizing RNA functions.
In Aim A, we will develop structured pool design approaches that allow generation of user-defined target structures with constant binding or catalytic motifs using a Monte Carlo simulation method.
In Aim B, we will computationally test the performance of designed pools using motif scanning and screening (e.g., PI's SVD/TNPACK software tools) methods. In addition, we will experimentally verify that designed pools are superior to random pools via a collaboration with Luc Jaeger, an expert on RNA in vitro selection and nanotechnology.
In Aim C, we will develop a computational approach to in vitro evolution by combining the motif scanning/screening methods, the nucleotide transition ("mixing") matrix approach, and the partial least squares method for accumulating beneficial mutations to model RNA motif selection and mutagenic PCR procedures;optimized RNA candidates from our computational in vitro evolution scheme will be experimentally tested by Jaeger's lab. With these algorithmic developments and experimental collaboration, we expect that our computational approaches to pool design and analysis and directed evolution will provide a comprehensive resource to assist experimentalists in designing better in vitro selection experiments and optimizing RNA functions for demanding biomedical applications such as discovering high-binding affinity aptamers targeting proteins in cancer and other diseases. Our project also provides continued excellent interdisciplinary training of students in computational biology, chemistry mathematics, and biomedicine.

Public Health Relevance

Project Narrative Synthetic RNAs discovered via in vitro selection and directed evolution experiments have wide-ranging biological and biomedical applications, including therapeutic RNAs that modulate disease-related proteins. Our work is based on the hypothesis that designed structured RNA libraries are better than random pools for discovering complex RNAs. Our project will develop and test computational approaches for designing structured RNA pools and enhancing directed evolution to assist experimentalists in discovering complex RNAs. We will interact with experimental biomedical researchers to exploit and extend our methods'capabilities for advancing the development of molecular tools for biomedical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081410-04
Application #
8327196
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Lyster, Peter
Project Start
2009-09-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
4
Fiscal Year
2012
Total Cost
$312,654
Indirect Cost
$104,383
Name
New York University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041968306
City
New York
State
NY
Country
United States
Zip Code
10012
Jung, Segun; Schlick, Tamar (2014) Interconversion between parallel and antiparallel conformations of a 4H RNA junction in domain 3 of foot-and-mouth disease virus IRES captured by dynamics simulations. Biophys J 106:447-58
Kim, Namhee; Zheng, Zhe; Elmetwaly, Shereef et al. (2014) RNA graph partitioning for the discovery of RNA modularity: a novel application of graph partition algorithm to biology. PLoS One 9:e106074
Kim, Namhee; Laing, Christian; Elmetwaly, Shereef et al. (2014) Graph-based sampling for approximating global helical topologies of RNA. Proc Natl Acad Sci U S A 111:4079-84
Jung, Segun; Schlick, Tamar (2013) Candidate RNA structures for domain 3 of the foot-and-mouth-disease virus internal ribosome entry site. Nucleic Acids Res 41:1483-95
Laing, Christian; Jung, Segun; Kim, Namhee et al. (2013) Predicting helical topologies in RNA junctions as tree graphs. PLoS One 8:e71947
Laing, Christian; Wen, Dongrong; Wang, Jason T L et al. (2012) Predicting coaxial helical stacking in RNA junctions. Nucleic Acids Res 40:487-98
Quarta, Giulio; Sin, Ken; Schlick, Tamar (2012) Dynamic energy landscapes of riboswitches help interpret conformational rearrangements and function. PLoS Comput Biol 8:e1002368
Izzo, Joseph A; Kim, Namhee; Elmetwaly, Shereef et al. (2011) RAG: an update to the RNA-As-Graphs resource. BMC Bioinformatics 12:219
Laing, Christian; Schlick, Tamar (2011) Computational approaches to RNA structure prediction, analysis, and design. Curr Opin Struct Biol 21:306-18
Kim, Namhee; Izzo, Joseph A; Elmetwaly, Shereef et al. (2010) Computational generation and screening of RNA motifs in large nucleotide sequence pools. Nucleic Acids Res 38:e139

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