The goal of the proposed program is to design and synthesize new fluorescent nucleoside analogs and implement them as probes for nucleic acids structure, dynamics and recognition. Advancing effective fluorescence-based tools for exploring nucleic acids and their interactions with ligands and potential therapeutic agents will further new diagnostic approaches and will facilitate drug discovery.
The specific aims of this project are:
AIM 1. To design, synthesize and incorporate new isomorphic fluorescent nucleoside analogs. The main design criteria include: (i) High structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties, (ii) Re shifted absorption spectrum to minimize overlap with the absorption of the natural bases, and (iii) Adequate emission quantum efficiency and long emission wavelengths (preferably in the visible range). Efficient synthetic pathways will be devised, providing the nucleosides and the necessary building blocks for automated and enzymatic oligonucleotide synthesis.
AIM 2. To photophysically and biophysically characterize the modified nucleosides and oligonucleotides. The photophysical characteristics (e.g., absorption and emission maxima, quantum yield and brightness, excited state lifetime, as well as susceptibility to environmental polarity and static and dynamic quenching by native nucleosides) will be rigorously evaluated and interpreted.
AIM 3. To implement the promising emissive analogs in biophysical and discovery assays. These assays will facilitate: (i) The discovery of new antibiotics targeting the bacterial ribosome (ii) The study of RNA helicases, ubiquitous motor proteins, which are involved in nearly every aspect of RNA metabolism, (iii) The monitoring of programmed ribosomal frameshifting, a processes which could be extremely detrimental to native protein synthesis, but, when programmed (e.g., in viruses) can maximize protein expression, (iv) The study of RNA deamination, an important posttranscriptional process, which diversifies mRNAs and the resultant proteins;it is linked to proper brain function and, when defective, to disease, and (v) The study of RNAi, a regulatory process induced by short interfering RNA (siRNA), which is also a powerful tool capable of altering cellular phenotypes, deciphering genetic pathways and identifying new therapeutic targets. Nucleic acids play central roles in cellular events and, as such, have immense impact on the emergence of diseases and, in turn, on human health. This necessitates the development of new effective tools for studying their recognition properties and alteration by exogenous agents. The emissive nucleoside analogs designed and prepared will be implemented in novel real time fluorescence-based assays. These investigations will further the fundamental understanding of key biological processes related to disease development and will have long-term impact on improving human health by advancing knowledge and facilitating drug discovery.

Public Health Relevance

Nucleic acids (DNA and RNA) play central roles in all cellular events and, as such, have immense impact on the emergence of diseases and, in turn, on human health. The goal of the proposed program is to design, synthesize and implement new emissive nucleoside analogs as probes for nucleic acids structure, dynamics and recognition. Novel real-time fluorescence-based methods for exploring nucleic acids and their interactions with potential therapeutic agents will further the fundamental understanding of key biological processes related to disease development and will have long-term impact on improving human health by advancing diagnostic tools and facilitating drug discovery.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
3R01GM069773-11S1
Application #
8845949
Study Section
Program Officer
Fabian, Miles
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Shin, Dongwon; Lönn, Peter; Dowdy, Steven F et al. (2015) Cellular activity of siRNA oligonucleotides containing synthetic isomorphic nucleoside surrogates. Chem Commun (Camb) 51:1662-5
Müller, Ulrich F; Tor, Yitzhak (2014) Citric acid and the RNA world. Angew Chem Int Ed Engl 53:5245-7
McCoy, Lisa S; Shin, Dongwon; Tor, Yitzhak (2014) Isomorphic emissive GTP surrogate facilitates initiation and elongation of in vitro transcription reactions. J Am Chem Soc 136:15176-84
Lane, Richard S K; Jones, Rosemary; Sinkeldam, Renatus W et al. (2014) Two-photon-induced fluorescence of isomorphic nucleobase analogs. Chemphyschem 15:867-71
Hopkins, Patrycja A; Sinkeldam, Renatus W; Tor, Yitzhak (2014) Visibly emissive and responsive extended 6-aza-uridines. Org Lett 16:5290-3
Fair, Richard J; McCoy, Lisa S; Hensler, Mary E et al. (2014) Singly modified amikacin and tobramycin derivatives show increased rRNA A-site binding and higher potency against resistant bacteria. ChemMedChem 9:2164-71
Noe, Mary S; Sinkeldam, Renatus W; Tor, Yitzhak (2013) Oligodeoxynucleotides containing multiple thiophene-modified isomorphic fluorescent nucleosides. J Org Chem 78:8123-8
Frolov, Ludmila; Dix, Andrew; Tor, Yitzhak et al. (2013) Direct observation of aminoglycoside-RNA binding by localized surface plasmon resonance spectroscopy. Anal Chem 85:2200-7
Liu, Wei; Shin, Dongwon; Tor, Yitzhak et al. (2013) Monitoring translation with modified mRNAs strategically labeled with isomorphic fluorescent guanosine mimetics. ACS Chem Biol 8:2017-23
Sinkeldam, Renatus W; McCoy, Lisa S; Shin, Dongwon et al. (2013) Enzymatic interconversion of isomorphic fluorescent nucleosides: adenosine deaminase transforms an adenosine analogue into an inosine analogue. Angew Chem Int Ed Engl 52:14026-30

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