Biomolecular labeling

Details Biomolecular labeling Biomolecular labeling

2000-03-01

2003-12-02

Riley, Jezia (Department: 1656)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S024300, C536S025300, C536S025320, C435S006120

Reexamination Certificate

active

06657052

ABSTRACT:

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
REFERENCE TO A MICROFICHE APPENDIX
Not Applicable.
DESCRIPTION OF THE INVENTION
This research is developing a synthetic method and in vivo designed reagent for the chemoselective, covalent modification of the phosphodiester group in nucleic acid polymers. Successful alkylation of phosphodiesters to form stable phosphotriesters has been accomplished through the development of chemistry based on the reactive paraquinone methide. The systematic analysis of model compounds is being used to add increasingly higher levels of selectivity to the alkylating methodology. The design of these model compounds is evolving to incorporate the necessary functionality to achieve the development of a final reagent which will be used for selective in vitro modification of a single phosphodiester group within a DNA or RNA target. This final reagent is designed to allow the delivery of a variety of reporter groups, drug agents or protein conjugates to DNA or RNA targets. This methodology would allow for the molecular level study of protein-nucleic acid interactions in such complex systems as the chromatin, where molecular level understanding is still very limited due to the complex, multi-protein machinery which operates at this level. This would also lead to an innovative approach for site-selective drug delivery to nucleic acid targets. Lastly, this methodology has the potential for transcription control through the site-selective delivery of transcriptional activators to genetic targets.
This proposal focuses on the synthesis and analysis of the model compounds and reactions which are being developed in the process of optimizing the reagent design. This research is providing a detailed understanding of various chemical reactions and molecular interactions. The knowledge gained and compounds produced in the process of this research are providing new synthetic methodology for nucleic acid chemistry and compounds for nucleic acid modification. This proposal describes the research necessary up to the total synthesis of a fully functionalized labeling reagent and its characterization.
1. Specific Aims
Rapid progress in sequencing the human genome
1
opens new doors for potential technological developments for studying and treating disease at the foundational genetic level. One area of such potential development is the in vivo chemical modification of genomic DNA for diagnostics, therapeutics, and the study of biological processes. This requires progress in several areas of biomedical technology. Advances in oligonucleotide delivery to cells
2
and sequence-specific recognition of DNA
3
are two key areas. Our research program is targeting an unexplored area for the development of an innovative, chemical means to covalently deliver a variety of reporter groups, drug agents, or proteins to DNA. The ability to site-specifically attach such moieties to DNA would allow various genetic-based, biological studies to be conducted
4
and provide a new means for efficient diagnostics
5
, therapeutics
6
and biological control at the genetic level
7
.
Covalent modification of the phosphodiester group would be of most interest as it is the common, repeating, nucleophilic functional group throughout nucleic acid polymers. Whereas covalent modification of the nucleic acid bases generally leads to strand cleavage through depurination and will disrupt base pairing by interfering with hydrogen bonding, modification of the phosphate will likely have less effect on nucleic acid structure and function (FIG.
1
).
This proposal will present the foundational research necessary for development of such a chemical reagent to accomplish this overall goal. This research is developing a variety of useful chemistry, synthetic methodology, and compounds in pursuit of the long-term goal.
The final in vivo designed reagent will covalently transfer attached molecules to a target phosphate group of a nucleic acid polymer (FIG. S
1
). The reagent is designed around a quinone methide with its bimodal electrophilic and a nucleophilic reactivity. The design features include:
An independently tethered delivering oligonucleotide and molecule to be transferred (1, loligo and R, respectively, FIG. S
1
). These are appended to a DNA synthesizer, machine-ready core reagent using standard automated, solid-phase, synthetic protocol for simplicity, efficiency and versatility.
Phosphate specificity through a guanidinium-phosphate complex (2). The guanidinium group will be substituted as needed to lower it nucleophilicity and prevent intramolecular reaction.
“Caged” reactivity initiated by proteolysis to afford the quinone methide precursor (3).
An intramolecular tertiary amine may be incorporated if it proves beneficial to assist 1,6-elimination
8
to afford the intermediate quinone methide (4).
Alkylation of the phosphodiester with the quinone methide resulting in the in vitro release of the delivering oligonucleotide through lactonization to accomplish the transfer step (4 to 5). The oligonucleotide tether is designed to be cleaved at a slower rate than 1,6-elimination occurs. The intramolecular conjugate acid will afford stability to the trialkylphosphate prior to lactonization.
Formation of a covalently stable trialkylphosphate upon lactone formation by trapping the ailcylated product (5) and preventing reaction reversibility.
The reagent is an affinity transfer alkylating reagent (ATAR) for labeling the phosphodiester of nucleic acids. The research program is designed to streamline development of the ATAR by optimizing the chemistry through independent model system studies. The final reagent will be suitable for general use by attaching any delivering oligonucleotide on an automated synthesizer followed by attachment of a desired reporter group
9
, drug agent
10
, or protein conjugate
11
on the solid support or post-synthetically. This provides a “user-friendly” reagent for use in modifying DNA, studying various nucleic acidprotein interactions, and for drug delivery applications.
The chemistry required for the ATAR is being developed through a variety of small molecule model studies. Each study requires minimal synthesis in order to independently investigate the various chemical aspects of the ATAR for optimization. The project has been designed so that the short-term model studies can be carried out by undergraduate research assistants. Progressively more complex studies are underway in order to coordinate the compatibility of the chemical reactions for optimal control of all aspects of the ATAR design. The chemistry necessary for the total syntheses of fully functionalized derivatives for incorporation into the ATAR on a DNA synthesizer is being developed in the course of these model studies. The multiple small component contributions required for this project make it an optimal training ground for short-term undergraduate research. This proposal maps out the investigations for development of the chemistry of the ATAR focusing on the model system syntheses and studies. Although the overall goal of this research is the envisioned applications of the ATAR mentioned above
4-7
, this will be beyond the time frame for which present funding is sought.
Several significant subset developments result from the pursuit of the overall research goal. One will be a simplified synthetic method
12,13
for site selective alkylation and peralkylation of oligonucleotide phosphodiesters to produce trialkylphosphate modified oligonucleotides for various uses.
14,15,5
This backbone modification affords enhanced hybridization properties
16
, antisense/antigene applications
17,18
and peralkylations of the phosphodiesters will alter an oligonucleotides solubility properties for use in large-scale solution phase oligonucleotide synthesis. This research has already afforded a useful synthetic method for modifying phosphodiesters with the formation, isolation and fall characterization of trialkylphosphates
19
. Some aspects of commercial potential for this methodology are being pursued with industrial

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