Nucleic Acid Mediated Electron Transfer

Inactive Publication Date: 2010-03-18
CALIFORNIA INST OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0030]Accordingly, it is an object of the invention to provide nucleic acids with electron transfer species covalently attached to a terminal base of the nucleic acid.

Problems solved by technology

Specificity, in contrast, remains a problem in many currently available gene probe assays.
It may be possible under some limited circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally very difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization.
Finally, the automation of gene probe assays remains an area in which current technologies are lacking.
This separation is generally achieved by gel electrophoresis or solid phase capture and washing of the target DNA, and is generally quite difficult to automate easily.
Unfortunately, the reliance of this approach on a chemiluminescent substrate known for high background photon emission suggests this assay will not have high specificity.
However, the fluorescence energy transfer is greatly influenced by both probe topology and topography, and the DNA target itself is capable of significant energy quenching, resulting in considerable variability.
In these systems, the electron donors and acceptors are not covalently attached to the DNA, but randomly associated with the DNA, thus rende

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of an Oligonucleotide Duplex with Electron Transfer Moieties at the 5′ Termini

[0174]In this example an eight nucleotide double stranded nucleic acid was produced, with each single strand having a single electron transfer moiety covalently attached to the 5′ terminal uridine nucleotide at the 2′ carbon of the ribose sugar.

Step 1: Synthesis of 5′-di(p-methoxyphenyl)methyl ether-2′-(trifluoroacetamido)-2′-deoxyuridine

[0175]2′-(trifluoroacetamido)-2′-deoxyuridine (2.0 g, 5.9 mmoles) prepared by minor modification of published procedures (Imazawa, supra) was repeatedly dissolved in a minimum of very dry CH3CN and rotary evaporated to dryness and then transferred to inert atmosphere vacuum line and further dried for a period of 1 hour. The following procedure for the synthesis of the material was adapted from Gait (supra): Under positive pressure argon, the material was dissolved in freshly dried and distilled pyridine and with stirring, 0.05 equivalents (wt.) of 4-dimethylamino...

example 2

Synthesis of Long DNA Duplexes with Electron Transfer Moieties at the 5′ Termini

[0179]In this example, an in vitro DNA amplification technique, PCR (reviewed in Abramson et al., Curr. Op. in Biotech. 4:41-47 (1993)) is used to generate modified duplex DNA by polymerization of nucleotides off modified primer strands (Saiki et al., Science 239:487 (1988)). Two oligonucleotides 18 bases in length and not complementary to each other are synthesized with amino-modification to the 2′-ribose position of the 5′ nucleotides, as in example 1.

[0180]A series of oligonucleotides of increasing lengths starting at 40 bases are chemically synthesized using standard chemistry. Each of the PCR templates shares a 5′ sequence identical to one modified 18mer. The 3′ end of the template oligonucleotide shares a sequence complementary to the other 18mer.

[0181]PCR rapidly generates modified duplex DNA by the catalysis of 5′-3′ DNA synthesis off of each of the modified 18mers using the unmodified strand as ...

example 3

Synthesis of Covalently Bound Electron Transfer Moieties at Internucleotide Linkages of Duplex DNA

[0183]In this example, alternative backbones to phosphodiester linkages of oligonucleotides are employed. Functional groups incorporated into these internucleotide linkages serve as the site for covalent attachment of the electron transfer moieties. These alternate internucleotide linkages include, but are not limited to, peptide bonds, phosphoramidate bonds, phosphorothioate bonds, phosphorodithioate bonds and O-methylphosphoramidate bonds.

[0184]The preparation of peptide nucleic acid (PNA) follows literature procedures (See Engholm, supra), with the synthesis of Boc-protected pentafluorophenyl ester of the chosen base (thymidine). The resulting PNA may be prepared employing Merrifield's solid-phase approach (Merrifield, Science, 232:341 (1986)), using a single coupling protocol with 0.1 M of the thiminyl monomer in 30% (v / v) DMF in CH2Cl2. The progress of the reaction is followed by q...

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Abstract

The present invention provides for the selective covalent modification of nucleic acids with redox active moieties such as transition metal complexes. Electron donor and electron acceptor moieties are covalently bound to the ribose-phosphate backbone of a nucleic acid at predetermined positions. The resulting complexes represent a series of new derivatives that are bimolecular templates capable of transferring electrons over very large distances at extremely fast rates. These complexes possess unique structural features which enable the use of an entirely new class of bioconductors and photoactive probes.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This is a continuing application of U.S. Ser. No. 09 / 306,749, filed May 7, 1999, which is a continuation of application Ser. No. 08 / 873,598, filed Jun. 12, 1997, now U.S. Pat. No. 5,952,172, which is an continuation-in-part of application Ser. No. 08 / 660,534, filed Jun. 7, 1996, now U.S. Pat. No. 5,770,369, which is a continuation-in-part of application Ser. No. 08 / 475,051, filed Jun. 7, 1995, now U.S. Pat. No. 5,824,473, which is a continuation-in-part of application Ser. No. 08 / 166,036, filed Dec. 10, 1993, now U.S. Pat. No. 5,591,578.FIELD OF THE INVENTION[0002]The present invention is directed to electron transfer via nucleic acids. More particularly, the invention is directed to improvements in the site-selective modification of nucleic acids with electron transfer moieties.BACKGROUND OF THE INVENTION[0003]The detection of specific nucleic acid sequences is an important tool for diagnostic medicine and molecular biology research. Gen...

Claims

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Application Information

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IPC IPC(8): C07H19/04C07H21/00C07H23/00C12Q1/68
CPCC07H21/00C07H23/00C12Q1/6818C12Q1/6825C12Q2565/607C12Q2565/401C12Q2563/137C12Q2563/113C12Q2565/101C12Q2565/1015Y02A50/30
Inventor MEADE, THOMAS J.KAYYEM, JON FAIZFRASER, SCOTT E.
Owner CALIFORNIA INST OF TECH
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