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GSJ: Received June 3, 2005:
http://wbabin.net/saba/saba48.htm
Isolation of Enantiomer-binding Proteins, via Microarrays or Beads, and Scintillation Proximity
James Saba
Antibodies have been successfully used to separate enantiomers, yet there is a serious limitation in that to select for an antibody, heretofore small molecules need to be conjugated to a support or to a carrier molecule.
Herein is a proposed a means of selecting polymers, particularly proteins such as antibodies, using non-conjugated target molecules. The screening process involves a support-affixed polymer library, a radioactively labeled target molecule, and scintillation proximity.
Figure 1 depicts the process using a microarrayed library of proteins (which defined herein includes peptides).
Only three loci are shown, and only one of multiple identical proteins at each locus is shown. Preferably the arrayed proteins are numerous, perhaps even 100,000 or more.
Particles such as encoded beads can also be utilized, as depicted in Figure 2.
Herein only one of each of multiple proteins on a bead is shown.
Beads could be randomly arrayed on a support such as a glass plate, perhaps with the beads dispersed in a thin layer of gel.
A microarray locus or bead may comprise the protein-encoding nucleic acid of a protein.
Polymers other than proteins are potentially useful, such as polynucleotide aptamers.
This invention is considered valuable and a US patent application is anticipated to be filed. However, in the mean time it is hoped that others with laboratory facilities will investigate its full potential.
The following provisional claims are an attempt to encompass important aspects of this invention, yet many other claims could be composed.
2) A in vitro process for isolating a polymer which selectively binds an enantiomer, and which utilizes this enantomer in a non-conjugated form.
3) A in vitro process for isolating a polymer which selectively binds an enantiomer, and which utilizes scintillation proximity.
4) Any process above wherein the polymers are proteins.
5) The polymer isolated from any process above.
6) The polymer of claim 5 utilized in an enantiomeric separation or in a biosensor.
2) A method of detecting the covalent or noncovalent binding of a radioactive molecule to a support-affixed polymer, which utilizes a photographic emulsion to record the radioactive molecule's disintegrations.
3) The method of claim 2 wherein support-affixed polymer is a member of a library of different support-affixed polymers.
4) The method of claim 1, 2 or 3 wherein the support is a microarray support or particle.
5) Any method above wherein the polymer is a protein.
6) Any method above useful in analysis of ligands which allosterically modulate receptor binding or enzyme catalysis.
7) Any method above useful in the isolation of enantiomer-selective polymers.
2) Efficient enantioselective separation of drug
enantiomers by immobilised antibody fragments.
Nevanen, et al J Chromatogr A. 2001 Aug
3;925(1-2):89-97
3) Antibody-based bio-nanotube membranes for
enantiomeric drug separations.
Lee, et al Science. 2002 Jun 21;296(5576):2198-200
4) Stereoselectivity of antibodies for the bioanalysis
of chiral drugs (review).
Got PA, et al Pharm Res.
1997 Nov;14(11):1516-23
5) Selective antibodies to propranolol enantiomers
produced from a new conjugate.
Sahui-Gnassi, et al Chirality. 1993;5(6):448-54
6) Synthesis of five enantiomerically pure haptens
designed for in vitro evolution of antibodies with
peptidase activity.
Wagner, et al Bioorg Med Chem. 1996 Jun;4(6):901-16
7) Enantioselective affinity chromatography of a
chiral drug by crystalline and carrier-bound antibody
fab fragment.
Vuolanto, et al Biotechnol Prog. 2004
May-Jun;20(3):771-6
8) Antibodies as chiral selectors for the
determination of enantioenrichment
Hofstetter, et al Enantiomer. 2001;6(2-3):153-8
9) Selective antibodies to methadone enantiomers:
synthesis of (R)- and (R,S)-methadone conjugates and
determination by an immunoenzymatic method in human
serum.
Chikhi, et al Chirality. 2001 May
5;13(4):187-92
The radioactive target provided to a microarray or beads could be racemic, and subsequent to locating binding proteins their specificity to a particular enantomer could be established.
The use of partitioned microarray supports and the process described in (1) could be utilized if the protein, subsequent to synthesis, became affixed to a scintillation proximity surface.
Likewise the process or distributing beads to a partitioned array (2) may also find utility in the above described processes.
If binding of radioactive target molecule by affixed protein is sufficiently strong such the unbound radioactive molecules can be washed away, then alternatively to scintillation proximity, a radiation-sensitive photographic emulsion could be layered over the array (3). If beads was used, subsequent to washing unbound radioactive molecules, the beads could be distributed within a thin layer of the emulsion. Since use of an emulsion gives a cumulative record via 'grains', it could be more sensitive than scintillation proximity.
2) Allosteric Drug Discovery Utilizing Microarrayed
Ligands.
Saba, JA Gen Sci J 2005 June 1
3) Radioactive labels: autoradiography and choice of
emulsions for in situ hybridization.
Brady, et al
(1990) In: In Situ Hybridization: Principle and
Practice (J. M. Polak & J. O'D. McGee eds.) Oxford
University Press.
4) Measurement of radioligand binding by scintillation
proximity assay.
Berry, et al Methods Mol Biol.
2005;306:121-37
Conceivably, labeling is not a necessity, as exemplified by the following references.
Label-free detection of nucleic acid and protein microarrays by scanning Kelvin nanoprobe. Thompson, et al Biosens Bioelectron. 2005 Feb 15;20(8):1471-81
Readout of protein microarrays using intrinsic time resolved UV fluorescence for label-free detection. Striebel, et al Proteomics. 2004 Jun;4(6):1703-11
High-density miniaturized thermal shift assays as a general strategy for drug discovery. Pantolianno, et al J Biomol Screen. 2001 Dec;6(6):429-40
A label-free optical technique for detecting small molecule interactions. Lin, et al Biosens Bioelectron. 2002 Sep;17(9):827-34
Pattern-Based Detection of Different Proteins Using an Array of Fluorescent Protein Surface Receptors Baldini, et al J. Am. Chem. Soc., 126 (18), 5656 -5657, 2004
The following simple process has been conceived as a means to integrate bead-based combinatorial synthesis with selecting for stereoselective binding molecules useful in enantiomer isolation.
Provisional claims
ii) screening this library for a library member which effects a desired response,
iii) utilizing a this library member as a support-affixed capture probe to screen an encoded library for members thereof which bind the library member, and
vi) isolating a bound encoded library member (receptor) and determining which enantiomer of the ligand or substrate it binds.
3) The process of claim 1 or 2 wherein the encoded library screened by said support-affixed capture probe is a phage display or cis-display protein (peptide) library.
Mass spectrometry could be utilized to identify those array loci wherein noncovalent binding of a drug or ligand to a protein has occurred.
2) Evaluation of automated nano-electrospray mass
spectrometry in the determination of non-covalent
protein-ligand complexes.
De Vriendt, et al Rapid
Commun Mass Spectrom. 2004;18(24):3061-7.