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Method using a nonlinear optical technique for detection of interactions involving a conformational change

a nonlinear optical and conformational technology, applied in the field of nonlinear optical techniques for detection of interactions involving conformational changes, can solve the problems of high cost to users, difficult to assign small measured changes in a particular polarization direction to conformational changes, and difficult to separate small changes in probe orientation from large fluorescent background that may be present in many biological samples

Inactive Publication Date: 2006-10-12
BIODESY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0121] The present invention provides advantages over fluorescence-based detection, such as reduced background, reduced photobleaching, simplified optical detection, and no need for labor- or time-consuming washing steps. These advantages are due to the low background of the nonlinear optical techniques and the fact that the method is a scattering rather than an absorption process.

Problems solved by technology

Current techniques that directly measure activation, such as patch-clamping techniques with an ion channel protein, are not amenable to high-throughput scaleup and require a skilled technician to operate, resulting in higher cost to the user.
Problems using fluorescence include the presence of a natural fluorescent background in many (non-labeled) biological samples as well as photobleaching.
Detection of orientational changes accompanying target binding is difficult to do using fluorescence as the technique is not very sensitive to label orientation: fluorescence polarization, not being a coherent technique, is sensitive to rotational motion during the fluorescence lifetime that makes it difficult to assign small measured changes in a particular polarization direction to conformational changes (rather than due to rotational motion).
It is also difficult to separate small changes in probe orientation from the large fluorescent background that may be present in many biological samples.
Wavelength-based changes due to changes in the microenvironment of the label as a result of conformational change can be followed, but not all conformational changes lead to a change in microenvironment and it may be difficult to assign relative changes due to microenvironement to the degree of conformational change that actually occurs.
Apart from various problems in the detection itself—concerning photobleaching, artifacts and background noise, these methods only provide indirect assays for the probe-target binding.
Surface-selective nonlinear optical techniques have previously been confined mainly to physics and chemistry since relatively few biological samples are intrinsically non-linearly active.
However, the stains intercalate into the membranes in either an ‘up’ or ‘down’ direction, thus reducing the total nonlinear signal due to destructive interference.

Method used

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  • Method using a nonlinear optical technique for detection of interactions involving a conformational change
  • Method using a nonlinear optical technique for detection of interactions involving a conformational change
  • Method using a nonlinear optical technique for detection of interactions involving a conformational change

Examples

Experimental program
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example 6.1

[0480] A Molecular Beacon analogue (MB analogue) oligonucleotide, coupled to a nonlinear-active dye, and purified, is purchased from a commercial source such as Midland Certified Reagent Company (Midland, Tex.). The nonlinear-active oxazole dye used is oxazole (SE) 1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.) attached via an amine group at the 3′ end.

[0481] The oligonucleotide is placed into the sample well of an EFISH cell. There are a variety of EFISH cells available in the art. The sample cell described in the publication by C. G. Bethea (‘Experimental technique of dc induced SHG in liquids: measurements of the nonlinearity of CH2I2”, Applied Optics 1975, 14, 1447) is used. The direction of the applied electric field is parallel to the electric field of the laser beam. A commercial femtosecond mode-locked system (Mira 900 and Verdi 5W) is used as the fundamental source. The fundamental is directe...

example 6.2

[0484] The β2 adrenergic receptor, a GPCR protein, is purified and detergent-solubilized according to well known procedures (e.g., Ghanouni et al., 98(11): 5997 PNAS). The protein is labeled at an endogenous cysteine (Cys-265) with 1-(2,3-epoxypropyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium trifluoromethanesulfonate (PyMPO epoxide, Molecular Probes), a nonlinear-active dye, at 1:1 stoichiometry following standard procedures and using the work of Ghanouni et al., 98(11): 5997 PNAS, as a guide. The nonlinear-active dye is attached to a part of the protein that undergoes a conformational change when the protein is activated. After separation of the non-covalently bound dye, the receptor is placed in a medium situated between two electrodes and through which passes a fundamental beam (e.g., output of ˜1 W avg. Power, ˜150 fs pulses from a Ti:Sapphire system such as the Verdi-Mira commercial system from Coherent Inc.). The apparatus and sample preparation (e.g., application of elect...

example 6.3

[0492] Oligodeoxyribonucleotides with suitable structures for molecular beacons are selected and synthesized according to procedures known to one of ordinary skill in the art with a primary amine at the 3′ end and a disulfide group at the 5′ end and a biotin group that replaces a dT. The following MB analogue can be used, for example: 5′-CCT AGC TCT AAA TCG CTA TGG TCG CGC(Biotin dT)AG G-3′ (SEQ ID NO: 6). The amine-reactive nonlinear-active oxazole dye: oxazole (SE) 1-(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.) is conjugated to the primary amine. In this coupling reaction, a 100 μl solution containing 100 μM oligonucleotide dissolved in 0.1 M sodium bicarbonate is reacted with 0.1 mg of the succinimidyl ester of the dye dissolved in 100 μl of dimethyl sulfoxide. The reaction mixture is stirred at room temperature for 2 hours. The reaction product is purified with a Sephadex column (NAP-5; Amersham Ph...

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Abstract

A nonlinear optical technique, such as second or third harmonic or sum or difference frequency generation, is used to detect binding interactions, or the degree or extent of binding, that comprise a conformational change. In one aspect of the present invention, the nonlinear optical technique detects a conformational change in a probe due to target binding. In another aspect of the invention, the nonlinear optical technique screens candidate probes by detecting a conformational change due to a probe-target interaction. In another aspect of the invention, the nonlinear optical technique screens candidate modulators of a probe-target interaction by detecting a conformational change in the presence of the modulator.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of international application number PCT / US01 / 2241 1, entitled “Method and Apparatus Using a Surface-Selective Nonlinear Optical Technique for Detection of Probe-Target Interactions”, filed Jul. 17, 2001, which in turn claims benefit of U.S. provisional applications No. 60 / 253,862, entitled “Method and Apparatus Using a Surface-Selective Nonlinear Optical Technique for Detection of Probe-Target Interactions”, filed Nov, 29, 2000; 60 / 260,249, entitled “Apparatus and Method for the Detection of Biological Reactions Using a Surface-Selective Nonlinear Optical Technique”, filed Jan. 8, 2001; 60 / 265,775, entitled “Apparatus and Method for the Detection of Biological Reactions Using a Surface-Selective Nonlinear Optical Technique”, filed Feb. 1, 2001; and 60 / 278,941, entitled “Apparatus and Method for the Detection of Biological Reactions Using a Surface-Selective Nonlinear Optical Technique”, filed M...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N33/53C07K14/705C07H21/04G01N33/543G01N33/58G01N33/68
CPCB82Y30/00Y10T436/143333G01N33/54373G01N33/583G01N33/6845G01N2333/726G01N2458/00G01N2500/02G01N2500/04G01N33/54313G01N21/31G01N33/74C12Q1/6825G01N33/587G01N33/5308G01N33/56966C12Q1/6816G01N21/636
Inventor SALAFSKY, JOSHUA
Owner BIODESY
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