AU2007100888A4 - Design and synthesis of streptavidin-DNA conjugates - Google Patents

Description

DESCRIPTION

TECHNICAL FIELD o The present invention is generally related to the techniques of conjugate a double o stranded DNA with streptavidin. More particularly, the present invention is related to conjugate a double stranded DNA with streptavidin covalently for immunoassays or PCR assays.

00 00 BACKGROUND OF THE INVENTION 00 SStreptavidin is a protein very closely related to a protein avidin, which provides a very stable noncovalent complex with vitamin D-biotin. Avidin itself is a very highly Sspecialized protein that is only rarely expressed. Streptavidin, on the other hand, is 0 readily expressed in Streptomyces species particularly in Streptomyces avidinii.

Streptavidin specifically binds a water soluble vitamin D-biotin (vitamin Similarly to avidin, it binds rapidly and almost irreversibly to any molecule containing unhindered biotin with a remarkably high affinity (Kd z 10 1 The cooperative biotin binding by streptavidin was described in J. Biol. Chem., 265:3369 (1990) Streptavidin, contrary to avidin, is carbohydrate free and thus more suitable, for example, for X-ray crystallographic studies or for various other detection techniques.

The comparative properties of avidins and streptavidins are described in Methods in Enzymology, 184:51 (1990). Isolation and properties of streptavidin, as well as its preparation, are described in Arch. Biochem. Biophys., 106:1 (1964) and in Methods in Enzymology, 184:51 and 80 (1990). Streptavidin has an apparent molecular weight of about 66,000 and is capable of binding one mole of biotin per mole of subunit. The thermal stability of streptavidin is remarkable. In the presence of SDS, streptavidin begins to dissociate to its monomers and dimers only at temperatures above 60 0

C.

Streptavidin has been extensively applied on enzyme linked immunoabsorbant assay (ELISA) J. Clin. Microbiol. 30:1667 (1992), J. Virol. Methods. 11:41 (1985). During ELISA detection of a protein, an antibody (specific to the target protein) binds to the target protein, which is fixed to a vessel. Non-bound antibodies are washed off while bound antibodies remain. A secondary antibody is then added (which is specific to the first antibody) that is conjugated to biotin. Then streptavidin that is conjugated to an enzyme that will produce a colour change in the presence of a special compound is added in. The colour change can be detected visually or via laser and the intensity can give a measure of target protein concentration.

Detection of the target protein would be limited in sensitivity to the minimum protein concentration of enzyme required to produce a detectable color change reaction. DNA can be detected and amplified by poly chain reaction (PCR) at very low copy numbers and therefore the DNA tagged antibody system could be used to detect the target protein at much lower concentrations i.e. higher sensitivity.

Therefore there exist a need for a technique for conjugating double stranded DNA with streptavidin, which can be applied on immuno-PCR to identify various entities such as viruses, bacteria and protein at much lower concentration in a sample. Method to conjugate DNA or oligonucleotides with streptavidin for immuno-PCR have been described in Nucleic Acids Res. 22:5530 (1994) and Nucleic Acids Res. 31: No. 16 O (2003), but in practical, the yield of streptavidin-DNA is less than 40%. This invention provides a method to conjugate double stranded DNA with streptavidin with high yield and also provide a method of purifying streptavidin-DNA o conjugate.

C SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide DNA-streptavidin 00 conjugate. In particular, the invention comprises streptavidin cross-linked by an end- 00 derivatized double stranded DNA. The conjugates can comprise streptavdin and the 00 DNA can contain one or more restriction enzyme sites. The invention provides Sgeneral techniques for preparing the double stranded DNA-streptavidin with high yield and for using them in immuno-PCR assays to detect antigen such as virus and Sbacteria in a sample.

DETAILED DESCRIPTION OF THE INVENTION This invention concerns streptavidin-DNA conjugates. Specifically, it provides a method to conjugate double stranded DNA to streptavidin. The described processes provide for significantly increased yields during conjugation. There is less aggregation of protein and a greater yield of conjugate when double stranded DNA is modified with sulfhydryl group. The purified streptavidin-DNA can bind biotin molecule with extremely high affinity. Any biological material containing unhindered biotin may be labelled with streptavidin-DNA, in which DNA may be amplified and detected by PCR at very low concentration from the biological samples.

Double stranded DNA may come from mammalian, bacteria and virus. The size of DNA of this invention is typically between about 400 nucleotides and about 1000 nucleotides. Size is not a critical factor. For a practical level of PCR reaction, the size of DNA should exceed 150 nucleotides. To minimize the cross-reaction, DNA from one spice should not be chosen to conjugate to streptavidin for analysing biological samples from same spice DNA from mammalian genomic DNA should not be used for analysing human samples). The DNA was prepared by PCR with a sulfhydryl modified primer as forward primer and a normal primer as reverse primer. After PCR reaction, the purified PCR product (double stranded DNA) has sulfhydryl modification in one chain, which can be covalently linked to streptavidin through a bifunctional coupling reagent.

There are couple of bifunctional coupling reagents available for cross-linking strepavidin with DNA such as N-[y-Maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS), N-[s-Maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS), m- Maleimidobenzoyl-N-hydroxyculfo-succinimide (sulfo-MBS), Sulfosuccinimidyl[4iodoacetyl]aminobenzoate (sulfo-SIAB), Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC), Sulfosuccinimidyl 4-[p-maleimidophenyl] butyrate (sulfo-SMPB). These hetero-bifunctional regents possess two different reactive groups, in which one is reactive to amino group on streptavidin surface and the other is sulfhydryl-reactive group, which is able to react with sulfhydryl modified double stranded DNA to link DNA with streptavidin covalently.

1. Preparation of thiol-reactive streptavidin O A streptavidin can be derivatised with 10-100 molar excess of bifunctional coupling reagent at 20-30 degree in aqueous buffer at pH 7.0. After incubation of 1-2 hours, the excess reagent is separated by gel filtration chromatography.

o 2. Preparation of sulfhydryl modified double stranded DNA A sulfhydryl modified double stranded DNA with size between 400 nucleotides and 1000 nucleotides can be prepared with standard PCR reaction with modified prime. After PCR reaction of 1-2 hours, DNA product is purified with size 00 exclusion chromatography.

00 00 3. Conjugation of streptavidin to DNA SPurified thiol-reactive streptavidin is reacted with sulfhydryl DNA with the molar t ratio of streptavidin to DNA at 1:1 to 1:10 in an aqueous buffer of pH 6.5-7.5 with 0 mM EDTA and 5-10 mM Tris [2-carboxyethyl] phosphine (TCEP). After an O incubation of 3-4 hours, the reaction is terminated with a 10-100-fold molar excess of N-ethymaleimide, and the conjugate is separated from free DNA by chromatography on Immoniblized Imminobiotin. Free streptavidin is then removed by size-exclusion

HPLC.

Once the DNA is cross-linked to the streptavidin, it is useful as a probe to bind to biotin containing molecules such as biotinylated antibodies and biotinylated proteins and being applied on immunoassays.

The present invention is described in detail by referring to the Examples below.

EXAMPLE 1 This example illustrates the method to conjugate 602 base pair DNA with streptavidin Preparation and purification of Streptavidin-DNA Conjugates MATERIALS AND METHODS Synthesis of 5' sulfhydryl modified double stranded DNA The 602 base pair double stranded DNA was synthesized using PCR with mouse leukaemia virus genomic DNA as template. J. Gen. Virol. 68 (PT 683-693 (1987).

The forward primer sequence is 5' (thiol CCCCACCATCAGGCTTAG (SIGMA GENOSYS and the reverse primer sequence is GGCTTTATTGGGAGCACGG (SIGMA GENOSYS The PCR reaction was conducted in a total volume of 25 [tL with 1.0 unit of Taq DNA polymerase (SIGMA in the presence of the PCR reaction buffer from SIGMA kit containing 200 [tM each of nucleotides dATP, dCTP, dGTP, and dTTP, 1.0 mM of MgCl 2 and 1 [tL of the solution of leukaemia virus genomic DNA (1.0 ug/mL) was added.

Amplification conditions were as follow: Denaturing at 950 C. for 15 seconds, annealing at 570 C. for 30 seconds, and elongation at 720 C. for 15 seconds for cycles. The PCR products obtained were analysed by electrophoresis on a 2% agarose gel to screen for the presence of the appropriate-size band using the fluorescent dye SYBR-Safe® (Invitrogen The PCR product then isolated by QIAquick PCR purification kit (QIAGEN to remove proteins and primers and the purified O sulfhydryl DNA was eluted into distilled water and concentration of DNA was determined with A 260 nm.

o Modification of Streptavidin with Sulfosuccinimidyl 4- (N-maleimidomethyl) 0 cyclohexane- 1-carboxylate (sulfo-SMCC) Sulfo-SMCC was dissolved into distilled water to make 5.0 mg/mL solution and uL of sulfo-SMCC solution was mixed and reacted with 100 uL of 10 mg/mL 00 streptavidin in 50 mM sodium phosphate buffer (pH=7.0) at room temperature for 2 00 hours and purify streptavidin-SMCC with HiTrap desalting column (Amershame 00 Biosciences. equilibrated with 50 mM phosphate buffer (pH=7.0) to remove Sunreacted sulfo-SMCC. The concentration of streptavidin-SMCC was determined with A 280 nm.

OConjugation of DNA to Streptavidin In separate reaction vessel, 10 ug of purified streptavidin-SMCC reacted with 20 ug sulfhydryl DNA in 50 mM phosphate buffer with 5 mM EDTA and 10 mM Tris [2carboxyethyl] phosphine hydrochloride (TCEP.HC1) (PIERCE It was reacted at room temperature for 4 hours.

Purification of streptavidin-DNA conjugate Streptavidin-DNA conjugate and unreacted DNA and streptavidin were loading on 2 mL Iminobiotin column (PIERCE which was equilibrated with binding buffer (50 mM ammonium carbonate buffer with 0.5 M NaCl pH=11.0). After washing with 10 mL of binding buffer to remove unreacted DNA and proteins, Streptavidin-DNA and streptavidin were eluted with 0.1 M acetic acid as shown in figure Then streptavidin-DNA and streptavidin were loading on size exclusion HPLC (Phenomenex Bio-SEC-3000 which was equilibrated with 0.1 M phosphate buffer with 200 mM NaCl pH=6.5. Streptavidin-DNA was eluted from 5.5 to 6.2 min and the result was shown in figure 6. As a control, streptavidin was loaded onto HPLC to indicate the elution time of streptavidin is greater than 9 min (red label in figure 6).

BIBLIOGRAPHY:

Bayer EA, Ben-Hur H, Wilchek M. Isolation and properties of streptavidin. Methods Enzymol. 1990;184:80-9.

Chaiet L, Wolf FJ. The Properties Of Streptavidin, A Biotin-Binding Protein Produced By Streptomycetes. Arch Biochem Biophys. 1964 Jul 20;106:1-5.

Gould EA, Buckley A, Cammack N. Use of the biotin-streptavidin interaction to improve flavivirus detection by immunofluorescence and ELISA tests. J Virol Methods. 1985 May;11(1):41-8.

Green NM. Avidin and streptavidin. Methods Enzymol. 1990;184:51-67.

Niemeyer CM, Sano T, Smith CL, Cantor CR. Oligonucleotide-directed self-assembly Sof proteins: semisynthetic DNA--streptavidin hybrid molecules as connectors for the o generation of macroscopic arrays and the construction of supramolecular bioconjugates. Nucleic Acids Res. 1994 Dec 25;22(25):5530-9.

Niemeyer CM, Wacker R, Adler M. Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA- 00 protein conjugates. Nucleic Acids Res. 2003 Aug 15;31(16):e90 00 00 Nikbakht KN, Boone LR, Glover PL, Myer FE, Yang WK. Characterization of a Smolecular clone of RFM/Un mouse chromosomal DNA that contains a full-length endogenous murine leukaemia virus-related proviral genome. J Gen Virol. 1987 0Mar;68 (Pt 3):683-93.

Ojeh CK, Tsunemitsu H, Simkins RA, Saif LJ. Development of a biotin-streptavidinenhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses.J Clin Microbiol. 1992 Jul;30(7):1667-73.

Sano T, Cantor CR. Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea. J Biol Chem. 1990 Feb 25; 265(6):3369-73.

O FIG. 1 is a diagrammatic representation of the method for synthesizing 5' sulfhydryl double stranded DNA.

0 FIG. 2 shows the nucleotide sequence of a 5' sulfhydryl double stranded DNA used to o cross-link streptavidin molecule. J. Gen. Virol. 68 (PT 683-693 (1987).

FIG.3 shows purification of 5' sulfhydryl double stranded DNA with QIAGEN PCR purification kit. Molecular mass of DNA ladders are shown left in figure and A, B, C 00 and D are four different batches of preparation of 5' sulfhydryl double stranded DNA.

00 The molecular mass of the DNAs is close to 600 base pairs, which is consistent to its size with 602 base pairs.

FIG.4 shows purification of melamide-modified streptavidin with HiTrap desalting Scolumn. The sample was loaded on HiTrap desalting column equilibrated with 50 mM O phosphate buffer pH 7.0 at flow rate 5.0 mL/min. Streptavidin-SMCC conjugates were collected from 14 to 26 seconds and unreacted sulfo-SMCC was eluted after 34 seconds as indicates in figure 4.

shows purification of unconjugated streptavidin and DNA-conjugated streptavidin with 2-iminobiotin affinity chromatography. Samples were loaded onto mL Immobilized 2-iminobiotin affinity column equilibrated with binding buffer mM ammonium acetate with 0.5 M NaCl pH=l 1.0) and after washing with 5 mL of binding buffer at 1.0 mL/min to remove unbounded proteins and DNA (peak 1), streptavidin and streptavidin-DNA were eluted with 0.1 M acetic acid (peak 2).

FIG.6 shows purification of DNA-streptavidin conjugates with HPLC gel filtration.

Samples were loaded on size exclusion HPLC equilibrated with 0.1 M phosphate buffer with 200mM NaCl pH 6.5 and streptavidin-DNA was eluted between 5.5 and 6.2 min. unreacted streptavidin was eluted after 9 min. As a control, streptavidin was loaded onto HPLC to indicate the elution time of streptavidin.

Claims (3)

1. 2. The conjugate of claim 1, wherein said the double stranded DNA is Sderivartised at 5' end with sulfhydryl group.
2. 3. The conjugate of claim 1, wherein said the double stranded DNA is 0 linked to said streptavidin by a covalent bond.
3. 4. The conjugate of claim 1, wherein said the coupling reagent being coupled in presence of Tris [2-carboxyethyl] phosphine (TCEP). The conjugate of claim 1, wherein said the double stranded DNA contain one or more restriction enzyme sites within.