WO1998051818A1

WO1998051818A1 - Method for treatment of deoxyribonucleic acid sequence by restrictase

Info

Publication number: WO1998051818A1
Application number: PCT/UA1997/000004
Authority: WO
Inventors: Volodymyr Mykolaiovych Khrapunov; Sergy Mykolaiovytch Khrapunov
Original assignee: Khrapunov Volodymyr Mykolaiovy; Sergy Mykolaiovytch Khrapunov
Priority date: 1997-05-14
Filing date: 1997-05-14
Publication date: 1998-11-19
Also published as: AU5237798A

Abstract

The present invention relates to chemistry and, more specifically, to gene engineering. Method for treatment of deoxyribonucleic acid sequence by restrictase, suitable for said treatment of practically any of said DNA sequence, comprises the following steps: binding co-valently a chemical compound capable to recognize specifically a concrete DNA site (component A) with a photoligand (component B) to obtain a new substance; adding the substance obtained to said DNA sequence to receive a complex; and illuminating by light the complex received with wave lengths selected from spectrum covered visible and ultraviolet ranges. The invention may be utilized for DNA site-specific restriction and site-specific mutagenesis in gene engineering, biotechnology, medicine, veterinary.

Description

METHOD FOR TREATMENT OF DEOXYRIBONUCLEIC ACID SEQUENCE BY RESTRICTASE

The present invention relates to chemistry and, more specifically, to gene engineering. Background of the invention

In the base of the invention the two fundamental positions are lied

There exist in nature or may be synthesized chemical compounds capable to recognize a large quantity of deoxyribonucleic acid (DNA) sequences, e.g. proteins, nucleic acids, polypepti- des, polynucleotides. (See, for example, B.Lewin Genes. John Willy and Son, Inc. 1983.).

There exist in nature or may be synthesized chemical compounds capable to absorb light of visible and ultraviolet ranges and to transmit the energy of that light to a substrat (matrix) with which these compounds (photoligands, photosencibilizators) are connected (See, for -example, Tyrrell R.M., Keyse S.M. New trends in photobiology. Invited review. The interaction of UVA radiation with cultured cells. // Journal of Photochemistry and Photobiology. B: Bilogy. -1990. -4. -P. 39-361 .

Moreover, a method for treatment of DNA sequence by restric- tase is known. This method consists in, that site-specific cleaving of DNA sequence is performed by means of enzymes (restricting endonucleases, restrictases). Every restrictase is strictly specific to one site(βhort sequence) of DNA. For instance, the EcoRI restrictase is capable to recognize the following site

5 ' i y

^{^} G A T T C ^'" C T T A A G

and cleaves the sequence at the places shown with arrows. ( See, for example, Roberts R. Restriction and modification enzymes and their recognition sequences. // Nucl. Acids Res. -1982. -10. -P. 117.).

Nevertheless,using the method known it is possible to be recognized by restrictases very short sites - of not more than 6 nucleotides which are met as parts of practically any long enough DNA sequence. Moreover, there exists a limited quantity of known rjsstrictases and therefore a limited quantity of DNA sequences recognized by restrictases.

The proposed method is free from these shortcomings and moreover the method may be realized for restriction and modification of practically any DNA sequence. The other aims and advantages of the method proposed will be clear from the following description, examples and claims.

Summary of the invention

Method for treatment of deoxyribonucleic acid sequence by res- trictase, suitable for said treatment of practically any of said DNA sequence, comprises the following steps: binding co-valently a chemical compound capable to recognize specifically a concrete DNA site (component A) with a photoligand (component B) to obtain a new substance; adding the substance obtained to said DNA sequence to receive a complex; illuminating by light the complex received with wave lengths selected from spectrum covered visible and ultra violet ranges; providing restriction of said DNA sequence or providing modification of said DNA sequence at place of said site recognized by said chemical compound.

It is possible to create a lot of modifications of the method for different purposes. For instance, restrictases include in- dentical A components ,. but various B components connected to them are bound to indentical base pare sequences of DNA but they may be switched on by different light sourses.

Examples of the realization of the method

Example . It was per ormed an appearance of breaks in DNA sequence illuminated by light in the range (spectrum) of 25 - 3 nm. The DNA was prepared from B.subtilis bacterii, then illumina- ted with a mercury-xenone lamp and restrictions were detected in extracted DNA sequence. The quantity of DNA sequence breaks was determined by means of centrifugation and calculation of molecular mass of DNA fragments. Centrifugation the illuminated cell lizates was performed in alkaline Saccharose gradient by Beckman centrifuge with rotor SW 50.1 of 35,000 rpm for 80 in.

It was determined that the action spectrum contained one main peak (less than 320 nm) and one minor peak of 350-400 nm. Taking into consideration that DNA did not absorb in the range of spec- tru more than 300 nm, the presence of breaks in the

when illuminating from 300 to 43 nm, meant the presence of natural photoligands specifically connected with DNA in cells.

Example 2. It was performed a photoinactivation of bacterio- phages T7. As photosensitizers (photoligands) 8-metoxypsoralen and andelicin were used. The bacteriophages were purificated and concentrated by centrifugation in CsCl gradient. An incubation of the bacteriophages in presence of furocumarines with followed illumination were performed in phosphate buffer, having concent- ration of 2 x 10 1? particles/ml of the bacteriophages. The quantity of the bacteriophages was determined after the first cycle of host E.coli cells growth by means of measurement of light scattering.

The effective photoinactivation of the T7 bacteriophages af- ter treatment with photoligands and illumination with a xenone lamp in the range of 310-380 nm proved, that natural photoligands from furocumarine group were effectively connected with bacterio- phage DNA and transferred to it the energy of the light absorbed. The analysis of modification of bacteriophage DNA after irradiation was done. It was shown the formation of DNA crosslinking photoproducts .

Crosslinking DNA was prepared with membrane filters and analysed by gel elecrophoresis. The yield of crosslinking photopro- ducts was up to 80 per cent under irradiation at the range of 320 - 380 nm.

Example 3. It was provided an action of light with the wavelengths of 450-600 nm radiated from a wolfram red-hot thread lamp to a substance consisted in a photoligand being co-valently bound with protein (antibody), capable to specifically connect to the bacterii of one species in mixture with bacterii of other specii. As a ligand, derivatives of fluoresceine were used taken in ratio to antibodies (fluoresceine/antibodies) as 4.0 .

Iodination of fluoresceine was performed by means of potassi- urn iodine adding to solution of the substance in phosphate buffer with pH 7.3 ♦ The iodinated substances were characterized by means of measuring their absorbtion spectra in visible and ultraviolet ranges. The cells mixed with fluoresceine-antibody substance were illuminated by light in the range of 410-620 nm, and their selective killing was determined based on E.coli cells capability of formation of the colonies to be restricted.

The effective selective killing the bacterii proved the possibility of protein usefulness ^{as a} specific component of the new substance , particularly of antibodies specifically connected to antigenes, and the possibility of using fluoresceine chemical derivatives as a photoligand.

Example 4. It was performed a hybridization of DNA sequence fragments with olygonucleotides, which were not in solution, but immobilized on a matrix of polyacrylamide gel. The matrix was formed from the elements of the gel (10 per cent of acrylamide, 0.3 per cent of bisacrylamide) of 80yi.m length, 80_j^m width and 15 _j^m thickness. The activation of the gel was performed by substitution of amide groups for hydraside groups. The hybridizati- on of the olygonucleotides immobilized on a single-strand DNA at 0°C in a buffer solution contained IM of NaCl at pH 7.0 for 15 minutes. Fluorescent labels were connected to the olygonucleotides. Iluumination of nucleic acids hybrides by light with wavelengths of 365, 404, 436, 546 nm was resulted in cleaving (rest- riction) of the DNA sequence.

Example 5- It was provided an attempt to find out stable pho- toproducts formed in DNA after irradiation in presence of photo- sensitizer - acetophenone.

The strain of E.coli 15(T~), a thymidine-requiring mutant,was grown in the media, contained 6 u g/ml of radioactive thymidine. The DNA was extracted with the help of Marmur (chloroform-isopro- panol) extracting procedure.

High concentrations of the DNA with 5 mM acetophenone added as photosensitizer were irradiated by light from a high-pressure Hg lamp. The wavelength of 365 nm was used for acetophenone excitation. The DNA was then lyophylizated and hydrolized in 90% formic acid.

The Dowex ion-change chromatography was used for determination of photoproducts. The chromatographic analysis demonstrated that thymine dimers production is the main modification of DNA with very low yields of other photoproducts.

Therefore, dependently on the type of photoligand and source of light selected, the following processes may be induced: 0

DNA sequence restriction, and

DNA sequence modification, for instance, formation of thymine dimers or products of crosslinking reactions.

Industrial utility

The present invention may be utilized for DNA site-specific restriction and site-specific mutagenesis in gene engineering, biotechnology, medicine, veterinary.

Claims

We claim :

1. Method for treatment of deoxyribonucleic acid (DNA) sequence by restrictase, suitable for said treatment of any of said DNA sequence, comprising the following steps: binding co-valently a chemical compound capable to recognize specifically a concrete DNA site with a photoligand to obtain a new substance; adding the substance obtained to said DNA sequence to receive a complex; illuminating by light the complex received with wave lengths selected from spectrum covered visible and ultra violet ranges ; and providing restriction of said DNA sequence at place of said site recognized by said chemical compound.

2. Method for treatment of deoxyribonucleic acid (DNA) sequence by restrictase, suitable for said treatment of any of said DNA sequence, comprising the following steps: binding co-valently a chemical compound capable to recognize specifically a concrete DNA site with a photoligand to obta- in a new substance; adding the substance obtained to said DNA sequence to receive a complex; illuminating by light the complex received with wave lengths selected from spectrum covered visible and ultra violet ran- ges ; and providing modification of said DNA sequence at place of said site recognized by said chemical compound.