Unit II Paper 10 MBT II for Msc BT

1. Prepared by,
K.P.Senthil Kumar.,M.Sc.,M.Phil.,ADAB

2. In biological systems,
 Photoreactive derivatives to study specific
interactions
 Ex:Receptor molecules with their ligands by
photoaffinity labeling
 Receptors are generally proteins e.g., enzymes,
immunoglobulins, or hormone receptors.
The ligands differ widely in their molecular
structure e.g., sugars, amino acids, nucleotides, or
oligomers of these compound.

3. The advantage
 Its Preferred over affinity labeling, or chemical
modification with group-specific reagents
 Photoactivatable nonreactive precursors can be
activated at will by irradiation .
These reagents do not bind covalently to the protein
unless activated.
 On irradiation of the precursors, highly reactive
intermediates are formed.
 That react indiscriminately with all surrounding
groups.
 After activation, a photoaffinity label, interacting
at the specific binding site, can label all the different
amino acid residues of the binding area.

4.  Today, aromatic azido compounds are mostly used
as photoactivatable ligand analogs.
 They form highly reactive nitrenes upon irradiation
because of the electron sextet in the outer electron
shell of these intermediates .
 In addition to the azido derivatives, photoreactive
precursors forming.
 Radicals or carbenes on irradiation can be used as
photoaffinity labels. All of these intermediates (e.g.
nitrenes), vigorously try to complete an electron octet
.

6. To produce covalent crosslinks between proteins and
DNA, various methods have been applied :
 Ultraviolet (UV) irradiation, γ-irradiation, chemical
methods, and even vacuum or extreme dryness.

7. To date, many successful attempts have been made to
photocrosslink proteins to nucleic acids using different
photoactivatable deoxynucleotides.
 5-bromo-,5-iodo-, 5-azido-, and
5-[N-(p-azidobenzoyl)-3-aminoallyl]-2'-deoxyuridine-
5'-monophosphate ,
4-thio-2'-deoxythymidine-5'-monophosphate ,
and 8-azido-2'-deoxyadenosine-5'-monophosphate (have
been incorporated into deoxyribonucleic acids to bind
DNA covalently to adjacent proteins

8. Methods
1. Synthesis of 8-N3dATP:
The synthesis of 8-N3dATP is performed principally by
analogy to the synthesis of 8-N3ATP .
In the first step, bromine exchanges the hydrogen at
position 8 of the adenine ring.
Then, the bromine is substituted by the azido group

9. 2. Characterization of 8-N3dATP:
Thin-layer chromatography (TLC). TLC is carried out on
silica gel plates F254 or cellulose plates F. The development is
performed in either isobutyric acid/water/ ammonia (66:33:1
v/v) or n-butanol/water/acetic acid (5:3:2 v/v).
Ultraviolet absorbance. Record the UV absorbance spectrum
of 8-N3dATP. It shows a maximum at 280 nm. The UV
absorbance of 8-N3dATP is pH dependent.

10. Photoreactivity. The photoreactivity of 8-N3dATP is tested
by two different methods. It can either be demonstrated by the
spectroscopic observation of the photolysis or by the ability of
the photolabel to bind irreversibly to cellulose on thin-layer
plates on UV irradiation prior to the development of the
chromatogram.

11. 3. Preparation of Azido-Modified DNA
Azido-modified and [32P]-labeled DNA are prepared by
nick translation.
 The detailed and exact composition of the reaction
medium depends strongly on the size as well as on the
amount of the DNA to be modified.
The optimal ratio of DNA, DNase I, and DNA
polymerase I (Kornberg enzyme) should be tested in
preliminary experiments

12. 4. Photocrosslinking
An ultraviolet lamp emitting UV light at wavelengths
of 300 nm and longer.
1. Prepare 20–30 μL aqueous solutions containing the
photoreactive DNA (0.5 pmol)
and the protein (1–25 pmol) to be cross-linked .
2. Incubate the reaction mixture for 10 min at 37 C in
the dark.
3. Expose the sample to UV irradiation .
The irradiation times can be chosen in a range
from 1 s to 60 min .
4. Keep the solutions in the dark before and after
photolysis .

13. 5. Analysis of DNA-Protein Adducts
 By polyacrylamide gel electrophoresis of the
irradiated samples followed by autoradiography.
SDS–polyacrylamide gel electrophoresis should
be performed immediately after photocrosslinking
according to Laemmli with some variations.
After the addition of 20 mg/mL of bromophenol
blue, the samples are loaded onto a SDS–
polyacrylamide gel of 5% polyacrylamide
(separating gel) with an overlay of 3.5%
polyacrylamide (stacking gel) containing 1% SDS.
After the electrophoretic separation, the gels are
silver-stained according to Adams and Sammons
,dried, and exposed to X-ray film at –70 C.

14. A quantitative determination of the DNA–protein
adducts is possible by densitometric measurement of the
autoradiogram .
Another possibility to detect the DNA–protein adducts is
the application of the nitrocellulose filter binding assay
according to Braun and Merrick .

15. Reference:
•Rainer Meffert, Klaus Dose, Gabriele Rathgeber, and
Hans-Jochen Schäfer. Ultraviolet Crosslinking of DNA–
Protein Complexes via 8-Azidoadenine. Chapter 23.
Methods in molecular biology. 2001(43) 323-335; Dna –
Protein Interactions Principles and protocols. Humana
Press Inc.