Fluorescent labeling of molecules Fluorescence methods in life sciences Ctirad Hofr 11 1 Fluorescent dyes - are added to the studied sample and bind to it covalently. They bind to proteins and nucleic acids through the amino or thiol groups and side chains. Fluorescent probes - bind to the studied sample non-covalently and after binding they change their fluorescence properties (e.g. intensity, position of emission maximum) Possibility of fluorophore introducing Covalent bond it uses a chemical reaction of fluorophore derivative, during which a covalent bond with biomolecule is created Non-covalent bond fluorophore binds to a biomolecule through non-covalent (e.g. electrostatic) interactions Fluorogenic reactions it uses a chemical reaction, during which a non-fluorescent precursor is changed to the fluorophore 11 3 o c R" OR' They are formed by reaction of carboxylic acid and alcohol R-C O + R'-OH R-C O + H90 O-H O-R' carboxylic acid alcohol ester 11 4 Amides are formed by reaction of ester and amine R-C O + hLN-R r f R-C O O-FT \ N-F ester amine amide Reaction of esters for covalent labeling of molecules with NH2 group 11 F F STP Ester Fťc-o-rí SucclnlmkJyl Ester TFP Ester R1C-NHR2 + HO Carboxamide SO, R1C—NHR2 Carboxamide It uses a reaction of ester with amine to form amide Reaction of carboxylic acid of dye Alexa 488 with NH2 group of protein Alexa Fluor® 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester (Alexa Fluor® 488 11 5-TFP) How to add NH2 group to DNA? Aliphatic chain terminated with NH2 group (amino-linker) is attached directly in the synthesis of oligonucleotide 0 II O-P-O—, H B Modification of the 5' end by the amino linker 0 1 e ® 0=P-0 H i 0 11 8 3CW SO£WH(CH2)5-C-0-N NH. Rhodamine Red™-X3 succinimidyl ester DNAwith „amino-linker DNA labeled with Rhodaminem Red-X 11 10 Other reactions for labeling through the amino group Reaction of an isothiocyanate with a primary amine R1N-C-S + R£NH, s 1 11 2 R NH-C—NHR Isothiocyanate Thiourea Reaction of sulfonic acid chloride with an amine r1so2ci + Sulfonyl chloride R2NH, R1^—NHR2 + HCI Sulfonamide 11 11 Connection through SH thiol group to form a thioether A R1 R2 • Thioether is similar to ester, except the O is replaced by S • Thioether is formed by reaction of alkylating agents (e.g. halogen, maleimide) with thiols (contain SH group) 11 Reaction for covalent labeling of molecules with SH group Reaction of thiol group with maleimide to form a thioether The double bond of the maleimide reacts with SH thiol group to form a thioether o o Maleimide Thioether Other reactions for labeling of molecules with SH group Disulfides r1s-sr1 + R2SH R1S-SR2 + R1SH Symmetric disulfide Mixed disulfide 11 14 Reaction catalyzed by Cu2+ Azide-alkyne cyclization chemistry for the detection of A) proteins and B) sugars Reaction partners A) L-homopropargylglycine (HPG) and Alexa Fluor 488 azide and B) A/-azidoacetylgalactosamine and Alexa Fluor 488 alkyne Partner on the left is introduced into proteins by de novo synthesis or posttranscriptional modifications DNA can be covalently labeled without connection of NH2 group in the synthesis It uses a reactivity of N7 guanine with platinum complexes Platinum complex contains a fluorescent dye which is covalently attached to guanine after the reaction 16 Separation of unbound dye Chromatography (protein) gel filtration Ultrafiltration Dialysis Precipitation (DNA) Peptid Target Prolin Tag Target Protein Flueragenic probe Target Pi *-rt n • A) Receptor-protein - fluorescent dye or probe is attached to a molecule that has high affinity to receptor (avidin - biotin) B) Binding mediated by the enzyme - fluorophore is attached to the substrate that can be covalently bound to a peptide "tag" of protein through another protein - enzym • C) Probe does not emit fluorescence originally. After its binding to "tag", activation of fluorescence occurs (fluorogenic reaction) Dragulescu-Andrasi, A., and Jianghong Rao, ChemBioChem, 2007 Donor HO /n wVo labeling of p-gal by a fluorogenic probe with spectral change. The labeling is proposed to take place in two steps. The first step involves O-galactoside bond cleavage, which generates an active intermediate quinone methide. This intermediate is susceptible to nucleophilic attack by a nearby amino acid residue, which leads to covalent attachment of the FRET donor (D) to the enzyme and displacement of the acceptor (A). - Target DNA Sequence complementary to target DNA I INI Mill I IT Hybrid Fluorophore Quencher 11 Beacon in English means signal fire (no bacon) DNA beacon consists of a DNA molecule that is able to form a hairpin structure DNA has a fluorophore attached at one end and a quencher at the other end In hybridization with a complementary DNA, the quencher is displaced and fluorescence is emitted subsequently 20 Structure and properties „DNA beacon" Increasing temperature leads to melting of hairpin structure, which results in increase of fluorescence intensity i 0" EDANS Dabcyl Note: In case of Dabcyl, it was found an unexpected ability to quench a wide range of fluorophores regardless of degree of spectral overlap. The reason for this universal quenching is probably formation of non-fluorescent complex of Dabcyl with fluorophore. Anyway, it is an advantage because one quencher can be used for a wide range of fluorophores. 5-C T 6GTCTT CTT-3' G-C A-T G-C C-G G-C In the case of hybridization with the DNA that differ only in one nucleotide, there is no increase in fluorescence signal 5-C T GGTGTT CTT-3' 5-C T GGT-TT CTT-3' _i_i_ 10 15 High sensitivity and the ratio on/off signal in the presence/ in the absence of complementary DNA TIME (minutes) Using of Dabcyl-Fluorescein couple for „DNA beacon" to monitor PCR Number of DNA beacon molecules in the sample Fluorescence intensity is directly proportional to the amount of amplified DNA 0 10 20 30 «0 50 CYCLE NUMBER 23 Donor beacon Acceptor beacon Exon G "Forget Exon 7 S -TGA C A ACTT TG GTATCGTGGA AGGAC TC ATG /A C C A CAGTC CATGC C ATCAC TGCC ACCC A -3' Dabcyl ^ACCATaGCACCTTCCTGAG^ GGTGTCAGGTACGGTAGT,, Cy3 FAM Acceptor Beacon Donor Beacon Qabcyl (A o z LU C_> (/> LÜ CC O 3 1.2 1.0 0.8 0.6 0.4 0.2 0 500 fam Donor and acceptor beacons, no target 0+T Donor beacon with target A+T Acceptor beacon with target Donor and acceptor beacons with target - / \p+t fam \ d+a+t d+a + t /d+a >^-**r—A+T _i_i_:_:-:-1 525 550 575 600 625 emission wavelength (nml 650 A preferred arrangement to reduce false positive results It use two hairpins One hairpin contains donor and the second contains acceptor of FRET Without target DNA, both fluorophores at the hairpin are quenched If the correct hybridization with target DNA occurs, FRET is observed If only one hairpin is hybridized, although the increase in intensity occurs, FRET is not increased This increases the specifity of the analysis 24 11 Optical and fluorescent display of kidney cells Increase in fluorescence intensity of "DNA Beacon" against the mRNA for p-actin demonstrated an increase in the concentration of mRNA and consequently an increase in production of p-actin 25 11 FISH (fluorescence in situ hybridization) Metaphase chromosomes DNA Probes Hybridization L is .1 J/7 34? /c if ffi$ /d tumescence detection DNA in the form of metaphase chromosomes is exposed to hybridization with fluorescently labeled DNA probe DNA probe has a sequence complementary to the target DNA on the chromosome After hybridization, a part containing the target DNA is localized based on fluorescence 26 Fluorescent deoxyribonucleotides for DNA synthesis of FISH probes Chromosomes hybridized with fluorescently labeled probes Chromosomal DNA was stained with DAPI nonspecifically s' ssDNA of Unknown Sequence 3' I I I I I 1 I I I I I g c g g T c c g I I I I I g c g c a 5 c g T N j DMA polymerase I + 4 dNTPt + L ddfJTPs I I I I Labeled S primer dd TP 4i e o < Initial sequencing used a radioactively labeled primer The primer was extended by DNA polymerase according to sequenced chain To a mixture of normal nucleotides (dNTP) there was always added one type of dideoxynucleotide (ddNTP) that prevents further elongation of synthesized chain This generates a mixture of DNA chains with variable length that always ends by a given ddNTP Each reaction mixture for a given ddNTP was analyzed in one electrophoretic well (total in 4 wells) The sequence was determined from the position of band for the corresponding ddNTP on the gel 5' o" o" er I I I "o-p-o-p-o-p-o-CH2 II 11 11 O 4' o o Phosphate group Not available for -phosphodiester bond formation H 3' O 0 Fluorescent Probe I Pyrimidine or Purine base Hi 1* H 2' Pentose sugar ddNTP H dNTP Hydroxyl group in DNA OH Dideoxynucleotide triphosphate ddNTP At position 3' of ribose, OH group is replaced by an H, which prevents further DNA chain elongation The labels are connected through an acetylene triple bond All labels are excited by one laser (488nm) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides JM Prober, GLTrainor, RJ Dam, FW Hobbs, CW Robertson, RJ Zagursky, AJ Cocuzza, MA Jensen, and K Baumeister Science 16 October 1987 238: 336-341 Fluorescent labeling accelerated the sequencing GCGGT CCG I sspNA of Unknown Sequence t I I I I I I J I II I I I I I I G C G C G C G T I I I I 3' S' Unlabeled primer DNA polymerase I + 4 ddNTPs + L fluorescent ddNTPs V u i < | G-505 5' ■ :- I T-526 T3P at u c Using four fluorescent labels allowed to use only one lane in the gel for identification of all nucleotides => 4-fold acceleration Fluorescence scanning enabled to complete genome sequencing projects of whole organisms in a significantly shorter time 3' 11 32 2 December 1999 A International weekly journal of science nature THE HUMAN XZji GENOME The most common sequencing primer is M13i 5'- gTA AAA CgA Cgg CCA gTg -3' http://www.wilev.com/colleqe/pratt/0471393878/stu dent/animations/dna sequencinq/index.html Lakowicz J.R.: Principles of Fluorescence Spectroscopy. Third Edition, Springer + Business Media, New York, 2006. Fisar Z.: fluorescence Spektroskopie v neurovedäch http://www1 - If 1 .cuni.cz/~zfisar/fluorescence/Default.htm Graphics from the book Principles of Fluorescence was for the purpose of this lecture kindly provided by Professor JR Lakowitzem.