MASARYK UMVB8TTY Design of PCR Primers gtča I lhj I i u I uuacuai (j ATCATGTTTT CCTTGATATC ATGT "GCTTCAACA CTCCAAATAC AGAGGTAATT AAATATTATT ATCA OATAATATG TTATTGATTT TTTGTTTGTG ATTTCATTTA GATTT "CTATGATTT CTTAGCATGA AATACAATTT ttgg AG AAA C AACT TTAAAAACA aaacttgaat TTTGAGAAAT TCAAAGATGT TATA" GTCAAAATT TAACAATTAT TCTTCTAAAT CATC CG G ATT CCGT BTACACATCT ACAATTTTCA ATTGAGGTAT TCTTGTTTTG ATGC 3ACGAATAGT TTGATTGATA AAAAAAATTC TAACCAATAT G ATA jTTTATTTTC I I M TGTCAA ACCATACTTT ATACTATGTA AC^TT BAGATTATTG AAAATAGTTT ATTTATAAAA TAGTAACCTA TTGT1 \AAAAAAAAA AAAAAATTGT AAATCGTGTT TGCAAACGAC ATG1 'CTTAGTTTJK AAACTVgcTg ATaTTcTFCA AATCGACTGT TCTT, ^ATCAACCflA ttaooatoaa t oa oaa*mAA TTGTAAACAC TTCA VTGGTGATTT TAAAGAATAT GTTTTACTTA TGTTATGAAC TATC" TGTGAAATA TTTCATAACT AATGTGGAAA ACTATATAAC CCCT VAAACGTAAG TAAAATTTAT GAAATCCTAT CATTTTTAAA GGTT> VTCAAAAAGT AATAATTCTT GGTACTTGCA ATAT I I I I GT CA1H7 "AGTTTATTA ATTTTATTTT GATTAAATGG TTTTAGATCC ATCAG íAGATCGCAG TTATAGCTGT AGACGATCCG AAGAAAGCAT TA1 AAAAATTCAA CGAGACAATA TAGATCTCAT AATCACAGAT TAT1 CTGGTATGAA CGGTTTACAA CTCAAAAAAC AAATCACTCA GGfi AATTTACCGG TCTTAG GTAA CAT I í I I I GT TCTTTACAAC TTAA Proteomics Core Facility CEITEC Central European Institute of Technology NCBR National Centre for Biomolecular Research Hana Konečná CG920 Genomics Lecture 12 OLIGONUCLEOTIDES • definition • aplications i • modifications • design of sequence • synthesis • rules • purification • software OLIGO 7 • quality control • example MASARYK uiwBsrrv oligonucleotide short single stranded structure DNAor RNA (also PNA) hydroxyl on both ends (no phosphate on 5-end as usual) orientation! polymerase 5' end H |]TT| MASARYK [lull uiwBsrrv Aplications of synthetic oligonucleotides • primers for synthesis of complementary DNA PCR, Real-Time PCR • gene synthesis and recombinant proteins • hybridisation probes for cloning • site directed mutagenesis • sequencing ang genetic profiling • diagnostics - tests and biosensors • gene arrays • blocation of gene expression antisense oligo • prospective therapeutics and DNA vaccines • NMR monitoring of DNA - protein interactions • structural X-ray analysis of NA 4 MASMYK UMVBSTTY • degeneration • end of sequence • bases • phosphate • carbohydrate • PNA [5J-conJugate|^ B heterocyclic base phosphodiester bridge B-o-deoxyribose ' 0 (sugar) 0=P-o" B V o -conjugate MASARYK uiwBsrrv Degenerated oligonucleotides Examples: ACG TAC GTA CGTACG TAC non-degenerated ACG TAM GTA CGT ACG TAC M = A/C ACG TAC GTA CDT ACG TAC D = A/G/T ACG TAC GTA CGT ACG NAC N = A/C/G/T MASARYK uiwBsrrv Degenerated oligonucleotides 2-deoxyinosin M AorC R AorG W AorT S C orG Y C orT K G orT V AorC orG H AorC orT D AorG orT B C orG orT N G or A orT or C X G or A orT or C MASARYK UMVBtSTTY postsynthetic modifications sequencing fragmenation analysis gene arrays Real-Time PCR Phosphorylation -► Amino group -► Thio group Digoxigenin -► Biotin Enzymes Psoralen Acridine Cholesterole -► Fluorescent dyes Quenchers 2,4- dinitrophenyl Spacer Branching Blocation MASARYK uiwBsrrv derivatized matrix Phosphate Thio group Amino group Spacer Acridine Biotin Fluorescent dyes Quenchers Cholesterole 2,4 dinitrophenyl 9 MASARYK uiwBsrrv rawwflRD pniMcn • 2x labeled probe • REPORTER • QUENCHER 2, Strand displacement: When the probe is intact, the reporter dye emission is quenched. r. a1 5_9, r I' 3, Cleavage: During each extension cycle, the DNA polymerase cleaves the reporter dye from the probe. SV J" —^ B' 9' 4, Polymerization completed: Once separated from the quencher, the reporter dye emits its characteristic fluorescence. MASARYK uiwBsrrv Other modifications Phosphorothioates <- backbone Phosphorodithioates H-phosphonates Methylphosphonates .... Modifications in 2'- position carbohydrate-- modification 11 MASARYK UMVBtSTTY TheraoeutiCS _* Nondegradable by nucleases! Modification of phosphodiester O 0 0 phosphorothioate methylphosphonate phosphoramidate S "CH2 "O i I 2 i CH2 H3C-N 0 = P-BH, 0 0 3-thioformacetal methylene(methylimlnio) boranophosphate 12 MASARYK UMVBsmr ANTISENSE oligonucleotide oligonucleotide or analogue complementary to segment of RNA or DNA inhibition of normal function due to coupling Antigone (triplex) Ribozymo Antisense Sense/Aptamer RNA with catalytic activity 13 MASARYK uiwBsrrv Peptidonucleic acid PNA DNA • uncharged molecule • binding with DNA/RNA N-(2-aminoethyl)-glycine-► i—NH 0 H2N— N-terminal """ ' \ ^ 5'-er^'^^ o=< o=p-o-RepeatJ 1 J \j L o=( o=p-o" 1 O 3 -end 0 C-terminal °K 0=^-0" PNA DNA 14 MASARYK UIW9STTY Peptidonucleic acid noncharged molecule binding with DNA/RNA N-(2-aminoethyl)-glycine- •NH O PH R,N- PNA MASARYK uiwBsrrv Why PNA • thermostable • Tm not depending on salts • high specificity • high affinity • resistant towards enzymes Gambari R. Expert Opin Ther Pat. 2014, 24(3):267-94. Peptide nucleic acids: a review on recent patents and technology transfer. 16 MASARYK UMVHWTTY I o=p-o Locked Nucleic Acid I • 2'-0, 4'-C methylene bridge • supressed flexibility of ribofuranose ring • structure is locked into rigid C3-endo conformation • enhanced hybridisation • outstanding biostability Molecular Therapy (2012); 20 8, 1590-1598. LNA-based Oligonucleotide Electrotransfer for miRNA Inhibition MASARYK uiwBsrrv design synthesis purification EXPEDITE 8909 18 MASARYK UMVBWTY Oligonucleotide Synthesis synthesis on solid matrix from 3'-end to 5'-end anhydrous environment O ii B, O n -O— P - o —, r t> n II - 0— p _0 o- N OH Cleevage/Deprotection ^1 B, °— P -O ^ J- I I \ OR Step 2. Coupling ^ O - HO- - o Stepi. Deblocking DMT- O Activation E ŕ - 0 — P - N(iPr), OR ♦ Tetrazole 19 MASARYK uiwBsmr Quality Control ISO.bia - 20.0U1 0.5 0.0 IS 20 Hln 25 30 35 HPLC perfusion chromatography anex RP 20 MASARYK UMV9STTY Perfusion chromatography 1341ong.bio - 20.0ul classical sorbent POROS 0.1JH 0,10- 0.05H 0.00 100 5 10 l's 20 ^2*5 30 35(^tg? Min 134.bio - 20.0ul 2 6 : a ■ 0.15H 0.1