Nucleosidtriphosphater - Fra Syntese til Biokemisk karakterisering
Summary
Den heri beskrevne protokol har til formål at forklare og forkorte de mange hindringer i vejen for den indviklede rute fører til modificerede nucleosidtriphosphater. Derfor denne protokol letter både syntesen af disse aktiverede byggesten og deres tilgængelighed for praktiske anvendelser.
Abstract
Den traditionelle fremgangsmåde til indførelse af kemiske funktionaliteter er brugen af fastfasesyntese ved at tilføje passende modificeret phosphoramidit forløbere for den spirende kæden. Men de betingelser, der anvendes under syntesen, og begrænsningen til temmelig korte sekvenser hæmmer anvendelsen af denne metode. På den anden side er modificeret nukleosidtriphosphater aktiveret byggesten, der har været ansat i den milde introduktion af talrige funktionelle grupper i nukleinsyrer, en strategi, der baner vejen for brug af modificerede nukleinsyrer i en bred palet af praktiske anvendelser såsom funktionel tagging og generering af ribozymer og DNAzymer. En af de største udfordringer ligger i den indviklet af den metode, der fører til isolering og karakterisering af disse nukleosidanaloger.
I denne video artikel præsenterer vi en detaljeret protokol til syntese af THESe modificerede analoger ved hjælp af phosphor (III)-baserede reagenser. Desuden er proceduren for deres biokemiske karakterisering videregives, med særlig vægt på primerforlængelsesreaktioner og TdT hale polymerisering. Denne detaljerede protokol vil være til nytte for skabelsen af modificerede dNTP'er og deres videre anvendelse i kemisk biologi.
Introduction
5'-nucleosidtriphosphater ((D) NTP) udgør en klasse af vitale biomolekyler, der er involveret i utallige processer og funktioner, der spænder fra at være den universelle valuta af energi til regulatorer af celle metabolisme. Ud over deres rolle i disse fundamentale biologiske forandringer, deres modificerede modstykker avanceret som en alsidig og mild platform for indførelsen af funktionelle grupper i oligonukleotider, en metode, pænt supplerer automatiserede fastfasesyntesen der normalt anvendes 1,2. Forudsat, at de (d) NTP'er kan fungere som substrater for RNA og DNA-polymerase 3, et væld af funktionelle grupper, herunder aminosyrer 4-13, boronsyrer 14,15, nornbornene 16 diamondoid-lignende rester 17, side-kæder til Organocatalysis 18 galdesyrer 19, og endda oligonukleotider 20 kan indføres i oligonukleotider.
_content "> Beyond repræsenterer en bekvem vektor til funktionalisering af nukleinsyrer kan modificerede dNTP'er være engageret i SELEX og andre beslægtede kombinatoriske metoder til in vitro-selektion til generering af modificerede katalytiske nukleinsyrer 21-30 og aptamere til forskellige praktiske anvendelser 10, 31-36. De yderligere sidekæder, der er indført ved polymerisation af de modificerede dNTPs menes at øge den kemiske plads, der kan udforskes i løbet af et valg eksperiment og supplerer temmelig dårlig funktionel arsenal af nukleinsyrer 37. Men trods disse attraktive træk og de seneste fremskridt i udviklingen af både syntetiske og analytiske metoder, ingen universelt anvendelig og højtydende procedure findes for skabelsen af modificerede nucleosidtriphosphater 2,38.Formålet med denne protokol er at kaste lys ind i (undertiden) indviklede procedurer, der fører to syntesen og biokemisk karakterisering af disse aktiverede byggesten (figur 1B). Vil blive lagt særlig vægt på samtlige syntetiske detaljer, som ofte er svære at finde eller er fraværende i eksperimentelle sektioner, men endnu afgørende for en vellykket gennemførelse af den syntetiske vej, der fører til isolation af rene (d) NTP'er (Figur 1).
Protocol
Representative Results
Discussion
Inddragelsen af ændringer i nukleinsyrer er af interesse for mange praktiske anvendelser, herunder udvikling af antisense-og antigenmidler 42,43, mærkning og funktionel tagging af oligonukleotider 41, og i bestræbelserne på at udvide den genetiske alfabet 44-46. Kemiske ændringer og funktionelle grupper er normalt indført i nukleinsyrer ved anvendelse af standard-og automatiserede fastfasesyntese protokoller. Men phosphoramiditfremgangsmåden byggesten har brug for, for at kunne mo…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af den schweiziske National Science Foundation (Tilskud n ° PZ00P2_126430 / 1 og PZ00P2_144595). Prof. C. Leumann er modtaget med tak for at give laboratoriet plads og udstyr, samt for hans konstante støtte. Sue Knecht er anerkendt for frugtbare diskussioner.
Materials
| tributylammonium pyrophosphate | Sigma Aldrich | P8533 | Hygroscopic solid, keep under Ar |
| 2-chloro-1,3,2-benzodioxaphosphorin-4-one | Sigma Aldrich | 324124 | Moisture sensitive |
| Pyridine | Sigma Aldrich | 82704 | Under molecular sieves |
| Dioxane | Sigma Aldrich | 296309 | Under molecular sieves |
| dimethylformamide (DMF) | Sigma Aldrich | 40248 | Under molecular sieves |
| Acetonitrile | Fisher Scientific | HPLC grade | |
| Triethylamine | Sigma Aldrich | 90342 | |
| Tributylamine | Sigma Aldrich | 90781 | |
| ddH2O | Milli-Q | deionized and purified water, autoclaved in the presence of Diethylpyrocarbonate (DEPC) | |
| Diethylpyrocarbonate (DEPC) | Sigma Aldrich | 159220 | |
| D2O | Cambridge Isotope Laboratories, Inc. | DLM-4-25 | |
| Biochemical reagents | |||
| g-[32P]-ATP | Hartmann Analytics | FP-301 | |
| Natural dNTPs | Promega | U1420 | |
| Vent (exo–) DNA polymerase | NEB | M0257S | |
| DNA polymerase I, Large (Klenow) Fragment | NEB | MO210S | |
| 9°Nm DNA polymerase | NEB | MO260S | |
| Terminal deoxynucleotidyl Transferase (TdT) | Promega | M828A | |
| Pwo DNA polymerase | Peqlab | 01 01 5010 | |
| T4 PNK | Thermo Scientific | EK0032 | |
| Acrylamide/bisacrylamide (19:1, 40%) | Serva | 10679.01 | |
| Agarose | Apollo Scientific | BIA1177 | |
| G10 Sephadex | Sigma | G10120 | |
| Urea | Apollo Scientific | BIU4110 | |
| Equipment | |||
| Jupiter semi-preparative RP-HPLC column (5m C18 300Å) | Phenomenex | ||
| Gene Q Thermal Cycler | Bioconcept | BYQ6042E | |
| PCR vials | Bioconcept | 3220-00 | |
| HPLC system | Amersham Pharmacia Biotech | Äkta basic 10/100 | |
| Oligonucleotides | |||
| All oligonucleotides were purchased from Microsynth and purified by PAGE | |||
| 5'-CAAGGACAAAATACCTGTATTCCTT P1 | |||
| 5'-GACATCATGAGAGACATCGCCTCTGGGCTAAT-AGGACTACTTCTAATCTGTAAGAGCAGATCCCTGG-ACAGGCAAGGAATACAGGTATTTTGTCCTTG T1 | |||
| 5'-GAATTCGATATCAAG P2 | |||
| More information on experimental procedures and equipment can be found in the following articles: | |||
| Chem. Eur. J. 2012, 18, 13320 – 13330 | |||
| Org. Biomol. Chem. 2013, DOI: 10.1039/C3OB40842F. |
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