Chain-terminating nucleotides

What is the property of the chain-terminating nucleotides that enables them to terminate the polymerizing DNA chain ... 

The most successful applications of CE-LIF detection have been in the analysis of carbohydrates and nucleic acids. Carbohydrates can be derivatized with agents such as aminopyrene trisulfonic acid, which introduces a fiuorophore and also confers electrophoretic mobility to the analyte. Double-stranded nucleic acids can be detected using intercalating dyes and by covalent attachment of nucleotides carrying fluorophores. The fluorescently labeled primers or chain terminating nucleotides which are widely used in automated slab gel-based DNA sequencers are also used in capillary-based sequencers. In one multicapillary sequencer, a novel sheath flow system is used for LIF excitation of separated bands with imaging of emitted fluorescence on a charge-coupled device (CCD) detector. 

Because dideoxynucleotides lack 3 -OH groups, these nucleotides cannot serve as acceptors for 5 -nucleotide addition in the polymerization reaction, and thus the chain is terminated where they become incorporated. The concentrations of the four deoxynucleotides and the single dideoxynucleotide in each reaction mixture are adjusted so that the dideoxynucleotide is incorporated infrequently. Therefore, base-specific premature chain termination is only a random, occasional event, and a population of new strands of varying length is synthesized. Four reactions are run, one for each dideoxynucleotide, so that termination, although random, can occur everywhere in the sequence. In each mixture, each newly synthesized strand has a dideoxynucleotide at its 3 -end, and its presence at that position demonstrates that a base of that particular kind was specified by the template. A radioactively labeled dNTP is included in each reaction mixture to provide a tracer for the products of the polymerization process. 

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

D Abramo CM, Cellai L, Gotte M (2004) Excision of incorporated nucleotide analogue chain-terminators can diminish their inhibitory effects on viral RNA-dependent RNA polymerases. J Mol Biol 337 1-14... 

Coelmont L, Paeshuyse J, Windisch MP, De Clercq E, Bartenschlager R, Neyts J (2006) Ribavirin antagonizes the in vitro anti-hepatitis C virus activity of 2 -C-methylcytidine, the active component of valopicitabine. Antimicrob Agents Chemother 50 3444-3446 D Abramo CM, Cellai L, GOtte M (2004) Excision of incorporated nucleotide analogue chain-terminators can diminish their inhibitory effects on viral RNA-dependent RNA polymerases. J Mol Biol 337 1-14... 

The terms first, second, and third nucleotide refer to the individual nucleotides of a triplet codon. U, uridine nucleotide C, cytosine nucleotide A, adenine nucleotide G, guanine nucleotide Term, chain terminator codon. AUG, which codes for Met, serves as the initiator codon in mammalian cells and encodes for internal methionines in a protein. (Abbreviations of amino acids are explained in Chapter 3.)... 

Mechanism of Action A nucleotide analog that inhibits HIV reverse transcriptase by being incorporated into viral DNA, resulting in DNA chain termination. Therapeutic Effect Slows HIV replication and reduces HIV RNA levels (viral load). Pharmacokinetics Bioavailability in fasted patients is approximately 25%. High-fat meals increase the bioavailability. Protein binding 0J%-12% Excreted in urine. Removed by hemodialysis. Half-life Unknown. 

One of the motive powers for the design of host molecules for nucleotides is their potential utility in chemotherapy. A number of nucleotide analogues exhibit antiviral activity in vitro [73] for example, 2, 3 -dideoxynucleosides are potent chain-terminating inhibitors of HIV reverse transcripase [74]. However, charged and hydrophilic as they are, they can hardly penetrate across cell... 

The NRTIs act by competitive inhibition of HIV-1 reverse transcriptase incorporation into the growing viral DNA chain results in premature chain termination due to inhibition of binding with the incoming nucleotide (Figure 49-4). Each requires intracytoplasmic activation via phosphorylation by cellular enzymes to the triphosphate form. Most have activity against HIV-2 as well as HIV-1. 

Tenofovir is an acyclic nucleoside phosphonate (ie, nucleotide) analog of adenosine (Figure 49-2). Like the nucleoside analogs, tenofovir competitively inhibits HIV reverse transcriptase and causes chain termination after incorporation into DNA. However, only two rather than three intracellular phosphorylations are required for active inhibition of DNA synthesis. 

Although initially and abortively developed for treatment of HIV infection, adefovir dipivoxil gained approval, at lower and less toxic doses, for treatment of HBV infection. Adefovir dipivoxil is the diester prodrug of adefovir, an acyclic phosphonated adenine nucleotide analog (Rgure 49-2). It is phosphorylated by cellular kinases to the active diphosphate metabolite and then competitively inhibits HBV DNA polymerase to result in chain termination after incorporation into the viral DNA. Adefovir is active in vitro... 

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