Guanine-rich DNA nanocircles for the synthesis and characterization of long cytosine-rich telomeric DNAs.
Chembiochem
; 6(8): 1458-62, 2005 Aug.
Article
en En
| MEDLINE
| ID: mdl-16052615
ABSTRACT
Short synthetic oligonucleotides derived from the human telomeric repeat have been studied recently for their ability to fold into four-stranded structures that are thought to be important to their biological function. Because telomeric DNAs are several kilobases in length, however, their folding might well be affected by cooperative or high-order interactions in these long sequences. Here, we present a new molecular system that allows for easy synthesis of very long stretches of the cytosine-rich strand of human telomeric DNA. Small circular DNAs composed of the G-rich sequence of human telomeres were prepared and used as templates in a rolling-circle replication mechanism. To facilitate the synthesis of the repetitive G-rich circles, an orthogonal base-protection strategy that made use of dimethylformamidine-protected guanine nucleobases was developed. Nanometer-scale circles ranging in size from 42 to 54 nucleotides were prepared. Subsequently, we tested the action of various DNA polymerases on these circular templates, and identified DNA Pol I (Klenow fragment) and T7 DNA polymerase as enzymes that are able to generate very long, C-rich telomeric DNA strands. Purification and initial structural examination of these C-rich polymeric products revealed evidence of a folded structure in the polymer.
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Bases de datos:
MEDLINE
Asunto principal:
ADN
/
ADN Circular
/
Telómero
/
ADN Polimerasa I
/
ADN Polimerasa Dirigida por ADN
/
Guanina
Límite:
Humans
Idioma:
En
Revista:
Chembiochem
Asunto de la revista:
BIOQUIMICA
Año:
2005
Tipo del documento:
Article
País de afiliación:
Estados Unidos