Tài liệu Báo cáo khoa học: Stem–loop oligonucleotides as tools for labelling double-stranded DNA pdf

Stem–loop oligonucleotides as tools for labelling

double-stranded DNA

Be´ne´dicte Ge´ron-Landre, Thibaut Roulon and Christophe Escude´

Laboratoire ‘Re´gulation et Dynamique des Ge´nomes’, De´partement ‘Re´gulations, De´ veloppement et Diversite´ Mole´ culaire’, Muse´um

National d’Histoire Naturelle, Paris

Triple-helix forming oligonucleotides (TFOs) repre￾sent an interesting tool for the sequence-specific

recognition of double-stranded DNA. They can be

used for the artificial modulation of DNA informa￾tion processing [1] and for other applications that

take place in vitro, such as double-stranded DNA

isolation, labelling or modification (reviewed in [2]).

Formation of DNA triple helices has been studied in

details for the past 15 years (reviewed in [3]). Two

different motifs of DNA triple helices can be

formed, depending on the base composition of

the TFO. Binding of the TFO occurs at oligopurineÆ

oligopyrimidine sequences. Pyrimidine-rich oligonucleo￾tides bind with a parallel orientation with respect to

the oligopurine strand, by forming TÆAxT and

CÆGxC+ base triplets, whereas purine-rich oligonucleo￾tides bind with an antiparallel orientation by forma￾tion of TÆAxT, TÆAxA or CÆGxG base triplets. The

conditions that favour triple-helix formation have

been well characterized. The pyrimidine motif is usu￾ally more stable at acidic pH, due to the require￾ment for cytosine protonation, whereas very stable

triple helices can be formed within the purine motif

at neutral pH, provided the target sequence contains

a high proportion of CÆG pairs and dications

are present. G-rich oligonucleotides often fold into

G-tetrad containing structures that can compete with

triple-helix formation, thereby limiting in practice the

use of this type of triple helix. Various strategies

have been developed that permit the recognition of

mixed sequence duplex DNA targets at physiological

pH [4,5].

Keywords

triple helix; DNA labeling; stem–loop

oligonucleotide; sequence specificity;

padlock oligonucleotide

Correspondence

C. Escude´, Laboratoire ‘Re´gulation et

Dynamique des Ge´nomes’, De´partement

‘Re´gulations, De´ veloppement et Diversite´

Mole´ culaire’, USM 0503 Muse´um National

d’Histoire Naturelle, CNRS UMR5153,

INSERM U565, Case Postale 26, 43 rue

Cuvier, F-75231 Paris Cedex 05, France

Fax: +33 14079 3705

Tel: +33 14079 3774

E-mail: [email protected]

(Received 23 June 2005, revised 17 August

2005, accepted 23 August 2005)

doi:10.1111/j.1742-4658.2005.04932.x

We report on a sequence-specific double-stranded DNA labelling strategy

in which a stem–loop triplex forming oligonucleotide (TFO) is able to

encircle its DNA target. Ligation of this TFO to either a short hairpin

oligonucleotide or a long double-stranded DNA fragment leads to the for￾mation of a topological complex. This process requires the hybridization of

both extremities of the TFO to each other on a few base pairs. The effects

of different factors on the formation of these complexes have been investi￾gated. Efficient complex formation was observed using both GT or TC

TFOs. The stem–loop structure enhances the specificity of the complex.

The topologically linked TFO remains associated with its target even under

conditions that do not favour triple-helix formation. This approach is suffi￾ciently sensitive for detection of a 20-bp target sequence at the subfemto￾molar level. This study provides new insights into the mechanics and

properties of stem–loop TFOs and their complexes with double-stranded

DNA targets. It emphasizes the interest of such molecules in the develop￾ment of new tools for the specific labelling of short DNA sequences.

Abbreviations

BQQ, (6-[3-(dimethylamino)propyl]amino-11-methoxy-benzo[f]quino-[3,4-b]quinoxaline); TFO, triplex forming oligonucleotide.

FEBS Journal 272 (2005) 5343–5352 ª 2005 FEBS 5343