Coexistence of G-quadruplex and duplex domains within the secondary structure of 31-mer DNA thrombin-binding aptamer

Abstract

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.

Keywords:

• thrombin-binding aptamer
• G-quadruplex structure
• noncanonical DNA
• circular dichroism
• UV melting

Acknowledgment

This work was supported by the Russian Foundation for Basic Research, grants No 11-04-01530 and 10-04-00645.

Notes

aG-quadruplex-forming motif is indicated by bold letters; the position of a single G residue substitution within G-quartet structural element is underlined.

aThe melting data obtained at 297 nm reflect G-quadruplex unfolding, while the data collected at 260 nm are related to DNA duplex denaturing.

bNot determined: 15 TBA and Hairpin-du do not contain the duplex-forming and G-quadruplex-forming motifs, respectively; 31TBA-du^-, which contains noncomplementary sequences flanking G-quadruplex-forming motif, unable to form DNA duplex structure; 31TBA(G¹⁴→A) does not fold into G-quadruplex conformation (according to CD data).

cMelting temperature value reported in this Table does not depend on oligonucleotide strand concentration (1.3, 2.1, 4.2, and 13 μM).