Abstract
BackgroundLocked nucleic acid (LNA) and 2'–O-methyl nucleic acid (OMeNA) are two of the most extensively studied nucleotide derivatives in the last decades. However, how they affect DNA quadruplex structures remains largely unknown. To explore their possible biological affinities for quadruplexes, we investigated how LNA- or OMeNA-substitutions affect G-quadruplex structure formation using a thrombin binding aptamer (TBA), the most studied extracorporal G-quadruplex-forming DNA sequence, which is frequently modified to increase its analytical performance.ResultsThe experimental results showed that when two or more nucleotides were substituted with LNA or OMeNA, the anti-parallel TBA structure was transformed into an unstructured random conformation in a 50 mM K+ environment; OMeNA appeared to have greater power to induce this transformation. However, the native TBA was unstructured in a 50 mM Ca2+ environment, whereas four or more LNA- or OMeNA- substitutions could convert this unstructured TBA into a parallel quadruplex structure. PAGE mobility measurements suggested that these TBAs might be a dimeric form.ConclusionLNA or 2'-OMeNA site-specific modifications induced G-quadruplex structural transformation of TBA, which enriched our understanding of the intrinsic G-quadrupex forming property and affinity of LNA and OMeNA modifications. This study demonstrates possible applications in the regulation of gene expression (i.e. manual intervention of gene therapy), genetic analyses, molecular diagnosis and the construction of nano-scale biostructures.
Highlights
Locked nucleic acid (LNA) and 2'–O-methyl nucleic acid (OMeNA) are two of the most extensively studied nucleotide derivatives in the last decades
Whether LNA- or OMeNAmodified sequences can form Hoogsteen hydrogen bonds or quadruplex structures is not clear. To explore their possible biological affinities for quadruplexes, we investigated how LNA- or OMeNA-substitutions affect a well-defined G-quadruplex structure formed by the thrombin binding aptamer (TBA), the most commonly explored extracorporal aptamer [10]
LNA- or OMeNA-modifications induced TBA structural transition from an antiparallel G-quadruplex to a random strand in a K+ environment To record quadruplex structure changes between native TBA and LNA- or OMeNA-modified TBAs, we used a circular dichroism spectropolarimeter to measure the sample structures in solution, as the anti-parallel Gquadruplex structure of native TBA in K+ solution can be identified conveniently by its CD spectrum characterized by a positive maximum at 295 nm and a negative minimum at 265 nm [13,14,15,16,17,18]
Summary
Locked nucleic acid (LNA) and 2'–O-methyl nucleic acid (OMeNA) are two of the most extensively studied nucleotide derivatives in the last decades. How they affect DNA quadruplex structures remains largely unknown. To explore their possible biological affinities for quadruplexes, we investigated how LNA- or OMeNA-substitutions affect G-quadruplex structure formation using a thrombin binding aptamer (TBA), the most studied extracorporal G-quadruplex-forming DNA sequence, which is frequently modified to increase its analytical performance. Among them, locked nucleic acid (LNA) and 2′–O-methyl nucleic acid (OMeNA) have attracted great attention and have been extensively studied [2,3], since they demonstrate many advanced properties, such as the ability to increase duplex thermal stability and to recognize mismatched base pairs [4]. A previous study showed that these nucleotide derivatives can form Watson-Crick base pairs in complementary double
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