Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme

Highlights

• G-quadruplex isomers which only differ in sequential orders of loops are designed.

• Loop transposition can determine the conformation of these G-quadruplex isomers.

• Hydrogen bonds from loops and flanking sequences cause the structural difference.

• Loop transposition enhances the activities of G-quadruplex/hemin catalyst robustly.

• Loop transposition alters the hemin binding affinity to G-quadruplex.

Abstract

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100 mM K⁺, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. ¹D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.

Introduction

DNA G-quadruplex structures are formed by repetitive guanine-rich sequences and stabilized by monovalent cations (such as, K⁺). G-rich sequences that have the potential to form G-quadruplexes are widely distributed in genomes [1] and appear to be involved in gene regulation [2], [3]. Due to the structural polymorphism and high thermal stability, G-quadruplexes are extensively used as scaffolds in DNAzymes [4], [5], [6], aptamers [7], biosensors [8], [9] and therapeutic agents [10], [11]. Unravelling the nature of the G-quadruplex folding is of fundamental and widespread importance.

There are two main components of G-quadruplex structure: the G-quartet cores and the loops. The G-quartet cores are composed of two or more stacked G-quartets with strands in parallel, antiparallel, or hybrid orientations [12], [13]. Loops are the sequences between two adjacent G-tracts. G-quadruplex structures are influenced by the environmental factors such as cation type and concentration [14], [15], dehydrating or crowding agent concentration [16], [17], targeting ligand [18], and DNA concentration [19], [20]. Besides the above external factors, G-quadruplex folding is determined by its primary sequence. G-quadruplex topology can be tuned by the length of G-tracts [21], [22]. The flanking sequence can contribute to or limit conformational heterogeneity [23], [24], [25], [26], [27], [28]. What's more, the influence of loop length on structural polymorphism and thermal stability has been extensively studied [29], [30], [31], [32], [33], [34], [35]. Moreover, Plavec and colleagues even designed novel G-quadruplex structure by optimizing the length of each loop individually [36], [37].

Loop transposition denotes that two or more G-quadruplexes share identical loops, G-tracts and flanking sequences but only differ in the sequential orders of their three loops. As far as we known, there is no evidence to show that the loop transposition can induce two G-quadruplexes into different conformations. For example, Fox et al. reported that d[(TG₃)₂T₄(G₃T)₂] and d[TG₃T₄(G₃T)₃], which differed in the sequential orders of T and TTTT loops, adopted parallel topologies equally [38]. The two T loops constrained the G-quadruplexes into parallel topologies [29], [30]. In addition, Mergny et al. reported that three terminal truncated sequences d[(G₃T₂)₂G₃T₃G₃], d[G₃T₃(G₃T₂)₂G₃] and d(G₃T₂G₃T₃G₃T₂G₃), sharing two TT and one TTT loops, also folded into predominantly parallel topologies without structural difference [30]. However, we demonstrated for the first time that the G-quadruplex conformations could be switched by the loop transposition alone.

DNAzyme was chosen to test whether the loop transposition could alter the functions through structural control. G-quadruplex-based DNAzymes (or DNA enzymes) are drawing more and more attentions from both chemists and biologists [39], [40]. The catalytic repertoire included porphyrin metallation [41], one-electron and two-electron (oxygen transfer) oxidation [42], [43], [44], [45], [46], photoreversion of thymine dimers [39], [47] and Aldol reactions [48]. Even more, the chiral catalysis, for instance, carbon-carbon bond formation [49], [50] and oxidation reactions [40], could be achieved by G-quadruplex based metalloenzymes.

The G-quadruplex/hemin catalyst is one of the most famous DNAzymes because of its versatility and accessibility in biological and chemical analyses [4]. It was accepted that parallel G-quadruplex promoted the peroxidase activity of hemin more efficient than that of non-parallel (hybrid or antiparallel) G-quadruplex [8], [51], [52], [53], [54]. Considering that loop transposition could alter the conformation of G-quadruplex, the relations between the loop transposition and catalytic activity of G-quadruplex/hemin DNAzyme were also investigated. Interestingly, loop transposition promoted the catalytic efficiency (k_cat/K_m) by increasing the hemin binding affinity to G-quadruplex.