G-quadruplexes are unusual DNA and RNA secondary structures ubiquitous in a variety of organisms including vertebrates, plants, viruses and bacteria. The folding topology and stability of intramolecular G-quadruplexes are determined to a large extent by their loops. Loop permutation is defined as swapping two or three of these regions so that intramolecular G-quadruplexes only differ in the sequential order of their loops. Over the past two decades, both length and base composition of loops have been studied extensively, but a systematic study on the effect of loop permutation has been missing. In the present work, 99 sequences from 21 groups with different loop permutations were tested. To our surprise, both conformation and thermal stability are greatly dependent on loop permutation. Loop permutation actually matters as much as loop length and base composition on G-quadruplex folding, with effects on Tm as high as 17°C. Sequences containing a longer central loop have a high propensity to adopt a stable non-parallel topology. Conversely, sequences containing a short central loop tend to form a parallel topology of lower stability. In addition, over half of interrogated sequences were found in the genomes of diverse organisms, implicating their potential regulatory roles in the genome or as therapeutic targets. This study illustrates the structural roles of loops in G-quadruplex folding and should help to establish rules to predict the folding pattern and stability of G-quadruplexes.
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 100mM 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.
