First Tandem Repeat of a Potassium Channel KCNN4 Minisatellite Folds into a V-Loop G-Quadruplex Structure

Abstract

The KCNN4 gene encoding a potassium channel protein whose expression has been correlated with tumor progression was found to comprise a guanine-rich minisatellite region with the ability to form a putative G-quadruplex (G4). Given the suggested regulatory role of G4s in gene expression, Gquadruplex formation for the polymorphic first repeat of the minisatellite was studied by nuclear magnetic resonance spectroscopy. A stable G-quadruplex of a truncated mutant sequence was shown to represent one of several coexisting species of the wild-type sequence. The high-resolution structure features a noncanonical G4 with a broken G-column and a V-shaped loop. The presence of a 3′-flanking thymidine interacting with the lateral loop preceding the V loop seems to be critical for the formation of this G4 topology. On the contrary, an additional 5′-flanking residue disfavored but still allowed folding into the V-loop structure. The latter may therefore serve as a putative therapeutic target in strategies for G4based modulation of KCNN4 expression.

Structural Aspects of Split G‐Quadruplexes in Quadruplex‐Duplex Hybrid Systems

“…An interesting observation concerns the accommodation of an additional 5’‐flanking sequence. Various studies on the latter reported inconclusive results and no detailed structural study has been performed [27,28,32] . The observation of nicely resolved imino resonances in the ODN2 : C‐ODN2‐Ov design suggests its expanded versatility and makes it amenable for a more detailed structural characterization.…”

“…Despite the formation of a parallel G-quadruplex, the vacancy-filling 3'terminal guanosine of the target strand is oriented in an antiparallel fashion and should thus widen the medium groove of the G-quadruplex. On the other hand, ODN2 follows the design of KCNN4 and chl1 sequences, [28,32] which were found to fold into a V-loop G-quadruplex containing a broken first Gcolumn. The first loop bridging a wide groove was cut and the 5'-overhang of the ODN2 elongated to complement the target strand.…”

“…[27] In contrast, a similar approach in another V-loop G-quadruplex destabilized the G-quadruplex to an extent that did not allow an unambiguous demonstration of its structural conservation. [28] Here, we study the formation of asymmetrically split Gquadruplexes of three different families either involving one vacancy (11 : 1, named ODN1), two vacancies (10 : 2, named ODN2), or three vacancies, i. e., with one missing G-column (9 : 3, named ODN3) in the quadruplex platform. The target strand was designed to form a total of eight Watson-Crick base pairs with a complementary flanking sequence in addition to guanine(s) at the terminus to fill the quadruplex vacant position(s).…”

Showcasing Different G-Quadruplex Folds of a G-Rich Sequence: Between Rule-Based Prediction and Butterfly Effect

“…Clearly, G4s with broken G-columns are far from being mere oddities but outcompete putative canonical topologies in many cases, being of potential significance in the design of G4 structures for various applications. Whereas such structures are heavily promoted by the lack of 5′-flanking residues, the latter were found to still allow folding into a V-loop G4. , Yet, given their noticeable decrease in stability when located in nontruncated sequences, the relevance of V-loop topologies within the genome seems an open question.…”

“…Whereas such structures are heavily promoted by the lack of 5′-flanking residues, the latter were found to still allow folding into a V-loop G4. 27,52 Yet, given their noticeable decrease in stability when located in nontruncated sequences, the relevance of V-loop topologies within the genome seems an open question.…”