High-Resolution Structures of DNA Minidumbbells Comprising Type II Tetraloops with a Purine Minor Groove Residue

Abstract

Minidumbbell (MDB) is a newly discovered DNA structure formed by native sequences, which serves as a possible structural intermediate causing repeat expansion mutations in the genome and also a functional structural motif in constructing DNA-based molecular switches. Until now, all the reported MDBs containing two adjacent type II tetraloops were formed by pyrimidine-rich sequences 5′-YYYR YYYR-3′ (Y and R represent pyrimidine and purine, respectively), wherein the second and sixth residues folded into the minor groove and interacted with each other. In this study, we have conducted a high-resolution nuclear magnetic resonance (NMR) spectroscopic investigation on alternative MDB-forming sequences and discovered that an MDB could also be formed stably with a purine in the minor groove, which has never been observed in any previously reported DNA type II tetraloops. Our refined NMR solution structures of the two MDBs formed by 5′-CTTG CATG-3′ and 5′-CTTG CGTG-3′ reveal that the sixth purine residue was driven into the minor groove via base−base stacking with the second thymine residue and adenine stacked better than guanine. The results of our present research work expand the sequence criteria for the formation of MDBs and shed light to explore the significance of MDBs.

A purine and a backbone discontinuous site alter the structure and thermal stability of DNA minidumbbells containing two pentaloops

“…Six additional sequences, namely 5ʹ‐CTTTG CGTTG‐3ʹ, 5ʹ‐CTTTG CATTG‐3ʹ, 5ʹ‐CTTTG CTGTG‐3ʹ, 5ʹ‐CTTTG CTATG‐3ʹ, 5ʹ‐CTTTG CTTGG‐3ʹ and 5ʹ‐CTTTG CTTAG‐3ʹ which contained a L2ʹ, L3ʹ and L4ʹ purine (G or A), respectively, were designed. In a recent study on MDBs containing two tetraloops, it was found that a purine residue in the 5ʹ‐tetraloop favoured the formation of dimeric conformers [21]. Therefore, sequences containing a L2, L3 or L4 purine in the 5ʹ‐pentaloop were not designed in this work.…”

“…Recently, we have found that replacement of a pyrimidine with a purine in MDBs containing two tetraloops lead to formation of a dimeric species under a high ionic concentration [21]. Accordingly, the solution structures of the above‐mentioned MDBs were characterized under a low ionic strength condition, that is 10 m m NaPi at neutral pH.…”

“…To study whether the MDB can embed a purine residue, we then designed six sequences by substituting the L2ʹ, L3ʹ or L4ʹ thymine with guanine or adenine, that is 5ʹ‐CTTTG CGTTG‐3ʹ, 5ʹ‐CTTTG CATTG‐3ʹ, 5ʹ‐CTTTG CTGTG‐3ʹ, 5ʹ‐CTTTG CTATG‐3ʹ, 5ʹ‐CTTTG CTTGG‐3ʹ and 5ʹ‐CTTTG CTTAG‐3ʹ. Our previous work showed that a purine in the 5ʹ loop lead to formation of dimeric conformers in MDBs containing two tetraloops [21], thus sequences having a L2, L3 or L4 purine in the 5ʹ‐pentaloop were not prepared in this work. To provide a deeper insight into the structure and stability of MDBs composed of a regular pentaloop and a quasi pentaloop, we designed four new sequences comprising a conserved regular CTTTG pentaloop, and a quasi pentaloop in which a backbone discontinuous site was introduced between L1ʹ and L2ʹ (C/TTTG), L2ʹ and L3ʹ (CT/TTG), L3ʹ and L4ʹ (CTT/TG), and L4ʹ and L5ʹ (CTTT/G), respectively.…”

“…Six additional sequences, namely 5ʹ-CTTTG CGTTG-3ʹ, 5ʹ-CTTTG CATTG-3ʹ, 5ʹ-CTTTG CTGTG-3ʹ, 5ʹ-CTTTG CTATG-3ʹ, 5ʹ-CTTTG CTTGG-3ʹ and 5ʹ-CTTTG CTTAG-3ʹ which contained a L2ʹ, L3ʹ and L4ʹ purine (G or A), respectively, were designed. In a recent study on MDBs containing two tetraloops, it was found that a purine residue in the 5ʹ-tetraloop favoured the formation of dimeric conformers [21]. Hydrophobic contacts (E) between the methyl group of T2 and the 2ʹ-methylene groups of C1/T7/T8/T9, (F) between the methyl group of T3 and the 2ʹ-methylene groups of C1/T2, and (G) between the methyl group of T9 and the 2ʹ-methylene groups of C6/T7/T8, as well as between the methyl groups of T8 and T9.…”

“…It was found that adenine methylation [16] and cytosine methylation/ acidic pH [17][18][19] could enhance the thermal stability of TTTA MDB and CCTG MDB, respectively, and an abasic site between two tetraloops could significantly stabilize both MDBs [20]. Besides, the MDB composed of two tetraloops could also sustain a purine as the second loop residue [21].…”