NMR Study On Spontaneous Reassembly Of Parallel Trimolecular G-quadruplex Via Novel Hoogsteen Strand Displacement Reaction

The strand displacement reaction(SDR)of nucleic acids conventionally refers to the displacement of one prehybridized strand from double-stranded DNA or RNA helices by one homologous single-stranded invading strand,depending on the WatsonCrick base pairing rule and the branch migration induced by the toehold motif.This DNA duplex-based SDR has been widely used in dynamic DNA nanotechnology.Guanine-rich nucleic acid fragments have a high propensity to fold into a noncanonical secondary structure,termed G-quadruplexe(GQ),which is formed by Hoogsteen type base pairing between four guanines and involves chelation of a metal ion,usually K+or Na+.GQ-forming motifs are widely distributed throughout the human genome.GQs have important biological implications in chromosome-end protection,replication,transcription,translation,and genomic regulatory processes.GQs often exist in many polymorphic forms.According to strand molecularity,a monomeric intramolecular GQ is self-folded by a single strand of G-rich oligonucleotides,whereas intermolecular GQs,mostly dimeric and tetrameric assemblies,are assembled by multiple G-rich strands.Up till now,very few trimolecular GQs(tri-GQ)have been reported.GQs commonly exhibit sufficiently high thermodynamic and kinetic stability at room temperature.In the conventional perception over time,these GQs that once formed essentially remain inert to SDRs with other G-rich invading strands of DNA or RNA fragments.The spontaneous reassembly of heteromolecular GQs via SDR is very rare.To further expand the molecular mechanism of new SDR,in this work,we explored whether a GQ can alternatively proceed in the Hoogsteen pairing-based SDR with other G-rich invading strands.The examined Tub10 sequence of d(CAGGGAGGGT)is a fragment from the G-rich promoter region of the human β2tubulin gene.We revealed that a parallel homomeric bimolecular GQ target of Tub10 as the starting reactant,although completely folded in K+solution and suficiently stable(melting temperature of 57.7 ℃),can still spontaneously accept strand invasion by a pair of short G-rich invading probes of P1 d(TGGGA)at room temperature.The final SDR product is a novel parallel heteromeric trimolecular GQ(tri-GQ)of Tub10/2P1 reassembled between one Tub10 strand and two P1 strands.Mainly by using solutionstate NMR with other experimental assistance of ESI mass spectrometry,dynamic light scattering,and PAGE gel migration,we presented the first NMR solution structure of such a discrete heteromeric tri-GQ of Tub10/2P1 and unveiled a unique 2:1 binding mode of two probes vs one target in mutual recognition among G-rich canonical DNA oligomers.After the examination on other sequence analogs of both G-rich probe P1 and GQ target Tub10,it revealed that G-rich probe P1 can specifically recognize the all-parallel target GQ,exhibiting an excellent selectivity on the folding topology of GQ target.In contrast,the traditional antisense probe with a long complementary C-rich oligonucleotide was unable to effectively distinguish those target GQs having similar sequences but completely different folds.In addition,the SDR efficiency of G-rich probe P1(5-nt)was much greater than that of antisense C-rich complementary probes having similar or slightly longer lengths of 5 to 7 nt.Moreover,in the "dual duplex invasion strategy",a single G-rich probe can have dual SDR modes,both Hoogsteen and Watson-Crick pairing-based,to associate with homologous G-rich and complementary C-rich strands of the DNA duplex.Our NMR results showed that short G-rich probe P1 indeed exhibited the potential to successfully capture G-rich target sequence Tub10 from the dsDNA context to form a hybridized GQ.In summary,our findings revealed a new GQ-specific,Hoogsteen pairing-based SDR and expanded our understanding of the heteromolecular interactions that play a critical role in biological systems.