Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength.
The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA that resides in the HDV genome and regulates its replication. The native fold of the ribozyme is complex, having two pseudoknots. Earlier work implicated four non-native pairings in slowing pseudoknot formation: Alt 1, Alt 2, Alt 3, and Alt P1. The goal of the present work was design of a kinetically simplified and maximally reactive construct for in vitro mechanistic and structural studies. The initial approach chosen was site-directed mutagenesis in which known alternative pairings were destabilized while leaving the catalytic core intact. Based on prior studies, the G11C/U27Delta double mutant was prepared. However, biphasic kinetics and antisense oligonucleotide response trends opposite those of the well-studied G11C mutant were observed suggesting that new alternative pairings with multiple registers, termed Alt X and Alt Y, had been created. Enzymatic structure mapping of oligonucleotide models supported this notion. This led to a model wherein Alt 2 and the phylogenetically conserved Alt 3 act as "folding guides", facilitating folding of the major population of the RNA molecules by hindering formation of the Alt X and Alt Y registers. Attempts to eliminate the strongest of the Alt X pairings by rational design of a quadruple mutant only resulted in more complex kinetic behavior. In an effort to simultaneously destabilize multiple alternative pairings, studies were carried out on G11C/U27Delta in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs approximately 60 min(-1)) kinetics to be realized. To account for this, a model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.
The Huck Institutes of the Life, The Pennsylvania State University, University Park, PA 16802, USA.
Trevor S
TS
Brown
Durga M
DM
Chadalavada
Philip C
PC
Bevilacqua
D020134:Catalytic Domain; D003698:Hepatitis Delta Virus / Q000737:chemistry* / Q000235:genetics; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D015233:Models, Statistical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010802:Phylogeny; D017510:Protein Folding; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D012964:Sodium / Q000737:chemistry; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000737:chemistry
D007477:Ions; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D016337:RNA, Catalytic; D012313:RNA; D014508:Urea; D012964:Sodium; D008274:Magnesium