How do functional RNAs work?

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme.

Biondi E*, Poudyal RR*, Forgy JC, Sawyer AW, Maxwell AW, Burke DH.

* co-first authors







The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu(2+), indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu(2+) binding by RNA; rather, these data establish that Cu(2+) enters the active site within a Cu(2+)•GTPγS or Cu(2+)•GTP chelation complex, and that Cu(2+)•nucleobase interactions further enforce Cu(2+) selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg(2+) with [Co(NH3)6](3+) significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg(2+) coordination is structural rather than catalytic. Replacing Mg(2+) with alkaline earths of increasing ionic radii (Ca(2+), Sr(2+) and Ba(2+)) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.



Redox sensitive RNA Aptamers that bind to Flavin

Poudyal RR et al. (In Prep)


I have been studying RNA aptamers that differentially recognize and bind to oxidized flavin (FAD or FMN) but not to the reduced form (FADH2 or FMNH2). Flavin is the workhorse of vitamin B2, and is used by many enzymes that are involved in electron transfer. Being able to distinguish between electron rich and electron deficient states of Flavin, is the key to building RNA based redox enzymes. 

Nucleobase modification by an RNA enzyme. (2016) Poudyal, R. R., Nguyen, P. D., Lokugamage, M. P., Callaway, M. K., Gavette, J. V., Krishnamurthy, R., & Burke, D. H. Nucleic Acids Research, gkw1199. (2016).









Ribozymes can catalyze phosphoryl or nucleotidyl transfer onto ribose hydroxyls of RNA chains. We report a single ribozyme that performs both reactions, with a nucleobase serving as initial acceptor moiety. This unprecedented combined reaction was revealed while investigating potential contributions of ribose hydroxyls to catalysis by kinase ribozyme K28. For a 58nt, cis-acting form of K28, each nucleotide could be replaced with the corresponding 2′F analog without loss of activity, indicating that no particular 2′OH is specifically required. Reactivities of two-stranded K28 variants with oligodeoxynucleotide acceptor strands devoid of any 2′OH moieties implicate modification on an internal guanosine N-2, rather than a ribose hydroxyl. Product mass suggests formation of a GDP(S) adduct along with a second thiophosphorylation, implying that the ribozyme catalyzes both phosphoryl and nucleotidyl transfers. This is further supported by transfer of radiolabels into product from both α and γ phosphates of donor molecules. Furthermore, periodate reactivity of the final product signifies acquisition of a ribose sugar with an intact 2′-3′ vicinal diol. Neither nucleobase modification nor nucleotidyl transfer has previously been reported for a kinase ribozyme, making this a first-in-class ribozyme. Base-modifying ribozymes may have played important roles in early RNA world evolution by enhancing nucleic acid functions.