# Structure-Activity Based Mechanistic Insights into Cleavage Chemistry by Self-Cleaving Nucleolytic Ribozymes

> **NIH NIH R01** · SLOAN-KETTERING INST CAN RESEARCH · 2022 · $336,300

## Abstract

ABSTRACT
Our mechanistic understanding of small (<150 nt) self-cleaving nucleolytic ribozymes that primarily use general-
acid base catalysis involving attack of the 2’-OH on the adjacent scissile phosphate to site-specifically cleave
the intervening phosphodiester backbone has initially focused on hammerhead, hairpin, glmS, hepatitis delta
virus and Varkud Satellite ribozymes, and more recently on twister (Twr), twister-sister (TSr), pistol (Psr) and
hatchet (Htr) ribozymes. Our overall goal as reflected from our structure-function studies of the Twr, Tsr, Psr and
Htr ribozymes is to expand on our current understanding of the catalytic versatility of RNA with the emphasis on
the contributions of active site organization, geometric constraints, activation of the 2’-OH nucleophile, the role
of transition state stabilization, protonation of the 5’-oxygen leaving group and the potential of Mg2+ cations in
mediating catalysis. One of the challenges in the field relates to whether self-cleaving ribozymes use a common
or diverse set of mechanisms, and the extent to which hydrated divalent cations catalyze cleavage chemistry.
We have an ongoing collaboration with the Ronald Micura lab (Innsbruck) to study catalytic mechanisms of self-
cleaving ribozymes by solving crystal structures of precatalytic, transition and product states in our lab, followed
by systematic multi-faceted studies of structure-guided selective catalytic mutants and analogs, as well as pKa
measurements of catalytic residues, and pH and temperature-dependence of catalytic rates by the Micura lab.
Aim 1: A recent biochemical genome-wide screen resulted in the identification of the naturally occurring self-
cleaving hovlinc ribozyme in humans. The sequence of the 168-nt hovlinc ribozyme and its 83-nt minimal
functional counterpart contained two pseudoknots with one of them embedded in the cleavage site. The cleavage
rate was shown to increase with pH and its inverse correlation with the pKa of divalent cations suggested the
catalytic participation of a hydrated divalent cation in cleavage chemistry. We propose to crystallize and
determine the structures of the precatalytic, vanadate transition-state mimic and product states of the minimal
functional hovlinc ribozyme and follow up with systematic functional studies with the Micura lab of structure-
guided modifications and rate measurements towards elucidation of its catalytic cleavage mechanism.
Aim 2. This Aim revisits structure-activity relationships of the Twr and TSr ribozymes to resolve discrepancies
in the published precatalytic structures and resulting mechanistic insights reported in the literature. The splayed-
apart orientation of bases at the cleavage site in a four-way junctional TSr ribozyme by our group contrasts with
the stacked bases at the cleavage site in a three-way junctional TSr from the David Lilley lab. We propose to
characterize vanadate transition-state mimics of the Twr and TSr ribozymes to resolve the existing...

## Key facts

- **NIH application ID:** 10421868
- **Project number:** 1R01GM145888-01
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** DINSHAW J PATEL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $336,300
- **Award type:** 1
- **Project period:** 2022-09-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10421868

## Citation

> US National Institutes of Health, RePORTER application 10421868, Structure-Activity Based Mechanistic Insights into Cleavage Chemistry by Self-Cleaving Nucleolytic Ribozymes (1R01GM145888-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10421868. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*