A Multisubunit Ribozyme That Is a Catalyst of and Template for Complementary Strand RNA synthesis

Abstract

Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.

RETRACTED: Replication of an RNA ligase ribozyme under alternating temperature condition

“…Among these, nucleic acids is an attractive system for self-replication because they can encode genetic information by recognizing their complementary partners and catalyze nucleic acid joining reactions [10][11][12]. Due to these properties, catalytic nucleic acids with a power to catalyze a broad set of chemical reactions greatly simplified the procedures needed to perform Darwinian evolution [13].…”

A self-replicating ligase ribozyme

“…With this broader notion of templating in mind, it was of interest to explore more complex nucleic acid polymers that would carry out self-replication mediated by secondary and tertiary interactions, rather than simple WatsonCrick pairing. In particular, it was of interest to examine a ribozyme-based system in which the structured nucleic acid components would add enzymatic activity to the self-replicating system.Nucleic acid polymers are attractive candidates for selfreplication because they can encode genetic information, recognize nucleic acid components with high specificity, and catalyze nucleic acid joining reactions (18)(19)(20)(21)(22). A system based on a ligase ribozyme was used to investigate the ability of catalytic RNA molecules to promote their own synthesis from component oligonucleotides.…”

“…Nucleic acid polymers are attractive candidates for selfreplication because they can encode genetic information, recognize nucleic acid components with high specificity, and catalyze nucleic acid joining reactions (18)(19)(20)(21)(22). A system based on a ligase ribozyme was used to investigate the ability of catalytic RNA molecules to promote their own synthesis from component oligonucleotides.…”

Rapid kinetic characterization of hammerhead ribozymes by real-time monitoring of fluorescence resonance energy transfer (FRET)

“…We used sophisticated instrumentation, a microtiter plate thermocycler and fluorescent reader+ It continuously records complete fluorescence spectra, and even more important, the spectral overlap of FAM and TAMRA signals are corrected by internal calibration of the software+ A simple fluorimeter lacks this feature+ No parallel readings are possible in a standard fluorimeter, but higher sensitivity is advantageous, as low as 1 nM FAM-labeled RNA can be used (Walter & Burke, 1997) instead of the 60 nM required for SDS 7700+ This limitation was overcome by taking advantage of the observation by us (Hanne et al+, 1998) and others (Tyagi et al+, 1998) that efficient quenching is possible with nonoverlapping pairs, like FAM and Cy-5 (or its equivalent BODIPY)+ It should be noted that introduction of dye labels affects kinetic parameters with further dependence on the actual dye moiety (Table 1)+ With unmodified RNAs, the observed values for k cat and k obs were in the normal range for hammerhead ribozymes, but they were lower than the high rates reported for similar format I/II constructs (Clouet-D'Orval & Uhlenbeck, 1996)+ However, we made the following changes in the substrate RNA: in stem I, an extra, unpaired U was added and in stem II, the 59-terminal G was replaced by dT+ These changes may seem minor, but as reported subsequently (Clouet-D'Orval & Uhlenbeck, 1997) the cleavage rate is very sensitive to length changes in stem I and stem II+ Interestingly, the addition of ethanol was useful in maintaining a constant FAM signal, but it had no effect on the hammerhead cleavage kinetics+ This is in contrast to significant rate enhancements observed for other ribozymes: cleavage with RNase P RNA (Kazakov & Altman, 1991) and ligation with group I intron (Doudna et al+, 1991; G+ Krupp, unpubl+)+ For each substrate, FRET efficiency and extent of possible cleavage will vary+ These data are not available from FRET analysis only+ The extent of FRET efficiency limits the signal difference between intact and cleaved substrate, but occurrence of fluorescent reporter signal will always be directly proportional to the fraction of cleavage products+ An initial signal increase will be followed by a more or less constant plateau+ But it is not justified to assume that the plateau is equivalent to 100% cleavage+ This value has to be determined by subsequent gel electrophoresis, either with radioactive RNA or with double-labeled RNA where the quencher dye can be excited directly+ For this purpose, we used Cy-5 or BODIPY as quenchers, as they can be quantitatively detected with STORM imager in the red mode+ A similar detection of the reporter (FAM) is not quantitative, as FRET also occurs in denaturing gels and its efficiency is unknown+ It should be noted that FRET can only monitor cleavage products that have dissociated from the ribozyme (Fig+ 3C)+ This limits its application to reactions in which product dissociation is faster than the cleavage step+ The presented method is useful for the wide range of studies where the same substrate RNA is used to characterize effects of changed reaction conditions (Burgin et al+, 1996;Clouet-D'Orval & Uhlenbeck, 1996;Murray et al+, 1998), addition of cellular proteins (Müller et al...…”