Ahead and behind: a small, small RNA world
Michael Yarus
Department of MCD Biology
University of Colorado
Boulder, CO 80309-0347
Is the RNA World hypothesis pertinent to the origin of life?
The discovery of RNA catalysis (Kruger et al. 1982; Guerrier-Takada et al. 1983) led quickly to the idea
that the first catalysts might have been RNA-like (Gilbert 1986). This notion maintains that phenotypes
(chemical capability) and genotypes (capability for replication) might be combined in a single RNA. In
this way, using a simplified definition of life (a replicator that chemically alters its environment), the
problem of life’s origin might itself be streamlined to the origin of a relatively familiar ribonucleotide
molecule.
However, the experimental demonstration of this process with specific RNA structures hits some thorny
obstacles. Nucleotides are difficult to synthesize and are unstable (for example, (Benner et al. 2012))
and their polymers are even more so (for example, (Bowler et al. 2013)). Even worse, related problems
seem inevitable, rooted in the basic combinatorics of the situation. For example, there are 1.3 x 1030
different sequences of the standard 4 nucleotides that are 50 nucleotides long. Supposing that a few
RNAs capable of replicating such RNAs might be among these 50-mers (but compare Wochner et al.
2011), how would the few replicators favor a staggeringly small minority of useful 50-mer molecules?
Actually, with random sequences one cannot assure that all sequences are present without having
multiple copies of each sequence. Five copies is sufficient to insure that at least one copy of all
sequences is present with probability 0.9933; but this requires 180,000 metric tons of 50-mer RNA!
While this problem can be softened by more congenial assumptions, it cannot be entirely avoided. For
example, if we are interested in 30-mer RNAs, ‘having them all’ requires 0.1 gram. Accordingly,
comprehensive RNA accumulations containing RNAs of usual ribozymic size (several tens of nucleotides)
seem unimaginable on the primitive Earth. To be more explicit, it does not seem likely that molecules of
this size are likely to appear under primordial conditions, to be replicated, or to be selected via their
(necessarily) extremely atypical chemical activity, even if they somehow do appear.
However, a possible solution is suggested by the same calculations that identified the problem. If
present-day ribozymes are too large, then consider something smaller.
Embodiments of the oligonucleotide ribozyme
A substantial argument for this point of view is that real, miniscule nucleotide structures do have
biochemical activities (Copley et al. 2007). For example, there is the RNA hydrolytic activity which
accompanies the binding of metal ions to RNA. For example, the fixation of Mn2+ within a tetramer
paired to a trimer helps extract a proton from the nearby ribose 2'OH, activating hydrolysis of the
tetramer (Kazakov and Altman 1992).
But there are also tiny RNAs that have more elaborate chemical capability; consider the nucleotide
coenzymes (Yarus 2011a), which mediate the reactions conducted by about 50% of modern “protein”
enzymes. These have varied structures (Richter 2013), but often are ribodinucleotides with AMP 5'-5'
linked to a second modified ribonucleotide structure with special chemical capabilities. These
coenzymes are often cited as a remnant of ancient RNA catalysts (White 1976; Yarus 2011a). Most
particularly, because they are composed of nucleotide-like moieties, they might be descended from
replicators. Modern enzymatic cofactors, like NAD+, would require only slight changes to lose their
replication, especially in view of their biological universality today. Such universality implies that
gigayears of evolution have affected the structures of similar enzymatic cofactors.
Moreover, pure standard oligonucleotides just above this dinucleotide size range are truly enzymatic.
The synthesis of aminoacyl-RNA from its biological precursor amino acid adenylate (aa-AMP), a required
step in translation, was first selected in 95-mer RNAs (Illangasekare et al. 1995). However, using an
improved, less-constrained selection, a much simpler ribozyme appears that binds this complex
substrate molecule in trans, and transfers the amino acid specifically, in emulation of this universal
translational reaction (Turk et al. 2010). The pentamer oligonucleotide 5' GUNMO 3' base pairs to
tetramer oligonucleotide 5' OMNU 3' (italics indicate complementarity; (Illangasekare and Yarus 2012)
to assemble a 3-nucleotide active site which transfers amino acid from an aa-AMP to the 2'OH of the
terminal U of OMNU. The small active center which makes OMNU-aminoacyl is regiospecific, utilizing
only the 2'OH, as do the natural class I protein aminoacyl tRNA synthetases (Eriani et al. 1990). The same
active center, composed of GU and U brought together by nonspecific base pairs (Illangasekare and
Yarus 2012), can be utilized in successive reactions that both activate the amino acid, using a
preselected ribozyme (Xu et al. 2014), and then transfer it to the tetramer. These latter reactions
therefore emulate both translational activation and acylation steps as performed by protein enzymes
today.
However, probably because the active center is too small to have significant contact with the amino acid
or the nucleobase of aa-NMP, it is not specific for the amino acid or the base (Turk et al. 2010). This is
likely to be an inherent property of small catalysts, not unique to this RNA (Yarus 2011b). When one can
span only a bond length or two from the atoms actually reacting, opportunity for specific contacts is
limited. Thus, small active centers cannot be as precise as when more architecture supports their sites.
Such catalysts may not even be able to spend full time in catalytic configurations, so they will also
accelerate reactions less than do active centers embedded in larger ribozymes. In fact, these tendencies
are evident in the reactions of the 5-nucleotide aminoacylator (Turk et al. 2011; Yarus 2011b). So,
though they are unlikely to be superior catalysts, five-nucleotide RNA enzymes with three–nucleotide
active centers are well-known.
The sporadically fed pool
Further, beyond the five-nucleotide aminoacylating ribozyme, we can be sure that oligonucleotides are
capable of solving most of the problems associated with the initiation of Earthly life (see the discussion
in (Szostak 2012)). This assurance comes from calculations on uncatalyzed RNA synthesis in the
“sporadically fed pool”, which implements a type of oligonucleotide synthesis which would likely be
practical, or perhaps even frequent, on a primitive planet.
A sporadically fed pool is one that gets small doses of activated and unactivated nucleotides at low
concentrations; and these substrates also arrive at uncontrolled random times. These nucleotides and
their possible oligomerized products immediately begin to decay at realistic rates. Surprisingly, in such a
pool, using rates for ribonucleotide reactions from the chemical literature, small self-complementary
RNAs readily appear and even replicate (Yarus 2012). Thus short RNAs can appear (Yarus 2011b) as soon
as there are activated nucleotides, in contrast to specific longer sequences which seem (in the first
paragraphs above) inaccessible. Further, replication needs nothing more exotic than ordinary solution
kinetics (Yarus 2012). Later, when the ability to make longer RNAs appears, small active centers can be
incorporated into the interiors of larger chains (Illangasekare and Yarus 2012), assuring their
evolutionary continuity.
However, this description leaves unmentioned the most interesting properties of a sporadically fed pool,
which emerge from its detailed kinetics (Yarus 2013). Because substrate arrival is unguided, all possible
reaction sequences are explored. Successful syntheses therefore can come from recurrent near-ideal
reactions, in which proper reagent addition and sequence is recurrently achieved with no external
guidance at all. Further, because the assumed dominant form of molecular decay (first order,
exponential) has a large variance (protracted tailing in time), intermediates persist and interact with
later substrate pulses. Thus, successful complex reaction sequences are more frequent than intuition
expects. Accordingly, a plausibly primitive reactor, the sporadically fed pool, can recurrently host
substantial reaction sequences, perform unexpectedly complex syntheses (Yarus 2013), and potentially
contribute these products to a descendant. The short oligonucleotides we have been talking about have
a possible route into a primordial world.
Conclusion – the promise of small oligonucleotides
In celebration of the 20-year anniversary of RNA, then, here is a prediction about its future. It will be
productive to examine the chemical abilities of short oligomers of varied structures (but surely including
2'-5', 3'-5', and 5'-5' connectivity) and varied nucleobase compositions. There are strong arguments for a
long-ago, small, small RNA world populated with Darwinian beings who were short oligomers; perhaps
we can recreate that first biosphere.
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