- Scripps Researchers ‘Evolve’ an RNA-Amplifying RNA Polymerase
- It’s Used for First Ever All-RNA Amplification Called “riboPCR”
- TriLink Reagent Plays a Role in this Remarkably Selective in Vitro Evolution Method
Those of you who regularly read my blog will recall an earlier posting on “the RNA World,” which was envisioned by Prof. Walter Gilbert in the 1980s as a prebiotic place billions of years ago when life began without DNA. That post recommended reading more about this intriguing hypothesis by consulting a lengthy review by Prof. Gerald Joyce. Now, Prof. Joyce and postdoc David Horning have advanced the hypothesis one step further by reporting the first ever amplification of RNA by an in vitro-selected RNA polymerase, thus providing significant supportive evidence for the RNA World. Following are their key findings, which were enabled in part by a TriLink reagent—read on to find out which one and how!
In Vitro Evolution of an RNA Polymerase
Horning & Joyce designed an in vitro selection method to chemically “evolve” an RNA polymerase capable of copying a relatively long RNA template with relatively high fidelity. The double emphasis on “relatively” takes into account that the RNA World would have many millions of years to evolve functionally better RNA polymerases capable of copying increasingly longer RNA templates with increasingly higher fidelity.
As depicted below, they started with a synthetic, highly structured ribozyme (black) wherein random mutations were introduced throughout the molecule at a frequency of 10% per nucleotide position to generate a population of 1014 (100,000,000,000,000) distinct variants to initiate the in vitro evolution process. Step 1 involved 5’-5’ click-mediated 1,2,3-trazole (Ø) attachment of an 11-nt RNA primer (magenta) partially complementary to a synthetic 41-nt RNA template (brown) encoding an aptamer that binds guanosine triphosphate (GTP). In Steps 2 and 3, the primer hybridizes to template and is extended by polymerization of A, G, C and U triphosphates (cyan).
Step 4 involves binding of aptameric structures to immobilized GTP (green), then photocleavage of the 1,2,3-triazole linkage in Step 5, followed by reverse transcription to cDNA and conventional PCR in Step 6 for transcription into ribozymes in Step 7. Twenty-four rounds of this evolution by selection were carried out, progressively increasing the stringency by increasing the length of RNA to be synthesized by decreasing the time allowed for polymerization. By the 24th round, the population could readily complete the GTP aptamer shown below. Subsequent cloning, sequencing and screening were then used to characterize the most active polymerase, which was designated “24-3.”
The TriLink “Connection”
The aforementioned in vitro evolution process actually involves tons of experimental details that interested readers will need to consult in the published paper, which is accompanied by an extensive Supporting Information section. In the latter, a subsection titled Primer Extension Reaction describes 3’ biotinylation of the template RNA strand (brown in above scheme) using TriLink “2’-azido-UTP” (more properly named 2’-azido-dUTP) and yeast poly(A) polymerase, followed by click connection of the RNA template’s 3’-terminal 2’-azido moiety to biotin-alkyne. This very clever functionalization of the RNA template strand allowed for subsequent capture of the double-stranded primer extension reaction products on streptavidin-coated beads, followed by elution of the desired nonbiotinylated strand for GTP aptamer selection (Step 4 above).
Properties of RNA Polymerase 24-3
Needless to say—but I will—enzymologists and RNA aficionados will undoubtedly be interested in musing over the kinetic and fidelity properties of RNA polymerase 24-3.
The rate of 24-3 polymerase catalyzed addition to a template-bound primer was measured using an 11-nt template that is cited extensively in the literature to evaluate various ribozymes. It was found that the average rate of primer extension by 24-3 is 1.2 nt/min, which is ∼100-fold faster than that of the starting ribozyme polymerase randomly mutagenized for in vitro selection.
The NTP incorporation fidelities of the starting and 24-3 ribozyme polymerases on this 11-nt test template, at comparable yields of product, are 96.6% and 92.0%, respectively. Horning & Joyce noted that the higher error rate of 24-3 is due primarily to an increased tendency for G•U wobble pairing.
Other longer RNA templates having various base compositions or intramolecular structures were also studied, with the stated “final test of polymerase generality” being use of 24-3 to synthesize yeast phenylalanyl tRNA from a 15-nt primer (in red right). The authors humorously describe the results as follows:
“Despite the stable and complex structure of the template, full-length tRNA was obtained in 0.07% yield after 72 h. This RNA product is close to the limit of what can be achieved with the polymerase, but is likely the first time a tRNA molecule has been synthesized by a ribozyme since the end of the RNA world, nearly four billion years ago.”
Exponential Amplification of RNA
PCR is the most widely used method for amplifying nucleic acids, and involves repeated cycles of heat denaturation and primer extension. The 24-3 RNA polymerase was used to carry out PCR-like amplification, but in an all-RNA system (named riboPCR by Horning & Joyce) using A, G, C, and U triphosphates and a 24-nt RNA template composed of two 10-nt primer-binding sites flanking the sequence AGAG. Somewhat special conditions were employed:
- The concentration of Mg2+ was reduced to minimize spontaneous RNA cleavage
- PEG8000 was used as a “molecular crowding” agent to improve ribozyme activity at the reduced Mg2+ concentration
- Tetrapropylammonium chloride was added to lower the melting temperature of the duplex RNA
Under these conditions, 1 nM of the 24-nt RNA template was driven through >40 repeated thermal cycles, resulting in 98 nM newly synthesized template and 106 nM of its complement, corresponding to 100-fold amplification. Sequencing of the amplified products revealed that the central AGAG sequence was largely preserved, albeit with a propensity to mutate the third position from A to G, reflecting the low barrier to wobble pairing.
Amplification of a 20-nt template (without the central insert) was monitored in real time, using FRET from fluorescently labeled primers, and input template concentrations ranging from 10 nM to 1 pM. The resulting amplification profiles shown in the paper are typical for real-time PCR, shifted by a constant number of cycles per log-change in starting template concentration. A plot of cycle-to-threshold vs. logarithm of template concentration, also shown in the paper, was linear across the entire range of dilutions indicating exponential amplification of the template RNA with a per-cycle amplification efficiency of 1.3-fold.
Implications for the Ancient RNA World
It would be an injustice to Horning & Joyce if I would try to paraphrase their concluding discussion of this investigation, so here is what they say:
The vestiges of the late RNA world appear to be shared by all extant life on Earth, most notably in the catalytic center of the ribosome, but most features of RNA-based life likely were lost in the Archaean era. Whatever forms of RNA life existed, they must have had the ability to replicate genetic information and express it as functional molecules. The 24-3 polymerase is the first known ribozyme that is able to amplify RNA and to synthesize complex functional RNAs. To achieve fully autonomous RNA replication, these two activities must be combined and further improved to provide a polymerase ribozyme that can replicate itself and other ribozymes of similar complexity. Such a system could, under appropriate conditions, be capable of self-sustained Darwinian evolution and would constitute a synthetic form of RNA life.
Applications for Today’s World of Biotechnology
The aforementioned report by Horning & Joyce has received wide acclaim in the scientific press and world-wide public media as supporting the existence of a prebiotic RNA World, billions of years ago, from which life on Earth evolved.
While the academic part of my brain, if you will, fully appreciates the significance of these new insights on “living” RNA eons ago, the technical applications part of my brain is more piqued by possible practical uses of all-RNA copying or all-RNA riboPCR.
I, for one, plan to muse over possible applications of such all-RNA systems in today’s world of biotechnology, and hope that you do too, and are willing to share any ideas as comments here.