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Abstract
In the RNA world, the exchange of sequence patches between two RNAs is an intriguing evolutionary concept, allowing generation of new RNA molecules with novel functionality. Based on the hairpin ribozyme (HPR) with its unique cleavage‐ligation properties, we here demonstrate RNA supported RNA recombination as a possible scenario for the emergence of larger RNA molecules with more complex functionality. A HPR variant designed for the purpose of recombination is capable of cleaving two different RNA molecules, one being a hammerhead ribozyme (HHR) and the other an aptamer (A), and to subsequently recombine and ligate the resulting fragments to a hammerhead ribozyme that is allosterically controlled (HHA) by a cognate ligand. Two such recombination processes involving aptamers for either theophylline or flavine mononucleotide (FMN) are demonstrated with yields of functional recombination product of up to 34 %.
Contaminated surfaces have been discussed as a possible source of severe acute respiratory
syndrome coronavirus-2 (SARS-CoV-2). Under experimental conditions, SARS-CoV-2 can remain
infectious on surfaces for several days. However, the frequency of SARS-CoV-2 detection on surfaces
in healthcare settings and the public is currently not known. A systematic literature review was
performed. On surfaces around COVID-19 cases in healthcare settings (42 studies), the SARS-CoV2 RNA detection rates mostly were between 0% and 27% (Ct values mostly > 30). Detection of
infectious SARS-CoV-2 was only successful in one of seven studies in 9.2% of 76 samples. Most of the
positive samples were obtained next to a patient with frequent sputum spitting during sampling.
Eight studies were found with data from public surfaces and RNA detection rates between 0% and
22.1% (Ct values mostly > 30). Detection of infectious virus was not attempted. Similar results
were found in samples from surfaces around confirmed COVID-19 cases in non-healthcare settings
(7 studies) and from personal protective equipment (10 studies). Therefore, it seems plausible to
assume that inanimate surfaces are not a relevant source for transmission of SARS-CoV-2. In public
settings, the associated risks of regular surface disinfection probably outweigh the expectable health
benefit
Abstract
The 10–23 DNAzyme is an artificially developed Mg2+‐dependent catalytic oligonucleotide that can cleave an RNA substrate in a sequence‐specific fashion. In this study, new split 10–23 DNAzymes made of two nonfunctional fragments, one of which carries a boronic acid group at its 5′ end, while the other has a ribonucleotide at its 3′ end, were designed. Herein it is demonstrated that the addition of Mg2+ ions leads to assembly of the fragments, which in turn induces the formation of a new boronate internucleoside linkage that restores the DNAzyme activity. A systematic evaluation identified the best‐performing system. The results highlight key features for efficient control of DNAzyme activity through the formation of boronate linkages.
Boronate esters formed by reaction of an oligonucleotide carrying a 5′-boronic acid moiety with the 3′-terminal cis-diol of another have been shown previously to assist assembly of fragmented DNAzymes. Here we demonstrate that boronate esters replacing the natural phosphodiester linkage at selected sites of two functional RNAs, the hairpin ribozyme and the Mango aptamer, allow assembly of functional structures. The hairpin ribozyme, a small naturally occurring RNA that supports the reversible cleavage of appropriate RNA substrates, is very sensitive to fragmentation. Splitting the ribozyme at four different sites led to a significant decrease or even loss of cleavage and ligation activity. Ribozymes assembled from fragments capable of boronate ester formation showed restoration of cleavage activity in some but not all cases, dependent on the split site. Ligation proved to be more challenging, no supportive effect of the boronate ester was observed. Split variants of the Mango aptamer also showed a dramatic loss of functionality, which however, was restored when 5′-boronic acid modified fragments were used for assembly. These studies show for the first time that boronate esters as internucleoside linkages can act as surrogates of natural phosphodiesters in functional RNA molecules.