Molecular evolution can be conceptualized as a walk over "fitness landscape", or the function of fitness (e.g., catalytic activity) space all possible sequences. Understanding requires knowing structure landscape and identifying viable evolutionary pathways through landscape. However, for any biomolecule is largely unknown. The RNA special interest because believed to have been foundational early life. In particular, an essential activity leading genetic code would reaction ribozymes with...

RNA editing in Trypanosoma brucei is a mitochondrial processing reaction that results the insertion and deletion of uridylate residues into otherwise untranslatable mRNAs. The process directed by guide RNAs which function to specify edited sequence. vitro requires protein extracts have been identified as part high molecular weight ribonucleoprotein complexes. Within complexes, are close contact with several proteins here we describe isolation cloning gRNA-interacting polypeptide from brucei....

Abstract The RNA world hypothesis describes a stage in the early evolution of life which served as genome and only genome-encoded catalyst. To test whether organisms could have used cyclic trimetaphosphate an energy source, we developed vitro selection strategy for isolating ribozymes that catalyze triphosphorylation 5′-hydroxyl groups with trimetaphosphate. Several active sequences were isolated, one ribozyme was analyzed more detail. truncated to 96 nt, while retaining full activity. It...

The guide RNA-binding protein gBP21 has been characterized as a mitochondrial RNA/RNA annealing factor. co-immunoprecipitates with RNA editing ribonucleoprotein complexes, which suggests that contributes its activity to the machinery. In support of this view, was found accelerate hybridization cognate (g)RNA/pre-edited mRNA pairs. Here we analyze mechanism gBP21-mediated reaction. Three possible modes action are considered: chaperone function, matchmaker function and product stabilization....

During an early step in the evolution of life, RNA served both as genome and catalyst, according to world hypothesis. For self-replication, organisms must have contained that catalyzes polymerization. As a first toward recapitulating laboratory, polymerase ribozyme was generated previously by vitro design. However, efficiency this is about 100-fold too low for self-replication because affinity its primer/template substrate. To improve substrate interactions colocalizing on micelles, we...

The emergence of catalytic RNA is believed to have been a key event during the origin life. Understanding how activity distributed across random sequences fundamental estimating probability that would emerge. Here, we analyze in vitro evolution triphosphorylating ribozymes and translate their fitnesses into absolute estimates for hundreds ribozyme families. analysis efficiently identified highly active estimated with good accuracy. evolutionary dynamics follow Fisher's Fundamental Theorem...

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for organisms. one these studies, catalytic RNAs (ribozymes) that catalyze triphosphorylation 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed more detail. TPR1 catalyzes reaction to rate 0.013 min-1 under selection conditions (50 mM Tmp, 100 MgCl2, 22°C). To identify faster triphosphorylation, and possibly learn about its...

Group I introns are pre-mRNA that do not require the spliceosome for their removal. Instead, they fold into complex three-dimensional structures and catalyze two transesterification reactions, thereby excising themselves joining flanking exons. These catalytic RNAs (ribozymes) have been modified previously to work in trans, whereby ribozymes can recognize a splice site on substrate RNA replace 5′- or 3′-portion of substrate. Here we describe new variant group intron ribozyme from Tetrahymena...

Group I intron ribozymes can repair mutated mRNAs by replacing the 3'-terminal portion of mRNA with their own 3'-exon. This trans-splicing reaction has potential to treat genetic disorders and selectively kill cancer cells or virus-infected cells. However, these have not yet been used in therapy, partially due a low vivo efficiency. Previous strategies improve efficiencies focused on designing testing individual ribozyme constructs. Here we describe method that selects most efficient from...

The RNA world hypothesis describes a stage in the early evolution of life which catalytic RNAs mediated replication organisms. One challenge to this is that most existing ribozymes are much longer than what may be expected originate from prebiotically plausible methods, or polymerization by currently polymerase ribozymes. We previously developed 96-nucleotide long ribozyme, generates chemically activated 5'-phosphate (a 5'-triphosphate) molecule, trimetaphosphate, and an 5'-hydroxyl group....

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has potential much broader application, including disease diagnostics, genotyping, expression studies. Current protocols microwells suitable enzymatic detection of products, but yields generally insufficient direct products using conventional fluorescent probes. Here, we quantitatively measure...

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis -splicing can be converted into trans ribozymes, which modify the sequence of a separate substrate RNA, both in vitro and vivo. Previous work on has mostly focused 16S rRNA group intron ribozyme Tetrahymena thermophila . Here, we test potential tRNA Ile bacterium Azoarcus This is only half size folds faster its active...

Synthesis of RNA in early life forms required chemically activated nucleotides, perhaps the same form nucleoside 5′-triphosphates (NTPs) as contemporary biosphere. We show development a catalytic (ribozyme) that generates triphosphate guanosine 5′-triphosphate (GTP) from and prebiotically plausible cyclic trimetaphosphate. Ribozymes were selected 1.6 × 1014 different randomized sequences by metabolically coupling 6-thio GTP synthesis to primer extension an polymerase ribozyme within 1016...

The group I intron ribozyme from Tetrahymena was recently reengineered into a trans -splicing variant that is able to remove 100-nt introns pre-mRNA, analogous the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution Escherichia coli cells. One clone with increased activity E. cells analyzed detail. Three its necessary mutations extended substrate binding duplexes, which led product formation and reduced cleavage at 5′-splice site. mutation conserved core...

How does a non-coding RNA evolve in cells? To address this question experimentally we evolved trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles evolution E.coli cells. Sequence variation was introduced during by mutagenic and recombinative PCR, increasingly active ribozymes were selected their repair an mRNA mediating antibiotic resistance. The most efficient contained four clustered mutations that necessary sufficient for maximum activity Surprisingly,...

Group I introns have been engineered into trans -splicing ribozymes capable of replacing the 3′-terminal portion an external mRNA with their own 3′-exon. Although this design makes potentially useful for therapeutic application, efficiency is usually too low medical use. One factor that strongly influences position target splice site on substrate. Viable sites are currently determined using a biochemical -tagging assay. Here, we propose rapid and inexpensive alternative approach to identify...

Polymerization of nucleic acids in biology utilizes 5'-nucleoside triphosphates (NTPs) as substrates. The prebiotic availability NTPs has been unresolved and other derivatives nucleoside-monophosphates (NMPs) have studied. However, this latter approach necessitates a change chemistries when transitioning to biology. Herein we show that diamidophosphate (DAP), one-pot amidophosphorylation-hydrolysis setting converts NMPs into the corresponding via amidophosphates (NaPs). resulting crude...

To explore how an early, RNA-based life form could have functioned, in vitro selection experiments been used to develop catalytic RNAs (ribozymes) with relevant functions. We previously identified ribozymes that use the prebiotically plausible energy source cyclic trimetaphosphate (cTmp) convert their 5'-hydroxyl group a 5'-triphosphate. While these were developed presence of Mg2+, we tested here whether lanthanides also serve as cofactors because are ideal cations for this reaction. After...

The RNA world hypothesis states that the early evolution of life went through a stage where served as genome and catalyst. replication organisms would have been facilitated by ribozymes catalyze polymerization. To recapitulate an in laboratory, series polymerase was developed previously. However, these polymerization efficiency is too low for self-replication, most efficient prefer one specific template sequence. limiting factor weak sequence-independent binding to its primer/template...