A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme

A bioinformatic covariation analysis of a collection of 119 novel variants of the antigenomic, self-cleaving hepatitis delta virus (HDV) RNA motif supported the formation of all of the Watson–Crick base pairs (bp) of the catalytic centre except the C19–G81 pair located at the bottom of the P2 stem. In fact, a novel Watson–Crick bp between C19 and G80 is suggested by the data. Both chemical and enzymatic probing demonstrated that initially the C19–G81 pair is formed in the ribozyme (Rz), but upon substrate (S) binding and the formation of the P1.1 pseudoknot C19 switches its base-pairing partner from G81 to G80. As a result of this finding, the secondary structure of this ribozyme has been redrawn. The formation of the C19–G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs. Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction

Development of the genetic code: insights from a fungal codon reassignment

The high conservation of the genetic code and its fundamental role in genome decoding suggest that its evolution is highly restricted or even frozen. However, various prokaryotic and eukaryotic genetic code alterations, several alternative tRNA-dependent amino acid biosynthesis pathways, regulation of tRNA decoding by diverse nucleoside modifications and recent in vivo incorporation of non-natural amino acids into prokaryotic and eukaryotic proteins, show that the code evolves and is surprisingly flexible. The cellular mechanisms and the proteome buffering capacity that support such evolutionary processes remain unclear. Here we explore the hypothesis that codon misreading and reassignment played fundamental roles in the development of the genetic code and we show how a fungal codon reassignment is enlightening its evolution.publishe

Computational generation and screening of RNA motifs in large nucleotide sequence pools

Although identification of active motifs in large random sequence pools is central to RNA in vitro selection, no systematic computational equivalent of this process has yet been developed. We develop a computational approach that combines target pool generation, motif scanning and motif screening using secondary structure analysis for applications to 1012–1014-sequence pools; large pool sizes are made possible using program redesign and supercomputing resources. We use the new protocol to search for aptamer and ribozyme motifs in pools up to experimental pool size (1014 sequences). We show that motif scanning, structure matching and flanking sequence analysis, respectively, reduce the initial sequence pool by 6–8, 1–2 and 1 orders of magnitude, consistent with the rare occurrence of active motifs in random pools. The final yields match the theoretical yields from probability theory for simple motifs and overestimate experimental yields, which constitute lower bounds, for aptamers because screening analyses beyond secondary structure information are not considered systematically. We also show that designed pools using our nucleotide transition probability matrices can produce higher yields for RNA ligase motifs than random pools. Our methods for generating, analyzing and designing large pools can help improve RNA design via simulation of aspects of in vitro selection

SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem–loops, extensive long-range interactions (LRIs) and a small, palindromic stem–loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem–loops are static structures, the 5′ and 3′ regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem–loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs

Prebiotically plausible mechanisms increase compositional diversity of nucleic acid sequences

During the origin of life, the biological information of nucleic acid polymers must have increased to encode functional molecules (the RNA world). Ribozymes tend to be compositionally unbiased, as is the vast majority of possible sequence space. However, ribonucleotides vary greatly in synthetic yield, reactivity and degradation rate, and their non-enzymatic polymerization results in compositionally biased sequences. While natural selection could lead to complex sequences, molecules with some activity are required to begin this process. Was the emergence of compositionally diverse sequences a matter of chance, or could prebiotically plausible reactions counter chemical biases to increase the probability of finding a ribozyme? Our in silico simulations using a two-letter alphabet show that template-directed ligation and high concatenation rates counter compositional bias and shift the pool toward longer sequences, permitting greater exploration of sequence space and stable folding. We verified experimentally that unbiased DNA sequences are more efficient templates for ligation, thus increasing the compositional diversity of the pool. Our work suggests that prebiotically plausible chemical mechanisms of nucleic acid polymerization and ligation could predispose toward a diverse pool of longer, potentially structured molecules. Such mechanisms could have set the stage for the appearance of functional activity very early in the emergence of life

Size, constant sequences, and optimal selection

Because the abundance of functional molecules in RNA sequence space has many unexplored aspects, we compared the outcome of 11 independent selections, performed using the same affinity selection protocol and contiguous randomized regions of 16, 22, 26, 50, 70, and 90 nucleotides. All affinity selections targeted the simplest isoleucine aptamer, an asymmetric internal loop. This loop should be abundant in all selections, so that it can be compared across all experiments. In some cases, two primer sets intended to favor selection of different structures have also been compared. The simplest isoleucine aptamer dominates all selections except with the shortest tract, 16 contiguous randomized nucleotides. Here the isoleucine aptamer cannot be accommodated and no other motif can be selected. Our results suggest an optimum length for selection; surprisingly, both the shortest and the longest randomized tracts make it more difficult to recover the motif. Estimated apparent initial abundances suggest that the simplest isoleucine motif was 20- to 40-fold more frequent in selection with 50- or 70-nucleotide randomized regions than with any other length. Considering primer sets, a pre-formed stable stem within fixed flanking sequences had a five-to 10-fold negative effect on apparent motif abundance at all lengths. Differing random tract lengths also determined the probable motif permutation and the most abundant helix lengths. These data support a significant but lesser role for primer sequences in the outcome of selections

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

CD19-specific chimeric antigen receptor (CAR19) T cells, generated using viral vectors, are an efficacious but costly treatment for B cell malignancies. The nonviral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic-resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that of using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems