Journal: bioRxiv

Article Title: Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

doi: 10.1101/2023.03.17.533225

Figure Lengend Snippet: Primer extension upon acid-denatured duplex, following delayed addition of varying amounts of substrates after neutralisation. Reactions were set up as in , but with different concentrations of triplet present upon neutralisation and freezing (shown are estimated eutectic phase triplet concentrations after neutralisation). After a delay of 5 minutes or 24 h, ribozyme was added (alongside remaining substrates to a final eutectic phase concentration of 25 µM) before continued incubation at −7°C. The proportional reduction in full-length product signal with a longer delay in each pair of lanes (each triplet concentration) is calculated by gel densitometry. Little reannealing appears to occur even down to low triplet concentrations. ppp NNN: Instead of just the triplet substrates in , all 64 triplet sequences were added as substrates; the strand coating effect was maintained.

Article Snippet: To identify TPR-synthesised RNA products from cycling in the absence of primers with ppp NNN, 3’ adapters were ligated to RNAs purified from the gel in (1× T4 RNA ligase buffer (NEB), 1.6 µM IllLigBio adapter pre-adenylated using a 5’ adenylation kit (NEB), 15% PEG, 16 U/µl of T4 RNA ligase 2 truncated KQ (NEB), 10°C 20 h, 65°C 12 min).

Techniques: Concentration Assay, Incubation

Journal: bioRxiv

Article Title: Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

doi: 10.1101/2023.03.17.533225

Figure Lengend Snippet: ( a ) Design of replication substrates and scheme for iterative replication of an N 18 RNA random-sequence library. To start, library template LibN 18 was mixed at 8 nM in replication buffer (including 0.9 mM KCl and 20 nM TPR) together with 20 nM each of the indicated primers (FITCrep, Cy5rep, ppp GUAGC, ppp GGACC) and 12 nM each of all 64 triplets ( ppp NNN). ( b ) Denaturing PAGE of FITCrep extension products from this reaction analysed at 5-cycle intervals before 3-fold serial dilution. ( c ) Quantification of overall amplification of ‘triplet register +5’-length products in part B, calculated as fold increase in band intensity since 5 cycles, multiplied by reaction dilution since 5 cycles. Exponential fits yielded observed per-cycle amplification efficiencies.

Article Snippet: To identify TPR-synthesised RNA products from cycling in the absence of primers with ppp NNN, 3’ adapters were ligated to RNAs purified from the gel in (1× T4 RNA ligase buffer (NEB), 1.6 µM IllLigBio adapter pre-adenylated using a 5’ adenylation kit (NEB), 15% PEG, 16 U/µl of T4 RNA ligase 2 truncated KQ (NEB), 10°C 20 h, 65°C 12 min).

Techniques: Sequencing, Serial Dilution, Amplification

Journal: bioRxiv

Article Title: Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

doi: 10.1101/2023.03.17.533225

Figure Lengend Snippet: ( a ) Replication reactions were set up with or without 0.1 µM of all 64 RNA triplets ( ppp NNN) in replication buffer (no primers, 20 nM TPR, 1.8 mM KCl) ± 20 nM N 20 random-sequence RNA template seed, and subjected to iterative cycles of replication and dilution as shown. ( b ) Denaturing PAGE of RNA samples at different stages (up to 73 cycles) stained with SYBRgold: both intensity and length of RNA products increased during cycling and serial dilution. ( c ) Estimated conversion of the total triplet substrate pool into RNA products. Lane intensities in (b) beneath the TPR bands were measured by densitometry, and the corresponding ppp NNN-free reaction backgrounds (and, if present, N 20 seed band intensities) were subtracted. These intensities were converted to RNA product yields using the N 20 seed band intensity as a reference ((b), left lane), treating fluorescence of stained RNAs as proportional to their length. The triplets needed to make these RNA yields were expressed as a percentage of the available triplet substrate. ( d ) In silico ‘translation’ using a reduced codon set of sequenced unseeded 73-cycle synthesis products (red) compared to a simulated pool of random sequences with matching lengths but unbiased composition (grey). For each sequence the longest stretch of family box codons is counted to show the maximum potential length of any encoded peptide at a primitive stage of genetic code development. ( e ) Ribozyme sequence complementarity in sequenced synthesis products from the unseeded 73-cycle reaction (left) and a simulated pool of randomised RNAs of identical composition (right). Data coloured by classification in (f) (see for criteria used); sequences with homology to ribozyme (+) strand are plotted separately . ( f ) Changes in proportions of sequence classes in 9-27 nt products from unseeded reactions during amplification. ( g ) As cycling progresses, the G-C base composition of sequences classed as having no ribozyme homology increases (data from N 20 -seeded reactions shown). ( h ) Mapping of ribozyme-homologous parts of 9-27 nt products from unseeded amplification reactions to the (+) and (−) strands of the TPR subunits 5TU and t1. Peak heights reflect the fraction of products homologous to that site, scaled by the product intensity in the corresponding gel lane in ( b ). Products with homology to multiple locations on one or both strands were randomly assigned to one. Note the prior emergence and build up of (−) strand TPR homology products, followed by (+) strand products (templated from (−) strand products).

Article Snippet: To identify TPR-synthesised RNA products from cycling in the absence of primers with ppp NNN, 3’ adapters were ligated to RNAs purified from the gel in (1× T4 RNA ligase buffer (NEB), 1.6 µM IllLigBio adapter pre-adenylated using a 5’ adenylation kit (NEB), 15% PEG, 16 U/µl of T4 RNA ligase 2 truncated KQ (NEB), 10°C 20 h, 65°C 12 min).

Techniques: Sequencing, Staining, Serial Dilution, Fluorescence, In Silico, Amplification

Journal: bioRxiv

Article Title: Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

doi: 10.1101/2023.03.17.533225

Figure Lengend Snippet: ( a ) Isolation of amplification products. We excised the indicated regions of the gel used to PAGE separate ppp NNN amplification products and eluted and precipitated the RNAs therein. To sequence these products we used a variant of the protocol in . There, alkaline phosphatase and polynucleotide kinase treatments generated 5’ monophosphates to enable 5’ adaptor ligation; here, RNAs were instead treated with a pyrophosphohydrolase to selectively convert 5’ triphosphates to monophosphates (to promote selective ligation and sequencing of ribozyme-synthesised products; nonetheless, a low level of TPR molecules/fragments were sequenced – see (f)). The named pools A-I sampled different stages of the N 20 seeded and unseeded amplifications with ppp NNN; the N 20 seed itself, lacking a 5’ triphosphate group, would not be sequenced. Pool A was derived from excising the corresponding 9-27 nt region of a separate equivalent 1-cycle seeded amplification. ( b ) Length distributions observed within the pools of sequenced RNAs. The strong triplet register bias confirms sequencing of ribozyme-synthesised RNAs. ( c ) Workflow for classification of the sequenced products. Only triplet-register products were analysed. Amplification products that exceeded stringent length-dependent sequence identity thresholds at any point when aligned along the (+) strand sequences of either 5TU or t1 TPR subunits (or their (−) strand complements) were classed as possessing ribozyme homology. These were further subdivided by the (+) or (−) strand they were matched to; some showed homology to both (unsurprising in a hairpin-rich RNA with internal complementarity) and were classed separately. Sequences that clearly did not align to the ribozymes or their complements at any point were also classed separately. Sequences between the indicated identity thresholds could not be easily categorised and were excluded from further analysis. ( d ) Levels of ribozyme homology within RNA product pools. Top: the fractions of each sequenced product pool that showed ribozyme homology as determined in part (c). A substantial fraction exhibits complementarity to ribozyme, though this decreases after many cycles. Bottom: absolute amounts of different product sequences. Here, total column heights are proportional to amounts of synthesised and triplet register RNA, based upon intercalator fluorescence in the excised region in part (a); columns are subdivided as in the top chart. All categories of product increase in absolute abundance over the course of cycling. ( e ) Reclassification of longer amplification products. Most of the > 27 nt sequences apparently exhibited no overall ribozyme homology (see part (d)). However, this was partly an artefact of aligning all along these longer sequences. Here, only the 4 th -12 th nt of each sequence was aligned, revealing ribozyme homology in a similar proportion of longer sequences (pools E & I) to those in the shorter fractions (pools D & H). Thus a substantial fraction of the longer amplification products exhibit local but not global ribozyme homology; they may have been generated by recombination or seeded from ribozyme-derived shorter sequences. Note that the 9 nt length window used here is too short to definitively class sequences as ‘not derived from ribozyme’ using our identity thresholds. ( f ) TPR identity amongst sequences classed as (+)-strand homologous. Shown are levels of identity amongst sequences meeting the identity threshold for (+) or (+) & (−) strand homology (with other sequences plotted in ), for both sequenced synthesis products from the unseeded 73-cycle reaction (left), and for a simulated pool of randomised RNAs of identical composition (right). The reaction products contain a population of sequences with complete or near-complete (+)-strand identity, exhibiting a reasonably uniform length distribution. These likely derive from background sequencing of ribozyme (or degradation products thereof, despite requiring a 5’ phosphate or triphosphate for recovery). A second population of products (<30 nt, and more abundant than in a pool of randomised sequences) has only partial (+) strand homology indicating a synthetic origin. ( g ) Negligible ribozyme homology is observed amongst scrambled pool sequences. 9 nt sequences, generated from random triplet assortments (matching the triplet compositions of the indicated pools) were classified as in part (c).

Article Snippet: To identify TPR-synthesised RNA products from cycling in the absence of primers with ppp NNN, 3’ adapters were ligated to RNAs purified from the gel in (1× T4 RNA ligase buffer (NEB), 1.6 µM IllLigBio adapter pre-adenylated using a 5’ adenylation kit (NEB), 15% PEG, 16 U/µl of T4 RNA ligase 2 truncated KQ (NEB), 10°C 20 h, 65°C 12 min).

Techniques: Isolation, Amplification, Sequencing, Variant Assay, Generated, Ligation, Derivative Assay, Fluorescence

Journal: Molecular Cell

Article Title: Mechanism of signal-anchor triage during early steps of membrane protein insertion

doi: 10.1016/j.molcel.2023.01.018

Figure Lengend Snippet:

Article Snippet: m7G(5')ppp(5')G RNA Cap Structure Analog , New England Biolabs , S1404L.

Techniques: Recombinant, Transfection, Software