RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox, we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit ‘+’ and ‘–’ strands in segments and assemble them into a new active ribozyme. Life as we know it relies on three types of molecules: DNA, which stores genetic information; proteins that carry out the chemical reactions necessary for life; and RNA, which relays information between the two. However, some scientists think that before life adopted DNA and proteins, it relied primarily on RNA.Like DNA, strands of RNA contain genetic data. Yet, some RNA strands can also fold to form ribozymes, 3D structures that could have guided life’s chemical processes the way proteins do now. For early life to be built on RNA, though, this molecule must have had the ability to make copies of itself.This duplication is a chemical reaction that could be driven by an ‘RNA replicase’ ribozyme. RNA strands are made of four different letters attached to each other in a specific order. When RNA is copied, one strand acts as a template, and a replicase ribozyme would accurately guide which letters are added to the strand under construction. However, no replicase ribozyme has been observed in existing life forms; this has led scientists to try to artificially create RNA replicase ribozymes that could copy themselves.Until now, the best approaches have assumed that a replicase would add building blocks formed of a single letter one by one to grow a new strand. Yet, although ribozymes can be made to copy straight RNA templates this way, folded RNA templates – including the replicase ribozyme itself – impede copying. In this apparent paradox, a ribozyme needs to fold to copy RNA, but when folded, is itself copied poorly. Here, Attwater et al. wondered if choosing different building blocks might overcome this contradiction.Biochemical techniques were used to engineer a ribozyme that copies RNA strands by adding letters not one-by-one, but three-by-three. Using three-letter ‘triplet’ building blocks, this new ribozyme can copy various folded RNA strands, including the active part of its own sequence.This is because triplet building blocks have different, and sometimes unexpected, chemical properties compared to single-letter blocks. For example, these triplets work together to bind tightly to RNA strands and unravel structures that block RNA copying.All life on Earth today uses a triplet RNA code to make proteins from DNA, and these experiments showed how RNA triplets might have helped RNA sustain early life forms. Further work is now needed to improve the ribozyme designed by Attwater et al. for efficient self-copying. The premise that some RNA sequences can catalyse and template their own replication - reciprocally synthesizing their own ‘+’ and ‘–’ strands - underpins current thinking about early genetic systems . Any ancIn a contrasting approach, RNA polymerase ribozymes (RPRs) have been developed that can use general monomer building blocks (ribonucleoside 5’ triphosphates (NTPs)) in RNA-templated RNA synthesis , akin toWe wondered whether this paradox might be avoided through a re-consideration of plausible building blocks for early RNA replication. Models of non-enzymatic polymerisation of all four activated ribonucleotides – the presumed source of the first RNA sequences – yield pools of di-, tri- and tetranucleotide etc. length oligonucleotides (in decreasing abundance) dominating the population alongside longer products . Here, wWe set out to explore the potential of short RNA oligonucleotides as substrates for RNA-catalyzed RNA replication. To do this, we required a ribozyme capable of general, iterative RNA-templated oligonucleotide ligation. Previously-described RNA polymerase ribozymes such as the ‘Z’ RPR  can use To be able to properly examine such RNA trinucleotide triphosphates (triplets) as replication substrates, we first sought to convert Zcore into an effective triplet polymerase ribozyme using in vitro evolution. We devised a selection strategy that required iterative templated triplet ligation by ribozymes to achieve their covalent linkage to a tagged primer . This en15 Zcore variants with a new random 3’ N30 region under eutectic phase conditions that increase RNA half-life and enhance ribozyme activity  receptive to activity enhancement by type 1 RNA . The catIn complex with type 5 , type 1 boosts polymerization of triplets (or longer oligonucleotides) to enable synthesis of long RNAs . Here, i+1’ , diversifying the previously-fixed 3’ domain reverse transcription primer binding sequence. This reselection yielded a shorter final heterodimeric triplet polymerase holoenzyme, hereafter termed ‘t5+1’ . This ro+1 activity, we revisited triplet-based RNA synthesis on structured templates. To provide a stringent test of template structure inhibition, we now examined hairpin-containing templates  with increasing RNA hairpin stability and estimated TMs of up to 93˚C (8S). The latter had previously strongly arrested even the most advanced mononucleotide RPRs at higher temperatures . Encouragingly, t5+1 could synthesise both a functional fluorescent ‘+’ strand of the 52 nt Broccoli RNA aptamer  could reduce tertiary structures  for synthesis of full-length α+ and ε+ segments  was similar to that observed when t5+1 used an unstructured model template . We found that this synthesis product could regenerate fresh δ- segment using t5+1 ribozyme-synthesised δ+ (left over from ribozyme assembly) as a template , efficiency of both segment synthesis and assembly will need to be increased significantly to realise a full self-replication cycle .At this point, we tested whether the broad oligonucleotide ligation capacity of t5+1  might al strands . Through strands , the ribtemplate , recapitTemplated ‘+’ strand self-synthesis is a central element of ribozyme self-replication. However, a limitation of our above strategy in the context of triplet-based self-replication is the continued requirement for some pre-synthesized longer oligonucleotides to act as primers and occasional substrates . In particular, some specific oligonucleotide substrates were required for efficient synthesis of α+ and ε+ segments to compete out inhibitory mutual hybridisation between ‘−’ strand template and corresponding ‘+’ strand unstructured elements in the t5 ribozyme . In vitrThe majority of specific oligonucleotides, however, were provided as primers to initiate syntheses, as required by all RPRs akin to the activity of replicative polymerases in biology. As a consequence of this, the priming sequence would effectively be excluded from evolution during replication. Furthermore, RNA oligonucleotides able to act as specific primers are unlikely to be prevalent in prebiotic substrate pools, and their depletion during successive replication cycles could lead to loss of sequence at genome ends. This ‘primer problem’ has previously been noted in the context of nonenzymatic replication  as one o+1 can extend primers bidirectionally, in both the canonical 5’−3’ as well as the reverse 3’−5’ directions , as exemplified here for the β+ segment  could be used as substrates by t5+1 in segment syntheses in place of defined triplet sets  and collated, which allowed an estimation of ribozyme misincorporation tendencies. On average, the starting Zcore ribozyme exhibited ~91% fidelity per position  synthesised by t5+1 using pppNNN indicated similar aggregate fidelity could be achieved during longer product synthesis excluding the final triplet  reverted the fidelity profile towards that of Zcore (Having established that accurate triplet-based copying is possible (in at least some sequence contexts), we sought to understand how the triplet polymerase ribozyme achieves it. Investigating the fidelity contributions of different t5of Zcore . Presencof Zcore . Overallof Zcore , though of Zcore . The ε dof Zcore .Tetrahymena group I intron folding  rendered the ε fidelity domain unable to discriminate mismatches . Previou folding  and the  folding .pppGCC:pppGGC (than pppACC:pppGGU) dimer formation would selectively reduce the effective concentration of free pppGCC vs. pppACC upon inclusion of their complementary pppGGC and pppGGU.An important contribution to triplet fidelity also appears to arise from unexpected behaviours of the triplet substrates themselves. We observed that in some direct pair-mispair triplet contests, inclusion of their complementary triplets caused a striking (~3 fold) drop in misincorporation errors . A potenpppNNN substrate pools  binding strength.These pairwise reductions were recapitulated in the presence of random te pools . Indeed,ror rate . Althougror rate . Dimer fpppNNN pool formulated with a reduced G content  with a robust ability to polymerize RNA trinucleotide triphosphate (triplet) substrates. Unusually, this triplet polymerase ribozyme comprises a heterodimer of a catalytic triplet polymerase subunit (t5) and a non-catalytic RNA cofactor (type 1), which enhances triplet polymerase activity and abrogates the need for template tethering. Such a quaternary structure - involving a heterodimer of a full-length and a truncated subunit - is reminiscent of the processivity factors of some proteinaceous polymerases such as the heterodimeric p66/p51 HIV reverse transcriptase holoenzyme , with particularly helpful stability contributions from intra-triplet base stacking . FurtherTogether these favourable molecular traits serve to pre-organize the template towards a double-stranded RNA duplex with triplet junctions poised for ligation. A triplet/template duplex presents a more ordered, regular target for sequence-general ribozyme docking (by e.g. the ε domain) than a single stranded template (variably prone to secondary structure formation or sequence-specific interactions with the ribozyme ). Such g+1 ribozyme fidelity is due to both a readout of cognate minor groove interactions by the ribozyme ε domain , suggestive of convergent adaptive solutions to the challenges of replication and decoding. It has long been speculated that the decoding centre of the small ribosomal subunit might have had its origins in an ancestral RNA replicase, but the implied triplet-based character of such a replicase was conspicuously discordant with modern mononucleotide-based replication . The utiIn conclusion, the unexpected emergent properties of triplets – including cooperative binding and unfolding of structured RNA templates, enhanced incorporation of AU-rich substrates, and error attenuation (resulting from triplet pool interaction networks) – argue that short RNA oligonucleotides may represent predisposed substrates for RNA-catalyzed RNA replication. Some of these benefits might also extend to codon/anticodon dynamics in early translation, and to the non-enzymatic replication of RNA , where d2 and 0.5 μl of 1 M tris•HCl pH 8.3  then added on ice, and left for >5 min to ensure folding. This was added to 5 pmol each of primer and template and 50 pmol of each triplet pre-annealed in 5 μl water, then frozen on dry ice (10 min) and incubated at −7˚C in a R4 series TC120 refrigerated cooling bath ) to allow eutectic phase formation and reaction.Standard ribozyme activity assays (modified where specified) comprise 5 pmol of each ribozyme annealed in 2.5 μl water , with 2 μl of 1 M MgCl2, 50 mM tris•HCl pH 8.3). Supercooled reactions  led to a higher final reaction volume, but eutectic phase equilibration restored standard operational concentrations, also applicable to the four-fold-diluted extensions with the fragmented ribozyme , with pre-annealed primer/template/substrates (0.5/0.5/5 pmol) added in 0.75 μl water. These reactions, and preparative syntheses , with urea to a 6 M final concentration and a 10–20 fold molar excess over template of complementary competing oligonucleotide see  to preve+/γδε+, +, δ+, ε+, + synthesised in + synthesis was denatured directly from the ligation reaction in 60% formamide with excess EDTA.To avoid using potentially confounding competing oligonucleotide when purifying extension products, reactions with a biotinylated primer or template (stopped as above) could be purified by bead capture using MyOne C1 (Invitrogen) streptavidin-coated paramagnetic microbeads (using 5 μg pre-washed beads per pmol biotinylated RNA) in 0.5 × − 0.8 × bead buffer , 1 mM EDTA, 0.1% Tween-20). After washing twice in BB to remove unbound components, beads were incubated (1 min) in 25 mM NaOH, 1 mM EDTA, 0.05% Tween-20 to denature the duplexes . To reco2) for separation by native PAGE  2) kept at 4˚C in a circulator bath for 6–8 hr at 10 W), then SYBR Gold stained as below.For gel mobility shift assays , ribozymFluorescent primer extension products were detected using the appropriate laser wavelength on a Typhoon Trio scanner  ); gel densitometry allowed quantification of RNA synthesis efficiency. Gel contrasts in figures were linearly adjusted to optimize display of bands of differing intensities.The gel in +) alongside known amounts of the positive controls indicated, followed by SYBR-Gold staining. To purify, bead-eluted products were run similarly, and excised using UV shadowing. Products were then eluted from the gel fragments in 10 mM tris•HCl pH 7.4, and Spin-X column filtrate ) precipitated in 75% ethanol with 1 μl 1% glycogen carrier (omitted for β+). Recovered full-length product yields were calculated similarly to reaction yields for αβ+/δε+/γδε+, or using A260s for β+, δ+, ε+.Full-length product yields in the +/γδε+ fragments were sequenced by first ligating a 3’ adaptor (10 U/μl T4 RNA Ligase 2 truncated KQ in 1 × RNA ligase buffer (New England Biolabs (NEB), ) with 15% PEG-8000 and 2 μM AdeHDVLig at 10˚C overnight). These reactions were bound to MyOne C1 microbeads (ThermoFisher (Invitrogen)), washed with BB to remove unligated adaptor, and reverse transcribed (50˚C 30 min) with 1 μM HDVrec primer using Superscript III (Invitrogen). Beads were washed again then PCR amplified  using GoTaq HotStart master mix ) and 0.8 μM each of primers P3HDV, and P5Xα8 or P5Xγ7, for high-throughput sequencing  MiSeq or HiSeq) after PCR product agarose gel purification. β+ syntheses’ cDNAs were amplified with P3HDV and P5Xβ6.Purified ribozyme- and TGK-synthesized αβpppNNN on templates encoding CCC-XXX-CCC, where XXX were 12 different triplet sequences evenly exploring base composition and distribution . The 12 pairs were combined in 0.27M MgCl2/67 mM tris•HCl pH 8.3 on ice to discourage primer-template assortment . 36 pmol of each triplet in pppNNN  were added to a reaction vessel in 10.8 μl water, to which 5.4 μl of the primer/template/buffer mix was added followed by 7.2 pmol of ribozyme pre-annealed  in 1.8 μl water . Reactions were frozen and incubated (7 days at −7˚C) as described above.Each primer/template pair (0.45/0.525 pmol per reaction) was annealed in 4 mM MgClReactions were stopped with 3.6 μl 0.44 M EDTA and 10.5 pmol of each template's competing oligonucleotide , denatured with 6 M urea, and urea-PAGE separated. After alignment with a fluorescence scan of the gel, a region of the sample lane corresponding to primers extended by +4 to +14 nt was excised (encompassing 2–4 triplet additions), and extension products were eluted, precipitated in 77% ethanol with 1 μl 1% glycogen carrier, washed in 85% ethanol and resuspended in water.th reverse transcription mix, 0.8 μM each of primers P3HDV and P5GGGX) for sequencing as above (yielding 2 × 105 – 4 × 106 sequences per ribozyme assay).These extension products were 3’ adaptor ligated as for fragment sequencing. Products were reverse transcribed  and then PCR-amplified  were combined for purification and sequencing as above, to allow isolation of the ribozyme-mediated errors resulting from inclusion of the other 62 (61 for ACC) triplets in the reaction . The counts of cognate triplet (C) and each error triplet (E) in the positive control (p) reduced error counts in the experimental samples (x) to yield ribozyme-mediated error counts (Er) thusly: Er = Ex - Ep*(Cx/Cp)  remained uncorrected).After processing and 3’ adaptor trimming, sequences corresponding to primer extended by CCC +1–3 additional triplets were collated for analysis. Variations in upstream primer sequences see  allowed For each template, counts were then collated at the first/second/third positions to yield base-specific mutation rates for each position . Across pppGU, encoding UAC yielded just pppAC) or use oligonucleotides generated during transcription to re-initiate .Triplets (and some other short oligonucleotides) were prepared from NTPs by T7 RNA polymerase run-off transcription of a 5’ single-stranded DNA overhang downstream of a DNA duplex T7 promoter sequence. In most cases, the 5’ overhang encoded (was the reverse complement of) the desired oligonucleotide. These oligonucleotides were short enough to synthesise during the abortive initiation stage of transcription, attenuating sequence constraints on the first bases of the transcript. However, T7 RNA polymerase exhibited tendencies to skip the first (or even second) base , and reduced recruitment as initiators of products with bases not complementary to the introduced first position template base .These tendencies could be subverted by encoding additional first bases . This initiated the oligonucleotide at the second position where skipping tendencies were lower  in 1 × MegaShortScript kit buffer with 1.5 μl MegaShortScript T7 enzyme (ThermoFisher). Also present were 15 pmol of each DNA oligonucleotide forming the transcription duplex target ).Each 30 μl transcription reaction contained 72 nmol of each desired product base as an NTP (Roche) ); incorporation and migration were similar between the two, and their concentrations were calculated from A260nm by comparison to the A260nm of mixtures of the component ribonucleotides with UTP vs. U2STP. Triplets with 2’-fluoro, 2’-deoxy positions could also be transcribed, with lower efficiency, by substituting the corresponding triphosphate (Trilink Biotechnologies). The biotinylated pppGAU–Bio triplet used in γ segment synthesis ), quantified via by comparison to the A290nm of mixtures of the component ribonucleotides.3’-deoxy triphosphorylated ‘terminator’ triplets were transcribed as above but using a 3’ deoxynucleoside 5’ triphosphate (Trilink biotechnologies) for the last position, migrating faster during PAGE than the equivalent all-RNA triplet. Triplets with 2-thiouridine residues were transcribed as for their corresponding U, replacing UTP with Uynthesis  was tranLonger triphosphorylated oligonucleotides used in ribozyme self-synthesis were generated similarly, but using ~200 ng of fully double stranded DNA as a template. Candidate product bands were purified and the desired oligonucleotide identified by ribozyme-catalysed in-frame incorporation and, for some, fragment sequencing.Transcriptions were performed on ~15 ng/μl dsDNA using MegaShortScript enzyme and buffer (ThermoFisher) with 7.8 mM of each NTP, or, to yield a 5’ monophosphate on the product to avoid aberrant ligation, 10 mM GMP (guanosine monophosphate) and 2 mM of each NTP (‘GMP transcription’).dsDNA templates for some of these in  were gen2) to extend 5’ biotinylated RNA primers (0.75 μM) on DNA templates (1 μM) using 2.5 mM of each NTP . Biotinylated products were bead-purified as above.Some 5’ biotinylated RNAs were synthesized using the TGK polymerase   cycloaddition (3’ biotinylation of RNAs was achieved in two stages: 3’ azidylation (at 2 μM with 25 U/μl yeast poly-A polymerase (ThermoFisher) and 0.5 mM 2’-azido-2’-deoxycytidine triphosphate (Trilink Biotechnologies) for 1 hr at 37˚C) with subsequent acidic phenol/chloroform extraction and 75% ethanol precipitation, then copper-catalysed biotin-(PEG)addition  with subRound one libraries were synthesised by mutual extension of 4 nmol of oligonucleotides 1baN30 and 1GMPfo or 1GTPfo at 1 μM each in 1 × isothermal amplification buffer (NEB) with 250 μM each dNTP, annealed  before addition of 0.4 U/μl Bst 2.0 (NEB) and 30 min 65˚C incubation.15 molecules) were transcribed in 5 ml transcription reactions (36 mM tris•HCl pH 7.9 (at 25˚C), 1.8 mM spermidine, 9 mM DTT, 10.8 mM MgCl2, 2 mM each NTP, 1% 10 × MegaShortScript buffer, 2% 1:9 MegaShortScript:NEB T7 RNA polymerase, 37˚C overnight). These were treated with DNase, acid phenol/chloroform extracted and 73% ethanol precipitated prior to urea-PAGE purification, elution, filtering (Spin-X) and re-precipitation, yielding the 1GTP Zcore selection construct  derived from amplification of recovered PCR products (see later).After purification, 375 μg of each DNA . DNA encoding type 5s amplified with AACAt5s was used to generate reselection libraries by PCR amplification using primers TriGAA7GAAM and T5ba13N/T5ba20N/T5ba28N; 5 pmol of the three dsDNA products were transcribed to generate reselection constructs.For round 8, 700 pmol DNA was formed, with Tri3CUUQ amplifying round seven merged output (50 pmol), round seven merged output recombined by StEP   or 2 μM HOCUG (Rounds 8–18) with 30 U/ml T4 RNA Ligase 2 (NEB)). After incubation at 4˚C for 1 hr, these were stopped with 2.2 mM EDTA and acid phenol/chloroform treated.At the end of incubation the reaction was thawed on ice. To link the primer 3’ hydroxyl to the 5’ monophosphate of GMP constructs, selection constructs were buffer-exchanged directly after thawing using a PD-10 column (GE) in a cold room, into 3 ml ligation mix (optimised to prevent ligation over gaps) (2 mM MgClConstructs were then precipitated with glycogen carrier and 0.3 M sodium acetate in isopropanol (55%) before resuspension and denaturation . For the reselection rounds, constructs were then treated with polynucleotide kinase (NEB) before denaturation to resolve the HDV-derived 2’, 3’-cyclic phosphates and allow later adaptor ligation.Constructs were urea-PAGE separated alongside FITC-labelled RNA markers equivalent to successfully ligated constructs. The marker-adjacent gel region in the construct lane was excised, excluding the bulk unreacted construct (judged by UV shadowing). Biotinylated (primer-linked) constructs were eluted overnight into BB with 100 μg MyOne C1 beads. After 30 μm filtering )) of the supernatant to remove gel fragments, the beads were washed in BB then denaturing buffer  to confirm covalent linkage of construct to primer, before further BB washing and transfer (to a fresh microcentrifuge tube to minimize downstream contamination). At this stage in the reselection, 3’ adaptors were then ligated to bead-bound constructs as above for 2 hr (with buffer/enzyme added after bead resuspension in other reaction components including 0.04% Tween-20), and beads BB washed and transferred again.Bead-bound constructs were now reverse-transcribed using 1 μM RTri (or HDVRec for the reselection) by resuspension in a Superscript III reaction with added 0.02% Tween-20 (50˚C 30 min). Beads were BB washed and the RNA-bound cDNA 3’ end blocked by incubation with terminal deoxynucleotidyl transferase (ThermoFisher) and 0.2 mM dideoxy-ATP (TriLink) with 0.02% added Tween-20 (37˚C 30 min), and beads were BB washed and transferred again.cDNAs were eluted (10 μl 0.1 M NaOH 0.1% Tween-20 20 min), neutralized and plus strands regenerated with 0.2 μM rescue oligonucleotide in an IsoAmp II universal tHDA kit (NEB) reaction (65˚C 60 min) to read through the structured product region. Whilst at this temperature, reactions were stopped with 5 mM EDTA and one volume of BB with 50–100 μg beads to bind the nascent biotinylated plus strands at room temperature. These beads were then BB washed, NaOH washed again to discard cDNAs (and recover only correctly-primed plus strands), and washed and transferred again.260nm quantified and added to construct synthesis PCR in 3 × molar amount of the anticipated recovered RNA (judged by test extensions) that yielded it.Each 50 μg of beads were then subjected to plus strand recovery PCR in a 100 μl GoTaq HotStart reaction with 0.5 μM each RTri (or HDVrec for reselection) and RecInt (rounds 1–5)/RecIntQ (rounds 6–9)/RecIntL /RecIntQL (rounds 15–18). The product was agarose size-purified, AThis final PCR for construct transcription in the subsequent selection round used 1 μM of the indicated construct synthesis primer, plus 1 μM RTri (or HDVrt for the reselection), in GoTaq HotStart reactions or (where indicated in source data) the GeneMorph II kit for mutagenesis ).Conditions for selections are included as source data 1 for  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Nebojsa Janjic (Reviewer #3).Thank you for submitting your article "Ribozyme-catalysed RNA synthesis using triplet building blocks" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The most important thing to improve is the written clarity of the manuscript. There was a lot of agreement between the reviewers' comments so we attach their full reviews below for your attention. All their concerns should be attended to.Reviewer #1:Attwater et al. report an advance in efforts to develop a robust self-replicating RNA by presenting an example of an RNA polymerase ribozyme (RPR) capable of transcribing RNA from structured RNA templates of substantial length with fairly good fidelity. This RPR uses a trinucleotide building block (in contrast to NTPs used by standard polymerases), that the authors speculate could have implications in the evolution of mRNA decoding by the ribosome.The major findings from this work are as follows:+1 which was used for downstream studies. This RPR could use structured hairpins as templates, even those with Tm >90oC that were intractable to previous RPRs that use NTPs as substrates. The authors demonstrate the efficiency of this RPR by transcribing both + and – strands of the Broccoli aptamer, a structured and fluorescent RNA.1) The authors exploit the ligase activity in the catalytic core of their previously reported RPR Z and evolve a suite of catalytic sequences capable of extending an RNA primer using RNA triplets as substrates. Through further iterations of in-vitro selection for triplet dependent RPRs, an RNA sequence emerged without any catalytic activity itself, but it enhanced triplet extension by these newly evolved RPR sequences (named t2 through t6) through a bimolecular interaction between the catalytically proficient sequence. Further rounds of selection using the cofactor t1 resulted in the most efficient triplet RPR, t5+1 RPR to replicate fragments of itself (both + and – strands), using triplet building blocks (and in some cases hexamers). The complete active RPR could be reconstituted from the five individual components of the RPR transcribed by the same RPR. Although this replicase ribozyme is still not able to replicate itself in its entirely, it does give rise to a functioning replicase by using building blocks as small as trimers, Previous efforts had primarily used the ligase activity of the RPRs to assemble oligonucleotides representing their sequence to assemble a 'replica' of itself. Considering the challenges of self-replication, even incremental advances in the right direction are significant.2) The authors used t5Overall this work introduces a new RPR system that brings the field closer to a self-replicating system, which is one of the cornerstones of Origin of life research. The authors explore properties of this replicase including fidelity and catalytic efficiency and explain the molecular basis for these properties. This work also introduces trinucleotides as plausible substrates on which ancient RNA-based replicases could have acted. Considering the relative ease of non-enzymatic polymerization of monomers into triplets, the presence of these triplets in a prebiotic setting seems plausible. The current work is detailed and the authors have explored multiple facets of the new system they have setup. This new RPR presents a major advance in terms of its ability to create a fully functional copy of itself . As such, this would be a great addition to the current understanding of RNA-based self-replication systems.1) Overall, the manuscript was very difficult to follow. It is up to the authors to make their presentation as simple and direct as possible. Given the availability of supporting information, there is no reason why the authors could not add schemes where appropriate to clarify their presentation, logic, and workflow.2) Regarding the dimerization, it is not clear how the authors implicated the 5'-hairpin to begin with, leading to the experiment that showing that a single mutation in that loop inhibits dimerization.3) The assertion that the two RNAs form a 1:1 complex is not quantitatively substantiated from Figure 3B.4) Considering their success in replicating structured hairpins and achieving primer free synthesis of an 18 nt strand (β+), it would be interesting (though not necessary for publication) to see how much of the Broccoli RNA could be transcribed without the addition of a primer sequence. If this is not possible, the authors could demonstrate the complete replication of a functional RNA of tractable length, like the Hammerhead ribozyme, to illustrate the ability of their trinucleotide RPR to replicate entire sequences of functional RNAs by exclusively using triplets as substrates.5) The fidelity analysis is based on testing 12 of the possible 64 trinucleotides, so one cannot be convinced of the generality of their observations and inferences. The authors need to underscore this in the discussion of fidelity. The authors show that increased concentrations of triplets help fidelity, however in the absence of the 'correct' triplet, it is likely that RPR will incorporate 'mismatch' triplets. The authors should comment on this aspect.Reviewer #2:1) In this paper, the authors demonstrate interesting and novel results that provide further evidence for RNA's capacity to act as prebiotic genetic material. The key feature associated with this study involved using trinucleotide triphosphates (triplets) rather than NTPs as substrates for a novel RNA polymerase ribozyme. The use of triplets allowed structured templates to be unfolded and in some cases avoided the need for primers, circumventing an ongoing obstacle to RNA self-replication. This study thus makes some significant steps towards identifying a truly self-replicating prebiotic biopolymer. Literature precedents are well-discussed, and the results are interpreted in a detailed and convincing manner. I would nonetheless encourage the authors to try to make these important results as accessible as possible to readers who are unfamiliar with in vitro evolution.Specific comments:2) Subsection “in vitro evolution of triplet polymerase activity”, last paragraph. The use of "in-ice evolution" (a technique developed by the authors) is reported. A sentence could be added describing why this technique is being used / why it is necessary.3) For certain experiments (e.g. subsection “Fidelity of triplet-based RNA synthesis”), random triplet pools were used. What kind of triplet pools were used in other sequence-copying experiments? Were they non-random? This needs to be more clearly articulated.4) "… we found that using modified substrates with a disrupted minor groove hydrogen bond acceptor at the 3rd position…" – What is the modification? This should be stated in the body-text.+1 ribozyme? Triplets reportedly incur a lower entropic cost than NTPs , but what would happen when a mixture of the two were used? Would both the NTPs and triplets be incorporated?5) What is the scope of substrates for the t5If these points are satisfactorily addressed, then I am in favour of acceptance.Reviewer #3:Attwater et al. used an in vitro evolution method to enhance the trace amount of ligase activity with short (3-nucleotide) substrates inherent in the truncated version of the Z RPR ribozyme . This was achieved by adding a random region of 30 nucleotides at the 3' end of Zcore and a template at the 5' end, followed by selection for variants able to extend a primer hybridized to the 5' template with two 5'-triphosphate triplets and ultimately ligate the extended primer to the 5' terminal triphosphate. After 7 rounds of activity enrichment, the authors identified a sequence that represented 25% of the activity-enriched pool called "type 0" that, upon truncation to its minimal active sequence ("0core"), was able to catalyze multiple cycles of templated ligation with triplet substrates, including the ability to extend thought double-stranded regions of the template by cooperative invasion through the secondary structure of the template .+1. t5+1 was able to catalyze the extension through templates with Tm values of 93 ⁰C, and was capable of generating both the functionally active Broccoli RNA aptamer and its complementary template. Finally, t5+1 was able to replicate its own t5 sequence split into five segments, mostly from triplets, with some help from longer substrates (hexamers) needed for two of the segments, thus falling short of having the capacity for a full replication cycle, but nonetheless, exhibiting impressive catalytic efficiency.After additional 14 rounds selection, the composition of the pool showed considerable changes: type 0 sequence had disappeared and was replaced by six new types of sequences, types 1-7. Remarkably, the most prevalent type 1 sequence (50% of the pool) was completely inactive and types 2-6 were less active than the activity enriched pool. This mystery was solved by the observation that a truncated version of the type 1 sequence, while being inactive, can form augment the activity of type 2-6 ribozymes by forming heterodimeric complexes. In the presence of type 1 sequence, the best stand-alone ribozyme, type 5, no longer required the template to be tethered to the 5' end of the type 5 ribosome, in a remarkable example of an intermolecular complex between the dimeric type 1:type5 ribozyme, the untethered primer template, and the triplet substrates. Further optimization of the type 5 ribozyme by re-randomization of the previously fixed 3' end sequences and reselection in the presence of minimal type 1 sequence  resulted in the final heterodimeric holoenzyme called "t5+1 can extend primers in both the canonical 5'-3' as well as the reverse 3'-5' direction raises the potential for primer extension from short sequences in a primer-free manner, therefore potentially solving the "primer problem" (depletion of longer but presumably sparse primers needed for replication in prebiotic conditions). Impressive overall positional fidelity of more than 97% was achieved by counter-selection for mispairing triplets with a clever inclusion of excess 3'-deoxy terminator triplets during late rounds of selection. Attwater et al. mapped the region responsible for fidelity to the 3' domain of t5. Further reduction in mis-incorporation comes from cognate triplet-triplet interactions have a self-correcting feature of countering the effect of triplets with strong base-pairing tendency.The surprising observation that t5eLife. [Editors’ note: this reviewer’s minor comments have not been included.]The authors make a strong case that triplets represent relatively short and yet powerful substrates able to invade highly stable structural features in RNA templates. The evolution of a heterodimeric ribozyme capable of generating functional RNAs with triplet substrates represents a major accomplishment that advances the plausibility of RNA-catalyzed RNA replication in prebiotic environment. Despite its considerable length, as well as its remarkable breadth and scope, the manuscript reads like a riveting novel. It is a tour de force scientific accomplishment, and as such, it will be of considerable interested to a wide audience of readers of  Reviewer #1:[…] 1) Overall, the manuscript was very difficult to follow. It is up to the authors to make their presentation as simple and direct as possible. Given the availability of supporting information, there is no reason why the authors could not add schemes where appropriate to clarify their presentation, logic, and workflow.We apologize for the poor presentation and have made several changes to enhance clarity. These include textual changes to reduce ambiguity and lay out our reasoning more thoroughly as well as explanatory schemes of ribozyme activity in Figure 1 and Figure 1—figure supplement 1. Furthermore, we have redesigned Figures 3 and 7, and now provide an additional dataset presenting substrate concentration-dependent error rates in a more straightforward manner in Figure 9—figure supplement 1.The manuscript structure is complicated by the multiple stages of RNA evolution and characterization involved in the project, and to help navigate the progress of our ribozyme development, we now provide the new Figure 4—figure supplement 1 detailing all the stages of evolution and engineering of the different triplet polymerase ribozymes to provide a “roadmap”.2) Regarding the dimerization, it is not clear how the authors implicated the 5'-hairpin to begin with, leading to the experiment that showing that a single mutation in that loop inhibits dimerization.We welcome the opportunity to clarify this. We had originally noticed the ‘cap-’ mutation in the most abundant isolated type 1 clones, and when testing this in different constructs observed that it quenched type 1 enhancement pointing towards the 5’ hairpin region as the critical interaction site. We now refer to this in the manuscript text.Our knowledge about this dimerization interface is currently limited and we have therefore generally refrained from speculation. However, it seems possible that the ‘cap-’ mutation was enriched in these type 1 sequences to diminish homodimerisation of type 1 that may interfere with heterodimerisation with active triplet polymerase in the selection pool.3) The assertion that the two RNAs form a 1:1 complex is not quantitatively substantiated from Figure 3B.We now include quantitative data on the stoichiometry of the complex as observed by gel shift (EMSA) titration in Figure 3B, and activity tests in Figure 3—figure supplement 1. These support a 1:1 stoichiometric complex in the active ribozyme as expected from the final selection pool composition.4) Considering their success in replicating structured hairpins and achieving primer free synthesis of an 18 nt strand (β+), it would be interesting (though not necessary for publication) to see how much of the Broccoli RNA could be transcribed without the addition of a primer sequence. If this is not possible, the authors could demonstrate the complete replication of a functional RNA of tractable length, like the Hammerhead ribozyme, to illustrate the ability of their trinucleotide RPR to replicate entire sequences of functional RNAs by exclusively using triplets as substrates.The reviewer highlights an important challenge that such ribozymes must ultimately face – copying without primers both strands of functional RNAs to achieve their complete replication using only short oligonucleotide substrates. With the current triplet polymerase ribozyme, while primer-initiated synthesis is almost general (as we show by synthesizing both + and – strands of the Broccoli RNA aptamer), we cannot currently achieve primer-free synthesis of all sequences. The latter includes Broccoli where yields of full-length RNA are poor using triplets alone. This may reflect inefficient initiation of synthesis or poor upstream inclusion of some 5’ terminal triplets.Nevertheless, the current triplet polymerase ribozyme can achieve this for some functional sequences, most relevantly from t5 itself. To demonstrate this, we have now included data on the primer-free synthesis and replication of the Υ segment. Specifically, we describe primer-free synthesis using triplets alone of the γ+ strand segment with sufficient yield to use this ribozyme-synthesized RNA as the template for primer-free γ- strand synthesis, demonstrating complete primer-free sequence replication using only triplets. These new data are now included as Figure 7C, replacing the (now in our view redundant) primer-free γ+ syntheses.5) The fidelity analysis is based on testing 12 of the possible 64 trinucleotides, so one cannot be convinced of the generality of their observations and inferences. The authors need to underscore this in the discussion of fidelity. The authors show that increased concentrations of triplets help fidelity, however in the absence of the 'correct' triplet, it is likely that RPR will incorporate 'mismatch' triplets. The authors should comment on this aspect.We measured fidelity of triplet incorporation with all64 triplet substrates present but in the context of 12 defined template triplet sequences flanked by conserved CCC incorporation sites. These were chosen to be compositionally balanced, including both GC-rich and AU-rich template triplets, to provide fair estimates of ribozyme fidelity, as well as an effective tool to compare the fidelities of different ribozymes.This assay was designed to allow us to separate incorporation fidelity (i.e. discrimination among 64 triplets for decoding a defined anti-triplet) from each ribozyme’s extension capabilities by providing a uniform, tractable sequence context.The important qualification regarding this assay, we feel, is therefore that while it provides an accurate representation of fidelity in a defined sequence context (triplet-triplet junction), it may not provide a comprehensive picture of triplet polymerase fidelity performance in all sequence contexts. We now provide this caveat in the text.Reviewer #2:1) In this paper, the authors demonstrate interesting and novel results that provide further evidence for RNA's capacity to act as prebiotic genetic material. The key feature associated with this study involved using trinucleotide triphosphates (triplets) rather than NTPs as substrates for a novel RNA polymerase ribozyme. The use of triplets allowed structured templates to be unfolded and in some cases avoided the need for primers, circumventing an ongoing obstacle to RNA self-replication. This study thus makes some significant steps towards identifying a truly self-replicating prebiotic biopolymer. Literature precedents are well-discussed, and the results are interpreted in a detailed and convincing manner. I would nonetheless encourage the authors to try to make these important results as accessible as possible to readers who are unfamiliar with in vitro evolution.As discussed above, we have made a significant number of changes to the manuscript in order to present our results more clearly.These include textual changes to improve clarity and remove ambiguities. We have also made an effort to lay out our experimental progress and reasoning more thoroughly, using explanatory schemes of ribozyme activity in Figure 1 and Figure 1—figure supplement 1, redesigning Figures 3 and 7, and providing an additional more straightforward dataset in Figure 9—figure supplement 1. We hope this will make the manuscript easier to read and more accessible to the general reader.Specific comments:2) Subsection “in vitro evolution of triplet polymerase activity”, last paragraph. The use of "in-ice evolution" (a technique developed by the authors) is reported. A sentence could be added describing why this technique is being used / why it is necessary.We have expanded our description of the mechanisms of in vitro evolution and discuss in more detail the reasoning behind our use of eutectic ice phases and in-ice evolution. As the properties of ice as a medium for RNA-catalyzed RNA synthesis, replication and evolution are discussed at length in the cited references, a fairly brief discussion must suffice. We hope nevertheless that this will now clearly illustrate our rationale in using this technique in our work.3) For certain experiments (e.g. subsection “Fidelity of triplet-based RNA synthesis”), random triplet pools were used. What kind of triplet pools were used in other sequence-copying experiments? Were they non-random? This needs to be more clearly articulated.We generally list the triplet substrates used for different experiments but apologise for any ambiguity.In general for preparative syntheses we have avoided using pools of random triplets, providing instead a set of defined triplets as specified by the template sequence to minimise substrate consumption. We have however verified that random triplet pools can be used for key triplet based RNA synthesis experiments including hairpin invasion  and ribozyme segment synthesis , as well as in experiments on ribozyme fidelity . We now explicitly state the substrate composition in the text and figure legends for all experiments described.4) "… we found that using modified substrates with a disrupted minor groove hydrogen bond acceptor at the 3rd position…" – What is the modification? This should be stated in the body-text.We apologize for this omission and have amended this. We now state in the text that uracil was replaced by 2-thio uracil in the triplets.+1 ribozyme? Triplets reportedly incur a lower entropic cost than NTPs , but what would happen when a mixture of the two were used? Would both the NTPs and triplets be incorporated?5) What is the scope of substrates for the t5This is an interesting suggestion considering that abiotically-generated substrate pools would likely include a mixture of different length substrates including mono- and dinucleotides. We have performed additional experiments to explore this and now include data on their incorporation during segment synthesis in Figure 6—figure supplement 5.+1 does incorporate both dinucleotide triphosphates and NTPs, but with reduced efficiency. Furthermore, they appear to serve as poor substrates for segment synthesis with triplets incorporated much more efficiently. Increasing amounts of NTP and dinucleotide substrates vs. triplets  do lead to diversification of extension products outside the initial triplet register, but do not notably affect the total amount of ligation performed.In summary, t5As seen in Figure 6—figure supplement 2, more length-heterogenous substrate pools (including longer oligomers) may offer both advantages and disadvantages, but we feel that a more systematic investigation of diverse substrate pools and their effect on yield and fidelity of RNA-catalyzed RNA replication goes beyond the scope of the current manuscript.


Exposure of the airway model to acidic solutions caused cellular damage and loss of viability, however, acid alone did not disrupt barrier integrity or instigate neutrophil trans-epithelial migration without pepsin. Gastric fluid from patients on PPI therapy exhibited only a slightly higher pH yet had significantly higher concentrations of pepsin and elicited more barrier disruption and neutrophil trans-epithelial migration compared to gastric fluid from patients off PPIs. Inflammatory and damaging responses observed with gastric fluid from patients on PPIs were largely driven by pepsin. These results indicate the potential for PPI usage to raise concentrations of pepsin in gastric fluid, which may enhance the pathological impact of micro-aspirations in children with extraesophageal reflux.Pepsin represents a potential biomarker for extraesophageal reflux disease when detected in airways, however a direct role for pepsin in lung dysfunction has not been clearly established. Children experiencing gastroesophageal and extraesophageal reflux are often prescribed proton pump inhibitors (PPIs) to reduce gastric acid associated damage to esophageal and airway mucosa. The potential of pepsin and gastric fluid, from children that were either on or off PPI therapy, to cause inflammation and damage using a human  Understanding the relationship between GER and airway dysfunction has been a challenge without sensitive biomarkers to effectively diagnose extraesophageal reflux disease. One of the proposed biomarkers is the proteolytic digestive enzyme pepsin2. Pepsin is produced in the stomach where it is autocatalytically cleaved from its proenzyme, pepsinogen. Since pepsinogen is uniquely released and processed to pepsin in the acidic environment of the stomach, the presence of pepsin in the lung suggests transfer of gastric contents to the airway, a consequence of reflux and micro-aspiration. Pediatric studies suggest that in patients with respiratory disease, up to 44% of patients can have detectable pepsin in bronchoalveolar lavage fluid or saliva suggesting an association between pepsin and extraesophageal symptoms4. Whether pepsin is simply a biomarker or if it plays a role in the pathophysiology of lung disease in children is not known. Pepsin, in the presence of acid, can cause the denuding of laryngeal epithelium with subsequent membrane leakiness as well as elicit the production of inflammatory cytokines5. Pepsin has also been shown, even in nonacidic environments, to promote macrophage differentiation and migration as well as induce inflammatory cytokines in tonsillar tissue6.Gastroesophageal reflux (GER) has been implicated as an exacerbating factor for lung disease in children8. Since pepsin is maximally activated at low pH, there is a theoretical benefit to raising the gastric pH using proton pump inhibitors (PPIs) in order to reduce acid related damage; however, PPI treatment does not mitigate reflux and micro-aspiration, it just changes the pH of the refluxate. Furthermore, it has been demonstrated that gastric fluid collected from adult patients receiving PPIs and exposed to airway epithelial cells stimulated a greater inflammatory response compared to gastric fluid from patients not taking PPIs7. Specifically, gastric fluid from PPI-treated patients triggered a more substantial release of the chemo-attractant IL-8, which is associated with neutrophil recruitment to the airway mucosa7.There is significant debate over the role of acid suppression in preventing pepsin-related damage to mucosal surfaces14. Evidence of pepsin exposure leading to airway epithelial damage, barrier leakiness, and/or neutrophil breach of the epithelium would suggest a potential for the capacity of pepsin to exert a pathological response, if present in the lungs. Additionally, gastric fluid was collected from children who were either taking or not taking PPIs and evaluated in the co-culture model to determine whether gastric fluid was capable of triggering airway responses and whether any such response was driven by pepsin and/or impacted by the PPI status of the donor.The study herein seeks to address the role of pepsin at various pHs in a co-culture model featuring neutrophils and airway epithelial barriers12. Pepsin was applied to epithelial cells at a range of pH to reflect the variability of pH found in gastric fluid. Consistent with previous studies15, neutrophils migrated across lung epithelial barriers in response to an imposed chemotactic gradient of fMLP or in response to apical infection with Pseudomonas aeruginosa (strain PAO1)  do not migrate across the epithelial barrier  Fig. . Neutropier Fig. . Pepsin,ier Fig. . Howeverier Fig. .Figure 1Since pepsin is a protease that works efficiently at low pH, it was next determined whether pepsin was capable of disrupting H292 airway epithelial barrier integrity in the absence of neutrophils. To assess barrier integrity, a horse radish peroxidase (HRP) flux assay was employed following H292 monolayer treatment with HBSS (pH 7.4), triton X-100 (Tx-100), and various concentrations of pepsin suspended in HBSS . After exposure to stimuli for 1 hour, the paracellular probe HRP was added to the basolateral side and the amount of HRP that transferred across the epithelial barrier to the apical side over the course of 2 hours was quantified. Buffer alone elicited minimal HRP flux, whereas treatment with Tx-100, a detergent that disrupts the epithelial barrier, caused substantial HRP flux Fig. . Pepsin Lung epithelial cells exposed to pepsin suspended in pH 3 buffer, as described in the above experiments, are likely rendered non-viable because of cellular exposure to extremely low pH for 1 hour. Consistent with this hypothesis, MTT conversion was not readily detected within H292 monolayers pre-exposed to pH 3 HBSS irrespective of pepsin concentration Fig. . MTT conSince pepsin treatment of H292 airway epithelial monolayers abrogated MTT conversion at pH 3 in a similar manner regardless of pepsin concentration, the impact of acid in the absence of pepsin was explored in several contexts. Firstly, the impact of H292 monolayer exposure to acid alone was explored and it was determined that exposure to pH 3 HBSS by itself caused complete abrogation of MTT conversion by 1 hour of exposure Fig. . Secondl18. Pepstatin A (20 µg/mL) was sufficient to block pepsin-induced neutrophil migration across H292 monolayers exposed to 50 µg/ml pepsin at pH 3  therapy exhibit more pepsin or less pepsin in their gastric fluid compared to individuals not receiving therapy, or whether any such differences would influence the inflammatory triggering potential of gastric fluid when encountering airway mucosal surfaces. Gastric fluid samples from twenty-four children that were either on (n = 12) or off (n = 12) PPI therapy at the time of gastric fluid harvesting were thus collected and processed to analyze a variety of properties , weight (p = 0.66), or gastric pH p = 0.17) . A representative experiment involving four patient gastric fluid samples demonstrates that the greatest magnitude of induced migration occurs at pH 3, with some activity occurring at pH 7.4, and very little induced migration detectable at pH 5   Fig. . HoweverNext the effects of Pepstatin A on neutrophil trans-epithelial migration induced by gastric fluid samples at both pH 3 and 7.4 was examined. Pepstatin A (20 µg/ml) incubation with pH 3 gastric samples during exposure to H292 monolayers resulted in a reduction gastric sample-induced neutrophil trans-epithelial migration Fig. . This ef14. Primary airway basal stem cells can be cultured on permeable Transwell filters at an air-liquid interface (ALI) to differentiate into a mature airway mucosa. The ALI cultures feature beating cilia, the capacity to secrete mucus, and differentiate as multiple epithelial cell types, which organize in a manner similar to the mature human lung mucosa23. Inverted ALIs cultured on 3.0 µm Transwell filters are a required modification to the conventional ALI Transwell model to permit the investigation of neutrophil trans-epithelial migration in a physiologically relevant direction and these cultures were fixed and stained with antibodies to detect acetylated α-tubulin (AcTub) and ezrin, which mark cilia and ciliated cells respectively  and does not occur when bacteria are suspended in buffer at pH 3 during infection. Further, bacterial induced neutrophil transepithelial migration is pepstatin A independent and requires epithelial 12-lipoxygenase activity19. The enzyme 12-lipoxygenase is likely non-functional after epithelial exposure to acidic conditions and pretreatment of the epithelium with an inhibitor of 12-lipoxygenase did not impact pepsin-induced neutrophil transepithelial migration.Lung dysfunction associated with extraesophageal reflux disease in children is widely reported, yet the inflammation and damage that occur within the airway resulting in the clinical presentation is not well understood. Several previous studies have explored this question by employing 28. Given the importance of pepsin and acid towards triggering airway inflammation  and damage  observed using the human airway mucosal in vitro model system, gastric fluid samples were collected from children undergoing endoscopy and evaluated in the same experimental system. Since treatment with proton pump inhibitors (PPIs) is administered to raise the pH in the gastric fluid, samples were collected from patients that were on PPI therapy or off PPI therapy to determine if any difference could be detected between these two cohorts. Surprisingly, there was only a slight increase in the pH of gastric fluid samples collected from patients on PPI therapy and this mean difference did not reach statistical significance. Interestingly, there was a statistically significant increase in pepsin concentration in the patients that were on PPI therapy and this difference correlated with significant increases in the ability of gastric fluid to induce neutrophil transepithelial migration and cause airway epithelial barrier disruption. Thus, at low pHs, gastric fluid from patients on PPI therapy caused significantly more inflammation and damage when compared with gastric fluid from patients off PPI therapy. Furthermore, the acid protease inhibitor pepstatin A significantly inhibited gastric fluid-induced neutrophil transepithelial migration at low pHs when gastric fluid was derived from patients on PPI therapy, suggesting that pepsin was a major driver of inflammation within the complex milieu of the gastric fluid derived specifically from this patient cohort. Collectively, these results suggest that pepsin in combination with acid cause inflammation and damage when applied to human airway mucosal models. Furthermore, these constituents appear to be important drivers of inflammation and damage by acidic gastric fluid, particularly in patients on PPI therapy, irrespective of the potential of PPIs to raise gastric pH.Gastric fluid contains a variety of substances, all of which can be refluxed and aspirated into the lung6. Similarly, we show that pepsin triggers neutrophil migration when in an acidic but not pH-neutral environment. Based on these results, we would expect that acid suppression would be beneficial to children with reflux related lung disease. However, when we looked at neutrophil migration across epithelium exposed to gastric fluid from PPI-treated patients, we surprisingly found that acid suppression may result in refluxate that is more destructive to the airway. Neutrophil migration was enhanced in magnitude following exposure to gastric fluid from PPI-treated patients compared with gastric fluid from untreated patients. These results raise the question that PPIs may, in fact, be more harmful than beneficial, possibly by indirectly increasing pepsin concentration in the gastric fluid as we observe herein that PPI-treated patients had higher gastric pepsin concentrations compared to untreated patients. The clinical implications of this are worth noting because PPIs are commonly used in the treatment of extraesophageal symptoms and our data suggest that while there may be a theoretical benefit to raising gastric pH, the benefit may be out weighted by the risk of increasing pepsin concentrations which may ultimately be more harmful.These results raise the possibility that pepsin in not just a biomarker but may have proinflammatory effects on the lung epithelium. A recent study of pepsin in tonsillar tissue reveals that pepsin can cause macrophage maturation, release of inflammatory cytokines, and triggers macrophage migration across an epithelial layer29. In a study by Mertens et al., bronchial epithelial cells were exposed to gastric fluid from adults on and off PPIs7. The authors first measured the gastric pH as well as concentrations of bile, pepsin, and endotoxin and then measured IL-8 production from the bronchial epithelial cells. The authors found that bronchial epithelial cells exposed to gastric fluid with higher pHs (less acidic) produced more IL-8 than cells exposed to gastric fluid with low pH. Interestingly, the authors of this study reported a relationship between PPI status and endotoxin levels. Filtered gastric fluid was found to exhibit a significant reduction in bronchial epithelial cell IL-8 production, which the authors interpreted as due to removal of food and bacteria, potent instigators of IL-8 production. There are several reasons why our results may differ from this study. First, our gastric concentrations of pepsin were significantly higher in the PPI treated patients than those found by Mertens et al. suggesting why we have observed a more prominent pepsin effect on inflammation than the prior study7. Second, the pHs of the gastric fluid in the Mertens et al. study were significantly higher than those in our study. Despite PPI use, only 1/12 treated patients in our study had a pH > 4 suggesting that pepsin activation may have differed in our patients and that the likelihood of gastric bacterial overgrowth in our patients may be much less.While we have shown that an acidic milieu and pepsin are significant triggers of inflammation, others have shown different mechanisms by which gastric fluid may trigger inflammationIn vivo, it is reasonable to speculate that exposure to aspirated products is likely to be episodic, lower in concentration, and briefer in duration as opposed to a single, high concentration, 1-hour duration exposure used in the in vitro model described here. Now that we have clarified that prolonged exposure of pepsin and acid on an epithelial model together have a synergistic negative effect, the next step would be to determine if there is a threshold effect for duration or concentration that might mimic a micro-aspiration model. This process is not well understood in vivo and therefore it is difficult to determine the ideal in vitro model to mimic human disease however we suggest that this study lays the groundwork for critical future studies, particularly as we show no benefit and potential harm to acid suppression therapy. In this study, we show that an acidic environment alone had a significant impact on the viability of the model and rendered the epithelium metabolically inactive. An important next step would be to consider designing a model that would allow for much smaller acid exposure through micro-aerosolization, which may more accurately represent what the airway surface would experience. Another issue observed in this study was that the pH of gastric fluid in children taking PPIs was surprisingly low. This may be related to differences in PPI metabolism compared to adults, genetic polymorphisms, delays in PPI dosage potentially related to NPO status at the time of endoscopy, or some combination of these factors30. This work raises the possibility that traditional PPI regiments in children fail to prevent some degree of acid aspiration while also increasing the amount of pepsin present.There are limitations to our study that merit discussion. We used both a lung epithelial model and an air-liquid interface model that were exposed to acid with or without pepsin or gastric fluid at varying pH. Despite these limitations, the results of this study are important because they suggest that pepsin may serve a pathological role in the lung rather than simply representing a biomarker of reflux-related lung disease. We further show that the concentration of gastric pepsin is higher in patients taking PPIs, thus raising the possibility that aspirated gastric contents in patients taking PPIs may have a greater proinflammatory and/or damaging effect and might contribute to a worsening of airway function in patients diagnosed with extraesophageal reflux.2 96-well 3.0 µm HTS TranswellTM filters  as described previously11. Wells were coated with 30 µg/ml rat tail collagen  prior to seeding. H292 cells were maintained in 10% FBS RPMI-1640 medium containing 1% penicillin-streptomycin and cultured at least eight days prior to use. Passages ranged from passage 4 to passage 35. Cultures are maintained with a volume of 235 µl media in the bottom chamber  and 80 µl media within the inner Transwell .The human lung epithelial cell line H292 was obtained from American Type Culture Collection  and grown on the underside of 0.14 cm23. These cultures were generated from basal stem cells isolated from a single donor without lung disease as previously described23. Basal cells were cultured using a recent modification of the ALI model whereby basal cells were seeded on the underside of individual 0.33 cm2 inverted 3.0 µm Transwells sized for 24-well plates (Corning product #3415) and cultured for 1 to 2 days prior to establishment of air-liquid interface, whereupon media was removed from the bottom well 14. This maneuver is necessary to conduct neutrophil trans-epithelial migration experiments in a physiologically relevant manner14. ALI cultures were grown for at least 9 days to generate a functional airway barrier but no longer than 34 days to avoid overgrowth or loss of barrier function. Media (200–300 µl) was added to the inner well of the Transwell  and changed every other day until differentiation was well established. Once ALI cultures reached maturity, cells were examined by confocal microscopy and neutrophil trans-epithelial migration assays were conducted. A voltmeter (EVOM2) was purchased from World Precision Instruments, Inc. to assess trans-epithelial electrical resistance (TEER) in evaluation of ALI cultures for inclusion in these studies in order to confirm epithelial barrier integrity prior to conducting assays. At room temperature, ALI cultures were fixed with 4% paraformaldehyde (PFA) for 10 min, washed, and permeabilized with PBS + 0.2% Triton X-100. Then, ALIs were treated with primary antibodies against cellular markers of various states of differentiation, followed by PBS + 1% bovine serum albumin (BSA) exposure for two hours at room temperature or 4 °C overnight (>16 hrs). After incubation, ALIs were washed with PBS + 0.2% Triton X-100 for 4 rinses and placed with secondary antibodies for a room temperature incubation of 1–2 hours. After rinsing 4 times with PBS that also has 0.2% Triton X-100, cells within ALI cultures were stained with 0.1 µg/ml DAPI  prior to mounting slides and recording images. ALI cultures stained as describe above were visualized using the IX81 inverted fluorescence microscope from Olympus. Several planes of focus were captured and combined using Olympus Soft Imaging Solutions, MicroSuite FIVE and Extended Focal Imaging (EFI) module to create a single in-focus image of ALI cultures22. Antibodies used in this study include acetylated Tubulin , Ezrin , ZO-1  for primary antibodies. Life Technologies provided the secondary antibodies conjugated with Alexa Fluor 594 and conjugated antibodies were diluted 1:500.Air-liquid interface (ALI) differentiated human primary cell cultures were generated as a model that physiologically reflects the airway mucosa, featuring functional cilia and goblet cells capable of secreting mucus4Cl lysis buffer. This technique allows for rapid isolation of functionally active PMNs (>98%) at 90% purity11.Neutrophils are polymorphonuclear cells (PMNs) that were derived from healthy adult human donors providing informed written consent prior to collection of blood. Blood was mixed with acid citrate/dextrose as an anticoagulant and centrifuged at room temperature with a speed of 1,000 x g and the break turned off to prevent agitation of separated layers. The majority of red blood cells (RBCs) were removed from separated blood layers by gravity sedimentation after addition of 2% gelatin. Prior to sedimentation, however, plasma and the thin white cell layer containing monocytes/lymphocytes were carefully removed by aspiration. Residual RBCs were then removed by lysis in cold NHEscherichia coli (MC1000) and pathogenic Pseudomonas aeruginosa (PAO1) were grown aerobically in 3 mL of Luria-Bertani broth at 37 °C overnight15. Prior to treatment of airway epithelial cells, 1 mL of the bacterial cultures were centrifuged for 5 minutes at 15,800 × g and the supernatant discarded. The pellet was washed with 1 mL of HBSS and centrifuged again. The supernatant was discarded, and the pellet was brought to a final volume of 500 µl and 600 µl for MC1000 and PAO1, respectively. For infection of airway epithelial cells, this concentrated solution of bacterial cultures was diluted 1:100 in HBSS and 25 µl of the 1:100 dilution was used to infect H292 monolayers cultured on 96-well 3.0 µm HTS TranswellTM filters and human primary airway basal stem cell derived ALI cultures14.Non-pathogenic 32.Human gastric fluid was collected from patients attending the laryngology outpatient clinic at Froedtert Hospital, Milwaukee, WI, scheduled to have a transnasal esophagoscopy for clinical indications. Approximately 5 to 10 mL of gastric fluid was aspirated from patients at the end of their scheduled procedure. Gastric fluid samples were pooled and pepsin 3b isolated by anion exchange chromatography. Gastric fluid was initially filtered through a piece of 113 V, 320-mm diameter Whatman filter paper  to remove mucoid and particulate matter. The filtrate was then dialyzed against 50 mmol/L sodium acetate buffer, pH 4.1 at 4 °C. Partial purification of pepsins was performed using a diethyl amino ethyl cellulose column  equilibrated with dialysis buffer at 4 °C. Negatively charged proteins, including pepsin 3b, were eluted from the DE52 column with salt (sodium acetate/1 mol/L NaCl at 2 mL/min). Fractions containing protein  were pooled and dialyzed against 50 mmol/L sodium acetate at 4 °C to remove all salt before performing the second high-resolution anion exchange chromatography with a Pharmacia Mono Q, 0.5 × 5 cm, column  developed with a linear gradient of 0.09 to 1.0 mol/L NaCl in 50 mmol/L sodium acetate, pH 4.1, over 30 minutes at 0.75 mL/minute. Pepsin 3b was identified by chromatography pattern, isolated, and stored at −20 °C in 50% glycerol  for evaluation of cough, esophageal esophagitis (EoE), gastroesophageal reflux disease (GERD), Celiac disease, or abdominal pain at Boston Children’s Hospital. Patients were included in the “on” treatment group if they had been taking PPIs for a minimum of three months and included in the “off” treatment group if they had been acid suppression naive or had been off PPIs for a minimum of three months (An exception to this was one patient who had been off PPIs for only three days) , which was calibrated with calibration buffer on ice. The gastric fluid was then aliquoted into three separate 1.5 ml sterile micro-centrifuge tubes and the pH was adjusted to approximately pH 3, 5, or pH 7.4 using 1N HCl or 1N NaOH. Sterile filtered HBSS at pH 3, 5, or 7.4 was used to correct for the dilution factor. Each pH range of a gastric fluid sample from a single patient was diluted to the same degree. Not more than 15% of the original volume was added to adjust pH of samples. Gastric fluid from each pH range was aliquoted into 50 µl aliquots to prevent multiple freeze-thaw cycles and stored at −80 °C  was selected and each sample was run in triplicate. All experiments were performed with positive and negative controls. Purified pepsin was diluted to a physiologically relevant concentration of 50 µg/ml2 for thirty minutes. H292 monolayers and ALI cultures were then inverted into a large petri dish and the apical side was treated with 25 µl of sample at the appropriate pH and allowed to incubate at 37 °C, 5% CO2 for one hour (H292 monolayer) or 3 hours (ALI cultures). After treatment, the cells were washed with warm HBSS twice before being placed in a receiver plate containing HBSS at pH 7.4. Either horseradish peroxidase (HRP) or isolated PMNs are then added to the basolateral side as described below.H292 monolayers and ALI cultures were washed with warm HBSS and allowed to equilibrate in HBSS at 37 °C, 5% COPepstatin A  was resuspended in DMSO at 10 mg/ml, aliquoted, and stored at −20 °C. Aliquots were diluted to a working stock of 1 mg/mL with an addition of filtered HBSS at the designated experimental pH of either 3 or 7.4. A final concentration of 0.2% DMSO +/− 20 µg/ml pepstatin A was established in all conditions by diluting (1:50) either the 1 mg/ml pepstatin A stock solution at the appropriate pH or 10% DMSO vehicle control solution at the appropriate pH directly into gastric fluid samples, pepsin, or PAO1 prior to treating H292 monolayers or ALI cultures. All final solutions being exposed to airway cells were evaluated using the Orion STAR A211 benchtop pH meter to verify pH prior to initiating treatment.5) in a volume of 40 µl HBSS were added to the basolateral side of each well and allowed to migrate for two hours at 37 °C, 5% CO2. Four blanks containing HBSS only in the absence of PMNs were included in each experiment. After incubation with PMNs for 2 hours, 96-well Transwell™ filter plates containing H292 monolayers were discarded and 12.5 µl of 10% Triton X-100 was added to each well in the 96-well receiver plate (containing transmigrated PMNs). A parallel plate containing a serial 2-fold dilution beginning at 2 × 105 PMNs also received 12.5 µl of 10% Triton X-100/well. PMNs from a single donor were used for each internally controlled experiment. PMN transmigration results are represented as number of PMNs that traversed the monolayer, calculated from a standard PMN dilution curve. Receiver plates were incubated at 4 °C for 20 minutes, while shaking, to fully lyse PMNs in detergent. A volume of 12.5 µl citrate buffer was then added to each well at room temperature. Finally, 250 µl ABTS  peroxidase substrate solution  was added to each well and allowed to develop at room temperature for 10 minutes, protected from light. Following incubation, optical density was determined at 405 nm. The number of PMNs that crossed the H292 monolayer in each sample was quantified by the myeloperoxidase (MPO) activity. The standard curve of known numbers of PMNs was used to convert OD@405 measurements of unknown wells to number of PMNs migrated12.After infection/treatment and washing with HBSS as described above, cells were placed into a receiver plate with HBSS pH 7.4 in the apical well. Wells containing 100 nM fMLP chemoattractant  served as a positive control to confirm that neutrophils were active and capable of migrating. PMNs (2 × 106) in a volume of 200 µl were added to the basolateral side of each well and allowed to migrate for two hours at 37 °C, 5% CO2. After incubation for 2 hours with PMNs, ALI cultures were discarded and migrated neutrophils were quantified similarly as described above with slight modifications to the volumes of Triton X-100, citrate buffer, and ABTS added as detailed previously14.After infection/treatment and washing with HBSS as described above, ALI cultures were then placed in wells containing HBSS. PMNs  using a standard curve of known HRP concentration and measurements at OD@40512.After 1 hour infection/treatment, H292 monolayers on 96-well Transwells were washed as described above and placed into a receiver plate containing HBSS. A concentration of 0.5 µg/ml horseradish peroxidase (HRP)  in a volume of 40 µl of was added to the basolateral side of each well and incubated for 2 hours at 37 °C, 5% COTM filters with polarized H292 monolayers were washed with HBSS and transferred to a new 96-well receiver plate. Receiver plates contained 115 µl HBSS and 55 µl of Component A (MTT) from the Vybrant® MTT Cell Proliferation Assay Kit 34. Two blanks containing no cells were included in a separate 96-well plate and both plates were incubated overnight at 37 °C, 5% CO2. The MTT Assay involves the conversion of the water soluble MTT -2,5-diphenyltetrazolium bromide) to an insoluble formazan. After overnight incubation, the formazan was then solubilized by the addition of 50 µl Component B (SDS) and incubated at 37 °C, 5% CO2 for four hours. Following incubation, 60 µl of the solubilized formazan in each well was mixed thoroughly by pipetting up and down and transferred into the corresponding empty wells in the plate already containing the two blanks. A volume of approximately 140 µl–160 µl was removed from the blank wells to equal the volume of the samples. Optical density was read at 570 nm.After measurement of HRP flux, all remaining supernatant was discarded and 96-well Transwell32. Briefly, Nunc-Immuno Maxisorp™ 96-well flat bottom microtiter plates  were coated with affinity purified rabbit anti-Hu3 antibody [3 μg/mL], produced against the N-terminus of mature human pepsin, in a volume of 100 μL 0.2 M sodium carbonate buffer, pH 9.6, per well, and incubated at room temperature for 18–20 hours. Plates were washed three times for two minutes per wash in phosphate buffered saline, pH 7.4 with 0.1% Tween-20 (PBS-T) and incubated with blocking buffer  for 90 minutes at 37 °C and 200 rpm on an orbital shaker . Plates were rinsed three times for two minutes per wash in phosphate buffered saline, pH 7.4 (PBS). Gastric specimens and pepsin standards  were prepared in sample diluent [1% bovine serum albumin  in PBS]. All gastric specimens were diluted at least 1:5 in sample diluent. Sample diluent alone was assayed in duplicate as a blank. After specimens and standards were applied to the microtiter plate in duplicate in a volume of 100 μL per well, the plate was incubated for one hour at 37 °C. Samples were removed from the plate by vacuum aspiration and wells were washed three times for two minutes per wash in PBS-T. Captured pepsin was detected by incubation with goat anti-human pepsin antibody diluted to 26 µg/ml in 1% BSA/PBS-T for 40 minutes at 37 °C and 200 rpm. The plate was washed three times for two minutes per wash in PBS-T and incubated with peroxidase-conjugated mouse anti-goat immunoglobulin G (Sigma-Aldrich) diluted 1:30,000 in 1% BSA/PBS-T. Enzymatic color development was carried out using 1-Step Ultra TMB ELISA (Sigma-Aldrich). Color development was stopped by the addition of an equal volume of 1N sulfuric acid and the wells read at 450 nm in the microplate reader . Duplicate readings for each standard, specimen and the blank were averaged and the average absorbance of the blank specimen was subtracted from average absorbance of specimens and standards. A standard curve was plotted using blank-subtracted mean absorbance and concentrations of pepsin standard dilutions. The concentration of pepsin in gastric specimens was calculated using a best fit curve model.The concentration of pepsin in specimens was determined by noncompetitive indirect sandwich ELISA as previously described with minor modificationsAll experiments were conducted on at least two separate occasions yielding similar results. Experiments from internally controlled experiments comparing mean of average values from clinical samples (12/group) derived from the “on” PPI therapy group versus “off” PPI therapy group are displayed as mean +/− SEM. Each data point in all other experiments represents the mean +/− SD. Differences were considered significant at p ≤ 0.05 using, in most cases, an unpaired two-tailed student’s T test with equal variance within an internally controlled experiment. Where different, specific statistical tests are described within the figure legends of each individual experiment.Four separate human studies protocols were approved by institutional review boards prior to collecting human samples for the investigations described herein. The protocol permitting human gastric fluid collection from patients for the isolation and purification of pepsin was reviewed and approved by the Institutional Review Board at the Medical College of Wisconsin, study # PRO 00004759. Human airway basal stem cells were isolated from discarded human airways harvested from the New England Organ Bank or Massachusetts General Hospital (MGH) Department of Pathology following approval of IRB protocol #2010P001354 by MGH. IRB Protocol #06-10-0439 was approved by Boston Children’s Hospital to obtain gastric fluid samples from children either taking or not taking proton pump inhibitors. Protocol #1999P007782 was approved by MGH to obtain blood from healthy adult volunteers to isolate neutrophils. Written informed consent was received from all participants and/or from parent/guardians, when applicable, prior to inclusion in each of these human study protocols. All methods were carried out in accordance with the relevant guidelines and regulations.LaTeX Supplementary File


This paper describes a novel hybrid approach of Taguchi and Genetic Algorithm to minimize number of iteration for optimization of a solution of the problem. A Genetic algorithm is used for global optimization. In GA initial population is selected randomly. Taguchi method gives a uniform combination of variables for the given search area. Hence, instead of selecting the initial populations by random search select the initial population by Taguchi design techniques. It will reduce the number of iteration to obtain a solution. This is explained with illustration.•It can be used for selecting initial population in an organized manner rather than random selection.•It can reduce the number of iterations.•It can be applicable to all optimization problems where Genetic Algorithm is used. FenαLoad anglenαNormal pressure anglecαCoast side pressure angledαDrive side pressure angleεContact ratioσFBending stressbEffective face widthFnSCritical section thicknessFehbending arm heighttFTangential loadiSpeed ratiokAsymmetry factortdSTip thickness at addendum circle on the drive sidetcSTip thickness at addendum circle on the coast sidetSTip thickness at addendum circlemModulenmNormal module1, T2TTorque on the pinion (1), gear (2)rAny radiusprPitch circle radiusbcrBase circle radius at coast sideHPSTCrRadius at HPSTCt1/rt2rAddendum circle radius on pinion (1), Gear (2)b1/rb2rBase circle radius on pinion (1), Gear (2)CCentre distanceFndθAngle at base circle radius coast side w.r.t y-axis on the drive sideFncθAngle at base circle radius coast side w.r.t y-axis on the coast sideHPSTCθAngle at HPSTC radius w.r.t y-axispzTotal tooth on the piniongzTotal tooth on the gearFYTooth form factorBYHelix angle factorSYStress correction factorFρNormal fillet radius at the root of a toothHPSTCHighest point of single tooth in contactSpecifications TableVarious methods are available to optimize a solution. The genetic algorithm is one of them and used for global optimization. GA is a work based on natural selection and fittest to survival. In GA initial population is required and it was selected by random search. GA gives solution after some iteration based on the complexity of problem .Taguchi design technique gives more variables with fewer experiments using orthogonal array method. It also gives a good combination of variables within the given search area .Various studies are available in the literature for hybridization of Taguchi method and Genetic Algorithm. In all hybridization, focus on a selection of initial population by Taguchi method is untouched. In this paper, instead of selecting initial population by random search, select the initial population by Taguchi design techniques. It will reduce the number of iteration to obtain a solution. This is explained with illustration.Gears are a very important part in power transmission. Durability and consistency in power transmission is the main focus in gear design. To improve higher durability in power transmission, current focus of in industry is on the failure occurring at a critical section of the tooth ,4.Failure at critical section of the tooth can be reduced by minimizing bending stress at critical section of the tooth. Higher pressure angle on the drive side is desirable to minimize bending stress at the root of a tooth ,6. It wiFor illustration, the parameters are given in In this illustration, the main aim is to minimize bending stress at the critical section. Objective function/fitness function has been considered from reference  to calcuObjective function/fitness function to minimize bending stress at the critical section with constraint:Nominal tooth root stress tS ≥ 0.25mConstraints are: d)α, Contact ratio (ε) and tip thickness (tS)are considered as a gene for chromosomes.9 chromosomes have been created using random search method. Parameters such as drive side pressure angle (t) are constraints for the same. Gear design standard procedures recommend that, the contact ratio should be higher or equal to 1.1  = Chromosome [1] =  = Chromosome [2] =  = Chromosome [3] =  = Chromosome [4] =  = Chromosome [5] =  = Chromosome [6] =  = Chromosome [7] =  = Chromosome [8] =  = Chromosome [9] =  = Chromosome [1] =  = Chromosome [2] =  = Chromosome [3] =  = Chromosome [4] =  = Chromosome [5] =  = Chromosome [6] =  = Chromosome [7] =  = Chromosome [8] =  = Chromosome [9] = [αAn initial population has been developed using Taguchi methods. This will help in achieving the best combinations. A population has been given in Section 4.1- step 5.As per reference , it is fFrom the illustration presented in the previous section, number of iterations are 15 when initial population selected by random search and number of iterations are 10 when initial population is selected using Taguchi method. Comparison of number of iterations obtained from GA, when initial population is selected by random search and initial population is selected by Taguchi method is given in It is observed that a number of iterations to solve a problem is reduced, when the Taguchi method is used to select the initial population instead of random search.


MDM2 oncogene copy number alterations in cases of chronic lymphocytic leukemia” (CLL) with great interest [MDM2 overexpression was examined at mRNA and protein levels, but amplification of the MDM2 gene at DNA level in CLL patients has been examined for the first time in our study” [MDM2 overexpression is an important pathological phenomenon seen in CLL [MDM2 overexpression can be useful in the diagnostic and therapeutic management of the patient. Nevertheless, we would like to point out that the present report by Darbaş et al. [MDM2 gene at DNA level in CLL patients. A previous report by Watanabe et al. [We read “Investigation of interest . Darbaş interest  stated: r study” . Indeed,ş et al.  is not te et al.  already  MDM2 oncogene copy number alterations in cases of chronic lymphocytic leukemia” [MDM2 in CLL patients. One of them was written by Watanabe et al. [MDM2 was amplified in 23 B-CLL specimens and found that none of the patients showed amplification. In the other study, Bixby et al. [MDM2 gene in 37 of 178 CLL patients using 50K SNP-array technology. After screening the literature in detail, we found two additional studies. Bueso-Ramos et al. [MDM2 gene using the Southern blot technique. Considering these four studies, amplification of the MDM2 gene at DNA level in CLL patients had been previously examined by Southern blot and array techniques, but we used the FISH technique in our study to demonstrate MDM2 amplification in CLL patients.We would like to thank the authors for showing interest in our letter, which was titled “Investigation of eukemia” . The auts et al.  reported