RNA-sequencing (RNA-seq) has emerged as one of the most sensitive tool for gene expression analysis. Among the library preparation methods available, the standard poly(A) + enrichment provides a comprehensive, detailed, and accurate view of polyadenylated RNAs. However, on samples of suboptimal quality ribosomal RNA depletion and exon capture methods have recently been reported as better alternatives.We compared for the first time three commercial Illumina library preparation kits  as representatives of these three different approaches using well-established human reference RNA samples from the MAQC/SEQC consortium on a wide range of input amounts (from 100 ng down to 1 ng) and degradation levels .R2 > 0.92) on intact RNA samples down to input amounts of 10 ng. For degraded RNA samples, Ribo-Zero showed clear performance advantages over the other two protocols as it generated more accurate and better reproducible gene expression results even at very low input amounts such as 1 ng and 2 ng. For highly degraded RNA samples, RNA Access performed best generating reliable data down to 5 ng input.We assessed the accuracy of the generated expression values by comparison to gold standard TaqMan qPCR measurements and gained unprecedented insight into the limits of applicability in terms of input quantity and sample quality of each protocol. We found that each protocol generates highly reproducible results  performs better than other methods on highly degraded and low amount samples.The online version of this article (doi:10.1186/s12864-017-3827-y) contains supplementary material, which is available to authorized users. In recent years, high-throughput RNA sequencing (RNA-seq) has become the method of choice to accurately probe the transcriptome of any biological specimens –6. This Several next generation sequencing protocols are currently available for the profiling of RNA samples, each with its own strengths and weaknesses. These methods use different strategies to reduce the representation of abundant ribosomal RNAs (rRNA) in RNA-seq libraries prior to sequencing. Poly(A) + enrichment using oligo-dT coated beads is the most common approach to quantify the polyadenylated RNA fraction of the transcriptome including coding mRNAs. However, this method fails at profiling other RNA populations (e.g. non-coding RNAs) and suffers from biases when applied to low quality or low quantity RNA samples –26. RiboAdiconis et al. [Numerous recent studies have compared different RNA-seq library preparation protocols. Some of these focused on degraded input RNA , 28, 29,s et al. ; howevers et al. .Despite these comparative efforts and in the light of new protocol developments, there are fundamental technical questions that remain still unanswered. For instance, how does the newly available RNA Access protocol perform on degraded samples? And, does it provide any advantages over ribosomal RNA depletion methods? Furthermore, how does the performance of these different approaches change when lowering the input RNA quantity even below the recommended amounts? These questions become especially relevant in the clinical context where, for instance, processing of human biopsies often results in low amount and very heterogeneous quality RNA samples.Here, we designed a study to evaluate the performance of the TruSeq, Ribo-Zero, and RNA Access library preparation kits on human reference RNA samples from the Microarray/Sequencing Quality Control consortium (MAQC/SEQC)  over a wTo evaluate the performance of RNA-seq methods in profiling non-optimal samples, we conducted a technical assessment of the three different RNA library preparation protocols mentioned above, namely TruSeq, Ribo-Zero and RNA Access, on two human reference RNA samples previously used in the MAQC/SEQC studies; these samples are the Universal Human Reference RNA (UHRR or SEQC-A) and the Human Brain Reference RNA (HBRR or SEQC-B) . Figure Overall we prepared a total of 222 sequencing libraries from 37 different combinations of degradation stage, input amount, and library preparation protocol each of which was applied to both samples in triplicate. Figure We initially examined the overall alignment rates to the human genome Fig.  and SEQCIn addition, the protocols showed marked differences in the percent of reads aligned to exons, introns, and intergenic regions. For RNA Access the percentages of intronic and intergenic reads were both ~1% across all input amounts and sample quality categories, thus indicating high efficiency of the exome pull down by the capture approach. For TruSeq the percentage of intronic reads decreased with the sample quality from ~6% for intact samples to 2–3% for degraded samples, whereas the percentage of intergenic reads increased from ~2–3% for 100 ng input to ~6–10% for 20 ng and less. For Ribo-Zero the proportion of reads aligned to exons was considerably lower and dependent on the sample as previously reported (10). As expected with a total RNA sequencing approach, we observed between half to two thirds of the reads mapping to exons while the rest mapped to mostly intronic regions (30–34%) and to some extent to intergenic regions in intact and degraded samples. For highly degraded samples, the percentage of reads mapping to exons, introns, or intergenic regions was much more variable across technical replicates and input amounts; for SEQC-A the percentage was similar to the intact and degraded sample qualities but for SEQC-B up to 80% of the reads were mapped to intergenic regions.All together these results indicate a consistent mapping performance of the RNA Access approach throughout all input amounts and degradation states, while the Ribo-Zero approach performed less well on highly degraded samples and at very low input amounts.We next measured the variation in 5′ to 3′ coverage along each transcript  as a gold standard reference. To this purpose, we compared the log fold-change values of the SEQC samples generated with the three RNA-seq methods to the TaqMan log fold changes since fold changes – as opposed to the direct comparison of absolute expression values – are a crucial parameter in the analysis of differentially expressed genes. In a differential expression analysis fold changes are usually accompanied by a measure of significance, such as a R2 value of 0.9 vs 0.89 for RNA Access and 0.88 for Ribo-Zero). The concordance with qPCR fold changes decreased consistently with lower input amounts for all three protocols on intact samples. The log fold change correlation for the RNA Access protocol remained stable until the low input amount of 5 ng and then dropped considerably at 2 ng (from 0.88 to 0.82). A similar behaviour was observed for TruSeq where the log fold change correlation was high at input amounts of 100 ng and 20 ng (0.89–0.9) and then dropped to 0.87 at 5 ng and to 0.84 at 1 ng. Instead, Ribo-Zero showed only a slight decrease in the correlation of log fold changes down to the lowest input amount of intact RNA (R2 = 0.86 at 1 ng).We performed pairwise comparisons of log fold changes between the individual technical replicates and the TaqMan qPCR fold change values Fig. . At recoR2 values for the RNA Access protocol again declined sharply at inputs of 2 ng and 1 ng, while similar to intact sample inputs the R2 values for Ribo-Zero went down gradually with decreasing input amount, being the best performing protocol at the lowest input amount (R2 = 0.85 at 1 ng).The correlation profiles for degraded sample inputs were similar to the intact RNA profiles Fig. . TruSeq R2 values at all tested inputs (down to 10 ng). The RNA Access approach, in comparison, performed much better on the highly degraded material. While the median R2 values decreased from 0.87 for 20 ng down to 0.85 for 5 ng and dropped further at 2 ng and 1 ng to an R2 value of 0.79, we found much less variability among the value distribution as compared to the Ribo-Zero method.Finally, when considering highly degraded RNA input, we observed a marked difference between the RNA Access and the Ribo-Zero protocols where the latter displayed a very high variability and lower median Overall our data suggest that all three protocols are robust and comparable on intact RNA samples and down to 10 ng of input. Going further down with the amounts, Ribo-Zero outperforms the other two protocols at 1 ng. In addition Ribo-Zero performs equally well on degraded RNA, closely followed by the RNA Access method. On severely degraded samples, where the RNA fragments are shorter than 200 nucleotides, Ribo-Zero reaches its limits and becomes much less reproducible. Here the RNA Access approach represents the best choice, still generating reliable data down to 5 ng input.R2 values) of the log fold changes of the SEQC samples for every combination of protocol, sample quality, and input amount  whereas the similarity drops significantly at 2 ng (0.68) and 1 ng (0.61). These results are similar to those observed for the correlation with the TaqMan qPCR data. There was almost no difference between the intact and degraded samples resulting in R2 values from 0.92 to 0.97 (for input amounts of at least 5 ng) – independent of which combinations were considered; however, there was a significant drop for the highly degraded samples with R2 values ranging from 0.79 to 0.89 (again for input amounts of at least 5 ng).Overall we found that the similarity between samples increases with the input amount, and independently of the protocol and the sample quality. We then assessed the sample similarity within each protocol separately. For RNA Access the results on intact samples for 20 ng, 10 ng and 5 ng are highly similar  for input amounts of at least 5 ng but the decrease in self-similarity to 2 ng (0.83) and 1 ng (0.76) was not as drastic as for RNA Access. Again, the R2 values of the intact and degraded samples were essentially interchangeable with respect to other Ribo-Zero samples as well as other protocols. But for the highly degraded samples only the log fold changes of the input amount 100 ng showed some agreement with the higher input amounts (100 ng and 20 ng) of the intact and degraded Ribo-Zero samples . In terms of self-similarity the achieved R2 values  were comparable to the self-similarity R2 values of the RNA Access protocols for input amounts that were an order of magnitude lower .Among Ribo-Zero samples the similarity values were also very high  and then a significant drop for 5 ng and lower with self-similarity R2 values from 0.82 down 0.61. Similar to the comparison with the TaqMan qPCR data, we again found an excellent agreement between the degraded and the intact samples despite the 3′ enrichment that we observed for the degraded sample.For TruSeq we found a very good agreement of the log fold change values for input amounts of 100 ng down to 10 ng -based TruSeq Stranded mRNA Kit (TruSeq) or the Illumina Ribo-Zero rRNA Removal Kit (Ribo-Zero). If the RNA quantity available for the experiment is significantly below the recommended starting amount of these kits , amplification methods like the NuGEN Ovation or the Clontech SMARTer can be applied to picogram amounts of the RNA samples. This extra amplification, however, invariably incorporates additional biases to those normally introduced by the library construction and sequencing procedures, and hence should be avoided if possible. On the other hand, if the RNA quality is low, the Ribo-Zero kit and the new Illumina RNA Access Kit (RNA Access) may represent valid options for the study but have not been comprehensively evaluated for accuracy and performance on low quantity and low quality RNA thus far.In this study, we performed a comprehensive and systematic assessment of two of the most commonly used commercial sequencing kits TruSeq and Ribo-Zero as well as the relatively new RNA Access kit covering a wide range of input amounts from 100 ng down to 1 ng and three sample quality stages from intact to highly-degraded. This input range is one of the most difficult to work with since the RNA amount is mostly below the sample requirements of the TruSeq and Ribo-Zero protocols, but still in an area where it would be ideal to avoid RNA amplification. We further aimed at understanding how the performance of these three protocols changes when the quality of the samples is not optimal or even heterogeneous across the RNA sample population. A summary of the results of the protocol assessment is presented in Table As already noted in previous studies , 27, 30,Adiconis et al. [If we consider degraded samples, then according to s et al. , the besThe capture based approach of the RNA Access kit, which enriches for reads mapping to exons performed very well even on severely degraded samples and at medium to low input amounts down to 5 ng Fig. . MoreoveIn summary, we conclude that RNA Access represents an attractive alternative for those studies in which the sample quality is severely compromised. For its broad application across the entire sample set, this protocol is also suited for the profiling of very heterogeneous RNA sample populations covering a wider range of low quantity and extremely low quality samples thus ensuring high accuracy and comparability of the results within the study.SEQC samples A and B were prepared by adding ERCC  spike-inTo obtain degraded RNA, aliquots of SEQC samples A and B were incubated at 94 °C for 30 min (degraded samples-size peak at ~800 bp) and for either 60 min (highly degraded Sample A-size peak ~200 bp) or 210 min (highly degraded Sample B-size peak <200 bp). Bioanalyzer profiles of the samples before and after degradation are shown in Additional file Poly-A enriched strand-specific libraries were generated with the TruSeq mRNA V2 sample preparation kit , ribosomal RNA depleted strand-specific RNA libraries with the TruSeq Stranded Total RNA LT sample preparation kit with Ribo-Zero Gold , and transcriptome capture based libraries with the TruSeq RNA Access Library Prep Kit . All protocols were performed following the manufacturer’s instructions. Recommended amounts of starting material were as follows: 100 ng of input RNA for TruSeq, 100 ng for Ribo-Zero, and 10 ng of intact RNA or 20 ng of degraded RNA for RNA access.Libraries were generated for different input amounts of total RNA  in triplicates. Ribosomal RNA depleted RNA was fragmented for 8 min (intact and degraded samples) or 2 min (highly degraded samples), different fragmentation times. qPCR was performed on unamplified libraries to evaluate the appropriate amplification cycles number of the large-scale PCR step in library preparation. The quality and yield of the prepared libraries was assessed using an Agilent 2100 Bioanalyzer .Sequencing was performed on a HiSeq2500 Instrument (Illumina) with 2 × 76 cycles, using either the Illumina TruSeq v3 or TruSeq Rapid SBS sequencing chemistry and following the manufacturer’s instructions. Images from the instrument were processed using the manufacturer’s software to generate FASTQ sequence files. Read quality was assessed by running FastQC (version 0.10) on the FASTQ files. Raw RNA-sequencing reads were deposited in the NCBI Short Read Archive under the accession number SRP097611.We used the Exon Quantification Pipeline 2.0  to alignFor the computation of fold changes (FC) of CPM values a pseudo count of 0.5 CPM was added to both values. The computation of the number of expressed genes was based on a FPKM cut-off of 0.3  and the R2) of the log fold change values, we first calculated the log fold changes and the corresponding R2 values of each of three technical replicates separately. We then averaged the individual R2 values of the three technical replicates to calculate the mean R2 values for each specific combination of input amount, degradation stage, and protocol.For the computation of the coefficients of determination (The TaqMan qRT-PCR data for the SEQC-A and B samples were downloaded from Gene Expression Omnibus under the accession number GSE5350.


OXC) and eslicarbazepine acetate (ESL) in terms of tolerability, or how to execute the change from OXC to ESL. We report the process of transitioning patients with focal epilepsy from previous OXC treatment to ESL due to tolerability problems. The rationale for change from OXC is reported, and the outcome with respective to this rationale is analyzed in terms of tolerability and efficacy.There is limited clinical evidence for comparison between oxcarbazepine  were recorded following the transition period. Subjects were classified by outcome in terms of AEs.The subjects were transitioned overnight from OXC to ESL. Fifteen patients OXC‐related AEs reduced significantly after transition. Particularly, most of (93%) the AEs presented in the morning resolved after transition to ESL. No patient had an increase in seizure frequency following the transition. The incidence of ESL‐related AEs was 39% at 1 month and 13% at 3 month follow‐up; however, all patients continued ESL throughout the study period.Twenty‐three subjects were transitioned from OXC‐related AEs improve in terms of tolerability after a switch to ESL with maintaining seizure control. This improvement is more pronounced if the OXC‐related AEs are most evident following morning dosing of OXC. Transition can be safely executed in an outpatient setting.This study demonstrates that patients suffering from  Monothe2+ channels , oral eslicarbazepine acetate (ESL) is approved in the EU as an adjunctive therapy with partial onset seizures with or without secondarily generalization and in the US as a monotherapy or adjunctive treatment of partial‐onset seizures . ESL is Eslicarbazepine acetate is efficacious and well tolerated as adjunctive therapy in drug‐resistant focal epilepsies at doses of 800 and 1,200 mg once‐daily  and efficacy (the secondary objective).2We identified all the patients (age at least 18 years) with focal epilepsy followed in the Department of Neurology in Tampere University Hospital, Finland from the patient registry. Following inclusion criteria were applied: (1) current treatment with OXC; (2) OXC‐related moderate or severe tolerability problems which affected daily life; (3) transition from OXC to ESL was performed due to OXC‐related AEs; and (4) transition was undertaken before 30 November 2015. The dosages of concomitant AEDs remained unchanged during the transition period. All subjects were on immediate‐release OXC as extended‐release OXC is not available in Finland. Information on the patient characteristics was obtained retrospectively from the medical records.Tolerability problems related to OXC were categorized as in recent meta‐analysis  were recorded and reported following the transitioning period. Mild was defined as a symptom not interfering with daily activities, moderate as interfering but not preventing daily life activities and severe as incapacitating at least part of daily activities. Patients were dichotomied by outcome in terms of AEs after switched from OXC to ESL.This was a noninvasive, retrospective study, which does not oblige ethics committee approval according to Finnish Law on Research. Access to patient records based on decision made by Head of Science Centre, Tampere University Hospital research and innovation services, Science Center.3We identified 23 patients, who were transitioned from OXC to ESL because of OXC‐related AEs. Demographic and medical characteristic of the subjects are presented in Table The most intolerable single OXC‐related AEs were somnolence, dizziness, and diplopia. Fatique, somnolence, tremor, diplopia, and nausea mostly resolved after transition. A clear tendency that some AEs would have been more persistent than others, especially in the morning, was not observed. However, dizziness and coordination problems were the commonest AEs that persisted after transition.The effects of the transition from OXC to ESL on seizure frequency are summarized in Table Changes in seizure frequency or duration were not observed during the transition period in a hospital inpatient setting. Two patients reported headache during the hospitalization, but at 1 and 3 month follow‐up this AE was not observed in these patients any more. None of those patients who had AEs during the follow‐up did report tolerability problems during the hospitalization.4The main objective of this study transitioning patients with focal epilepsy from OXC to ESL due to typical OXC‐related AEs was to evaluate the tolerability during sufficiently long follow‐up period. Our study demonstrates that patient satisfaction improved significantly in terms of reduced AEs after switching from OXC to ESL in 65% of the subjects without increase in seizure frequency. This finding is similar to a previous study demonstrating that 15 of 26 patients who were transitioned from OXC to ESL due to OXC‐related AEs no longer had AEs after the change  of the patients when substituted with ESL, whereas if these symptoms exist either after evening dose or without diurnal variation the substitution is less helpful. This association is so strong that the current number of patients is sufficient to provide the conclusion.In conclusion, our findings support the notion that patients currently receiving OXC and experiencing intolerable AEs benefit from switching to ESL in order to maintain seizure control and improve AED tolerability. This is particularly true if these AEs are most evident following morning dosing. Our data also suggest that transition from OXC to ESL can be performed safely in an outpatient setting instead of overnight hospitalization for cost effectiveness and patient comfort.Jussi Mäkinen has received support for travel congresses from Biogen‐Idec, Boehringer‐Ingelheim, Eisai, and Orion Pharma; received speaker honoraria from Boehringer‐Ingelheim; received research funding from Finnish Epilepsy Association; and participated in advisory board for Eisai. Sirpa Rainesalo has received speaker honoraria from FennoMedical, Orion Pharma, UCB and received support for travel to congresses from Abbvie and UCB. Jukka Peltola has participated in clinical trials for Eisai, UCB, and Bial; received research grants from Eisai, Medtronic, UCB, and Cyberonics; received speaker honoraria from Cyberonics, Eisai, Medtronic, Orion Pharma, and UCB; received support for travel congresses from Cyberonics, Eisai, Medtronic, and UCB; and participated in advisory boards for Cyberonics, Eisai, Medtronic, UCB, and Pfizer.


PANDAR (Promotor of CDKN1AAntisense DNA damage Activated RNA) is a lncRNA that is transcribed in a p53-dependent manner from the CDKN1A promoter and is involved in the regulation of proliferation and senescence. Overexpression of PANDAR has been observed in several tumor species and correlated with a poor prognosis for patient survival rate. Depending on the cellular state, PANDAR is known to interact with proteins such as the nuclear transcription factor Y subunit A (NF-YA) and the scaffold attachment factor A (SAF-A). However, a comprehensive analysis of the PANDAR interactome was missing so far. Therefore, we applied peptide nucleic acid (PNA)-based pull-downs combined with quantitative mass spectrometry to identify new protein binding partners. We confirmed potential candidates like U2AF65 and PTBP1, known to be involved in RNA processing. Furthermore, we observed that overexpression of PANDAR leads to a reduced level of the short pro-apoptotic BCL-X splice variant (BCL-XS) which is regulated by PTBP1. Simultaneous overexpression of PTBP1 was able to rescue this effect. Overall, our data suggest a role for PANDAR in the regulation of splicing events via its interaction partner PTBP1.Interactions of long non-coding RNAs (lncRNA) with proteins play important roles in the regulation of many cellular processes.  Those ncRNAs have a low potential to be translated into proteins and can be sub-divided according to their size into small (<200 nt) and long non-coding RNAs . The function and cellular role of lncRNAs is diverse due to the various different interaction mechanisms and partners5. Their expression profile can vary within different cell types, developmental states and diseases7. Therefore, the distinct expression profile of lncRNAs is often used as biomarker for disease type and stage. Considering the plethora of identified lncRNAs, there are only a few well-characterized species, yet. This makes the functional analysis including the involved complexes and binding partners an essential task for research11.Non-coding RNAs (ncRNAs) regulate different cellular mechanisms by interacting with all kind of biomolecules, such as other nucleic acids, proteins or small molecules and co-factors, thereby modifying their subsequent functionsPANDAR (Promotor of CDKN1AAntisense DNA damage Activated RNA) comprises 1506 nt and is transcribed in antisense direction from the CDKN1A promotor. When bound to protein complexes, PANDAR is able to modulate different cellular effects: the interaction with the NF-YA protein, for instance, leads to the inhibition of apoptotic gene activation12. Also, the scaffold attachment factor A (SAF-A) interacts with PANDAR and plays an important role within the regulation of cellular senescence13. In proliferative fibroblast cells, PANDAR and SAF-A recruit the polycomb repressive complexes PRC1 and 2 to inhibit the transcription of pro-senescent genes. Contrary to this, in senescent fibroblast cells, PANDAR interacts with NF-YA thereby sequestering it from promotor regions of proliferation responsible genes14.The lncRNA PANDAR levels compared to adjacent healthy tissue. This correlates with increased tumor size and tumor-nodus-metastasis (TNM) stage15. In contrast to this, several cancer types like gastric cancer, hepatocellular carcinoma and bladder cancer revealed increased PANDAR levels that correlated with a higher TNM stage and poor prognosis18. Further in vitro analysis of bladder cancer revealed that PANDAR upregulation leads to proliferation, while PANDAR knockdown results in apoptosis18. Given the variety of tightly regulated expression levels and involvements within various cellular stages, the detailed interaction network of PANDAR and its mechanisms behind needs further investigations.The investigation of patient derived non-small cell lung carcinoma samples revealed decreased PANDAR interacting proteins using a peptide nucleic acid (PNA)-based pull-down combined with quantitative mass spectrometry. Subsequent functional characterization of interacting candidates revealed a potential role of PANDAR in pre-mRNA splicing regulation via one of its newly identified binding partners polypyrimidine tract-binding protein 1 (PTBP1).Herein, we identified PANDAR bound proteins, we used the combination of a peptide nucleic acid (PNA)-based PANDAR pull-down followed by quantitative mass spectrometry after stable isotope labeling in cell culture (SILAC). Therefore, we designed, synthesized and analyzed PANDAR specific PNA probes  as relative enrichments over unspecific background of the eGFP-negative control. Additionally, we included NF-YA and SAF-A for the analysis, as these proteins were known to interact with PANDAR13. A threshold for most promising interaction partners was set to exceed the median absolute deviation (MAD) from the median of all candidates by a factor of three (z-score ≥ 3). SAF-A, PTBP1 and splicing factor U2AF 65 kDa subunit (U2AF65) fulfilled these criteria so that our subsequent analyses focused on these candidates  compared to an empty vector control  or large (BCL-XL) variant. While the interaction of Serine/arginine splicing factor 1 leads to anti-apoptotic BCL-XL, its displacement by PTBP1 leads to the pro-apoptotic BCL-XS variant19. Based on this, we investigated if PANDAR interaction modulates the alternative splicing of BCL-X which is regulated by PTBP1. We could show that overexpression of PANDAR leads to a reduced BCL-XS mRNA. This could on the one hand be due to a decoy mechanism of PANDAR, on the other hand the function of other binding partners might be altered, which could cause this effect. However, PTBP1 alone rescues the effect and leads to a normal BCL-XS mRNA level, which strengthens the role of PTBP1 in this PANDAR induced splicing regulation.As PTBP1 is our strongest validated candidate Figs , we focuPANDARs 3′-end . A secondary structure model of PANDAR . A size of 15 bases each was chosen to give enough affinity for the target RNA: PNA_P1 (C-TTGGAGCTGGAGTCA-N) and PNA_P2 (C-CGTCTGTGTTACCGA-N). The synthesis was performed by solid phase synthesis with a NovaSynTGR resin with PNA building blocks (Fmoc-G(Bhoc)-aeg-OH, Fmoc-C(Bhoc)-aeg-OH, Fmoc-A(Bhoc)-aeg-OH, Fmoc-T-aeg-OH) purchased from Link Technologies. Each PNA was coupled to a PEG5 linker (Iris Biotech) and depending on the purpose of use, additionally to either biotin or FITC (Sigma Aldrich). PNA was purified and binding affinity of PNA to PANDAR RNA was analyzed by fluorescence polarization (FP) . Therefore, in vitro transcribed full length PANDAR RNA (1506 nt) was serial diluted against 10 nM of FITC-PNA_P1 or FITC-PNA_P2 in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.01% Tween20. After 3 h of incubation on ice the FP was measured and KD was analyzed.Peptide nucleic acids (PNA) were specifically designed against the 2 at 37 °C.Human osteosarcoma cells (U-2 OS) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (PAN Biotech) and incubated in a humidified chamber with 5% COPANDAR overexpressing U-2 OS cells were incubated for 3 h at 4 °C on a rotating wheel in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM EDTA. Next, PNA-RNA complexes were added to Streptavidin magnetic beads (NEB) and incubate for 1 h at 4 °C on a rotating wheel. After magnetic separation and washing, the bound RNA was reverse transcribed into cDNA  and subsequently used for quantitative PCR (RT-qPCR)  in an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). A PANDAR coding plasmid was used as a standard.To analyze the binding of PNA to RNA from total isolated RNA, pull-down experiments were performed. First 50 pmol biotinylated PNA and 5 µg total isolated RNA from stable PANDAR binding candidates within osteosarcoma cells, cells were cultivated in specialized SILAC medium  without arginine and lysine supplemented with either “light” arginine and lysine  or “heavy” arginine and lysine  for 5 passages as described before31. After incorporation of amino acids into the proteins, cell lysates were spiked with in vitro transcribed PANDAR full length RNA and pulled down with biotinylated PNA pull-down probes (PNA_P1 or PNA_P2). In detail, 50 pmol biotinylated PNA was bound to Streptavidin magnetic beads for 1 h at 4 °C on a rotating wheel. The unbound PNA in the supernatant was removed and 100 pmol in vitro transcribed PANDAR full length RNA was incubated for 1.5 h at 4 °C. The control contained only magnetic beads. Cell pellets were lysed with lysis buffer  SDS, 1% (v/v) NP40, 1 mM PMSF, 1x complete EDTA free protease inhibitor cocktail (Roche)) for 30 min on ice. To get rid of cell debris, the suspension was centrifuged at 15000 × g for 10 min at 4 °C. To get rid of unbound RNA, PNA-RNA complexes bound to the beads were washed with wash buffer ) and the soluble supernatant of the lysates was added to the respective samples. After incubation , samples were magnetically separated and washed three times. After the first wash, samples were combined for forward and reverse experiments respectively in new tubes  and Arg10/Lys8 lysate with spiked PANDAR (referred to as “for” experiment) and in parallel Arg0/Lys0 lysate with spiked PANDAR and Arg10/Lys8 lysate without spiked PANDAR (beads only) (referred to as “rev” experiment). Afterwards, samples were subjected to quantitative mass spectrometry.To determine 32. Digested samples were separated on a Thermo Fisher Scientific EASY-nLC 1000 HPLC system  using a two hours gradient from 5–60% acetonitrile with 0.1% formic acid and directly sprayed in a quadrupole Orbitrap mass spectrometer  via a nano-electrospray source33. The Q Exactive was operated in data-dependent mode acquiring one survey scan and ten subsequent MS/MS scans34. Resulting raw files were processed with the MaxQuant software (version 1.5.2.18) using the uniprot human database for the search giving deamidation (NQ), oxidation (M) and acetylation at the N-terminus as variable modifications and carbamidomethylation (C) as fixed modification35. A false discovery rate cut off of 1% was applied at the peptide and protein levels. For quantitation we filtered for at least two SILAC ratio counts per protein.Samples were reduced, alkylated and digested directly on beads with LysC/Trypsin and prepared for LC-MS/MS analysis as previously described36. All other potential PANDAR binding candidates were sub-cloned from cDNA into a pcDNA3 mammalian expression vector (Thermo Fisher Scientific), which was modified with an N-terminal eGFP-coding sequence and to be compatible for gateway cloning (Thermo Fisher Scientific). Clones were sequenced and used for transient transfection with Lipofectamin2000 (Invitrogen) for 24 h in a stable PANDAR overexpressing U-2 OS cell line. For each experiment, a eGFP empty vector negative control (pcDNA3-eGFP) was used. After checking the transfection efficiency, cells were washed twice with ice cold PBS and harvested by scraping at 4 °C in lysis buffer  SDS, 1% (v/v) NP40, 1 mM PMSF, 1x complete EDTA free protease inhibitor cocktail (Roche)). Suspension was incubated on ice for 30 min and shred by using a sterile syringe (24G). Cell lysates were centrifuged for 10 min at 20000 rpm at 4 °C. The supernatant was added to equilibrated GFP-MA-Trap Beads (ChromoTek) and incubated for 2 h on a rotating wheel at 4 °C. After magnetic separation, the flowthrough was harvest to analyze the saturation of eGFP-protein binding to the beads. Afterwards, beads were washed three times with ice cold buffer  and split for RNA and protein analysis. Binding of the eGFP-tagged proteins was confirmed by western blotting using an anti-GFP antibody (Table pDest-eGFP-CUGBP1 and pDest-eGFP-MBNL1 were gifts from Nicolas Charlet-Berguerand (Addgene plasmid #61276 and #61277), pcDNA3-HtrA2-eGFP was a gift from L. Miguel MartinsPANDAR RNA, samples were subjected to RT-qPCR and absolute values determined via a PANDAR coding plasmid. Relative enrichments were calculated over eGFP background control. Promising interaction partner should exceed the median absolute deviation (MAD) from the median of the population by a factor of three (z-score ≥ 3).RNA samples were purified by phenol-chloroform extraction. To analyze the bound PANDAR enrichment were validated by pull-down from osteosarcoma cell lysate utilizing in vitro biotinylated PANDAR RNA. Therefore, in vitro transcription was performed with T7-transcription kit (Thermo Fisher Scientific) and biotin RNA labeling mix (Roche) for 2 h at 37 °C and DNase treated for 45 min at 37 °C. The RNA was purified with RNA Clean and Concentrator5 (ZymoResearch). The RNA was heated up to 50 °C for 5 min, chilled for 2 min on ice and 15 min at room temperature in 10 mM Tris-Cl pH 7.0, 100 mM KCl, 10 mM MgCl2. Equilibrated streptavidin magnetic beads  were incubated with the biotinylated PANDAR RNA for 1.5 h at 4 °C on a rotating wheel. To rule out unspecific binding of proteins to streptavidin magnetic beads, beads without RNA were used. The supernatants of cell lysates (as described before) were incubated with either PANDAR coated or empty beads for 2 h at 4 °C on a rotating wheel. After magnetic separation, beads were washed four times with ice cold buffer and resuspended in 1xSDS sample buffer for further western blot analysis . All PANDAR fragments  where sub-cloned via Gateway cloning (Thermo) into a Gateway compatible pcDNA3-GW vector backbone. Osteosarcoma cells (U-2 OS) were co-transfected with pcDNA3-TcYPF-PANDAR (or PANDAR fragment containing pcDNA3 plasmids) and either pcDNA3-eGFP-PTBP1 or an empty control plasmid (pcDNA3-eGFP) for 48 h using Lipofectamin 2000 (Invitrogen). Total RNA was extracted , reverse transcribed into cDNA  and subsequently used for quantitative PCR  in an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). Utilized primer pairs are listed in Table −ΔΔCT method was used with GAPDH as reference gene37.The All data generated or analyzed during this study are included in this published article and its .Supplementary Information


The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5′-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process. Enzymes speed up chemical reactions that are essential to life. Most enzymes are proteins, but some are molecules of ribonucleic acid or RNA. Like DNA, RNA is made from a chain of building blocks called nucleotides. In modern organisms, protein-based enzymes build RNAs by linking nucleotides together, while the building blocks of proteins are linked by an RNA-based enzyme at the heart of a structure called a ribosome. The earliest life on Earth most likely relied only on RNA-based enzymes, but during the emergence of life, scientists believe that RNA molecules must have replicated spontaneously before dedicated RNA-based enzymes had evolved.How RNA could replicate without enzymes has been a puzzle for decades. Recently, scientists discovered a previously unsuspected chemical intermediate that forms during the process, and hypothesized that this molecule’s special structure is what enables the chemical reaction that adds new nucleotides to a growing strand of RNA.To test this hypothesis, Zhang et al. diffused free RNA nucleotides into a crystalized complex containing template strands of RNA attached to short pieces of RNA called primers, which kick-start replication. Then, the crystals were frozen at various intervals and viewed using X-rays. This allowed Zhang et al. to observe the structural changes that occurred over time as the compounds reacted. The approach first revealed that the free nucleotides had paired with complementary nucleotides on the RNA template strands. Then, pairs of free nucleotides reacted with each other to form the intermediate. Finally, the intermediate reacted with the primer, forming a new bond that connects the RNA primer to one of the nucleotides of the intermediate, while the other nucleotide of the intermediate was released as a free nucleotide.This experiment confirms that the specific structure of the intermediate molecule promotes RNA replication without help from enzymes. These findings will benefit chemists and biologists who study how RNA evolves and replicates. Future research building upon this work will deepen scientific understanding of the environmental conditions that were required for life to appear on Earth. Many lines of evidence point to a central role for RNA in the emergence of life on Earth . As partCrystallography is a powerful tool for gaining atomic-level understanding of the mechanism of nonenzymatic RNA polymerization. However, the widely used nucleoside-5′-phosphoro-imidazolide substrates are too labile to use directly in crystallographic studies. We have instead utilized stable phosphonate analogs, co-crystallized with a primer template duplex, to help model the structure of the reaction site . These s2+ ion that catalyzes the rate-limiting step of the reaction  residues, denoted by italics. The LNA modification locks the sugar into the C3′-endo A-form conformation characteristic of a canonical RNA duplex. The two-nucleotide template overhang is composed of 5-methyl cytidine LNA to rigidify the backbone and facilitate crystallization. This duplex was co-crystallized with 2′-deoxyguanosine-5′-monophosphate (dGMP), to bind the template before soak-in experiments phosphonate (PZG) and guanosine 5′-(4-methylimidazolyl) phosphonate (ICG), and the stable intermediate analogue GpppG . Our atteriments . The useCC template in a well defined conformation . A third water molecule coordinated to the presumed Mg2+ is within hydrogen bonding distance of O6 of the first bound monomer, and is also hydrogen bonded to a fourth water molecule that appears to be hydrogen bonded to N7 of the second bound monomer . These proposed Mg2+-mediated electrostatic and hydrogen-bonding interactions bridge the two bound monomers and the primer, and thus potentially stabilize the template-bound structure of dGMP.In the crystal structure of the RNA duplex with dGMP, we also clearly observed electron density near the N7 of the first template-bound dGMP that appears to represent a Mgd waters ; Mg2+ wa2+-guanosine interaction, we replaced Mg2+ with Sr2+ in our crystallization buffer, to increase the electron density of the bound metal cation. We obtained crystals of the same RNA duplex in 20 mM Sr2+ with the RNA nucleotide guanosine-5′-monophosphate bound to the template region, with the same overall structure as the other complexes. In this structure, much stronger electron density was present near N7 and O6 of the first template-bound GMP, indicating the presence of Sr2+ . The individual RNA-monomer double helices are slip-stacked with one another end-to-end, and groups of three adjacent duplexes form a triangular prism-like structure . We soaked the crystals in buffer containing Gp-AI-pG for various times to allow the intermediate to diffuse into the crystal. No Mgperiment . In the -pairing , with hy-pairing .The two guanosine-5′-phosphate residues of Gp-AI-pG are linked by the 2-aminoimidazolium bridge. The electron density corresponding to the phosphate-aminoimidazole-phosphate bridge suggests that its structure is stabilized by hydrogen bonds between the 2-amino group of the imidazolium moiety and the non-bridging oxygen atoms of the flanking phosphates . UnfortuA comparison between template-bound dGMP and Gp-AI-pG reveals similar overall structures . A key d2+ might lead to formation of the intermediate, possibly followed by reaction with the primer in the crystal. We therefore grew crystals of the RNA duplex in buffer at pH 7.0 containing dGMP and 20 mM Mg2+. The RNA-dGMP crystals were then transferred to a new drop of the same pH 7.0 crystallization buffer, containing 20 mM 2-AIpG and 20 mM Mg2+. Crystal soaking was terminated by equilibrating the crystal in cryo-protectant  for 2 min followed by storage in liquid nitrogen prior to analysis. In the crystallization buffer, only 8% of the 2-AIpG decayed in 3 hr as determined by 31P NMR, indicating that the majority of the monomer remains activated during these experiments  was mixed with an equal volume of dGMP (50 mM) and heated to 80o C for 2 min before being slowly cooled to room temperature. All crystals were grown at 18o C. The Nucleic Acid Mini Screen Kit, Natrix High Throughput Kit and Index High Throughput Kit  were used for screening crystallization conditions by the hanging drop or sitting drop vapor diffusion methods. Optimal crystals grew in a crystallization buffer of 5% v/v (+/-)−2-methyl-2, 4-pentanediol, 20 mM sodium cacodylate pH 7.0, 6 mM spermine tetrahydrochloride, 40 mM sodium chloride, and 20 mM magnesium chloride. Crystals used for soak-in of Gp-AI-pG were obtained under identical conditions except that magnesium chloride was omitted. For crystallization of the RNA duplex with GMP, 20 mM SrCl2 was added in place of MgCl2. To soak 2-AIpG or Gp-AI-pG into crystals of the RNA duplex with dGMP, the preformed crystals were first stabilized in a drop containing crystallization buffer for 20 min. Then, the crystals were transferred to a drop containing crystallization buffer containing 20 mM Gp-AI-pG or 2-AIpG and incubated for various amounts of time to allow diffusion of the molecules into the crystal. As with the crystallization procedure, Mg2+ was omitted for soaking Gp-AI-pG into the crystal. After incubation with the activated substrates, the crystal was dipped into cryo-protectant (35% MPD) for 2 min and then placed in liquid nitrogen.The oligonucleotide 5′-All crystal diffraction data were collected under a stream of nitrogen at 99 K. The data sets were obtained at the SIBYLS beam lines 821 and 822 at the Advanced Light Source, Lawrence Berkeley National Laboratory. The distance from the detector to the crystal was set between 200–300 mm, and the collecting wavelength was set to 1 Å. The crystals were exposed for 1 s per image with one degree oscillations, and 180 images were taken for each data set. These data were processed using HKL2000 . All theThe structures at 1.5 and 2 hr were refined as mixtures of the intermediate state and product state.31P NMR. These samples were placed in a Shigemi tube with a co-axial insert containing D2O for locking the NMR signal. All NMR spectra were taken on a Varian INOVA 400 MHz NMR spectrometer at 25°C and analyzed by integration of peaks using MestReNova software. 31P NMR signals are referenced to internal trimethyl phosphate added after the experiments. The NMR spectra were shown in 2-AIpG and Gp-AI-pG were synthesized and purified as previously described . To char 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 Timothy Nilsen as Reviewing Editor and Michael Marletta as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Gerald F Joyce (Reviewer #1); Steven Benner (Reviewer #2); John Chaput (Reviewer #3).Thank you for submitting your article "Crystallographic observation of non-enzymatic RNA primer extension" 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.Summary:Zhang et al. have obtained a series of X-ray crystal structures that capture the reaction pathway for non-enzymatic, RNA-templated addition of an activated mononucleotide to the 3' end of an RNA primer.eLife. Please address these points as thoroughly as possible before resubmitting.All of the reviewers agreed that the manuscript presented exciting and important work. They further agreed that only minor revision was required before the work could be accepted by Reviewer #1:eLife, subject to minor revision.Zhang et al. have obtained a series of X-ray crystal structures that capture the reaction pathway for non-enzymatic, RNA-templated addition of an activated mononucleotide to the 3' end of an RNA primer. The activating group is the long-studied 5'-phosphoroimidazolide, which only recently was shown to form an imidazolium-bridged dinucleotide that is the true reactive intermediate. The manuscript includes the structures of a non-reactive template-bound monomer (dGMP), a pre-reactive template-bound dimer (imidazolium-bridged diguanylate), and, most significantly, a time series of complexes that progress from the reactive template-bound monomer, through the reactive intermediate, to the extension product. "Seeing" this reaction for the first time brings chills. Even if the 2-aminoimidizole activating group is not literally what was employed at the dawn of the RNA world, visualizing the non-enzymatic copying of genetic information is a major accomplishment. This manuscript is highly suitable for publication in 1) It would be helpful to include in Figure 1 the paired structure of the template-primer complex, shown as a homodimer and indicating the sites on both strands for binding the two guanylate residues.2) Clearly it would have been preferable if the template were all-RNA, rather than containing LNA residues. The authors state that this was done "to rigidify the backbone and facilitate crystallization". I presume that attempts were made to use all-RNA molecules, in which case this should be stated explicitly. It also should be noted that the LNA residues are "locked" into the C3'-endo conformation, which helps enforce an A-form duplex structure. Including this information at the outset will prepare the reader for the discussion in the following paragraph.2+ was included in the crystallization or soak buffer for these experiments, so at least with regard to metal ions it is not surprising they were not observed.3) The authors state: "Surprisingly, no metal ion or water molecules are observed close to the N7 positions". My understanding is that no Mg2+, attaining the proper solvation state, or other factors. The fact that the reaction is slower in the crystal does not diminish the significance of the results, and indeed was helpful for visualizing the reaction trajectory. Clearly that trajectory is qualitatively the same as for the reaction in solution.4) Figure 4 is simply "Wow!"; the payoff of decades of research. To gild the picture just a bit, it should be possible to make a quantitative estimate of the fraction of imidazolium-bridged diguanylate vs. extension product for the 1, 1.5, 2, and 3 hour time points. This assumes one can fit the electron density to a two-state model that corresponds to the fraction reacted at each time. These data in turn could be used to estimate the reaction rate within the crystal. As the authors note, this rate is much slower than in solution, which may be due to constraints of crystal packing, but could also be due to access to the catalytic MgReviewer #2:The problem that this manuscript addresses, the uncatalyzed template-directed synthesis of RNA, is one of the six or seven central problems in the origins of life.Further, the result that the research reported in this manuscript has achieved, allowing crystallography to follow dynamic processes, here chemical reactions, has long been a "Holy Grail" goal of crystallography, dating back to the early 1980s when cryo-enzymology achieve this for a few cases, notably ribonuclease in the laboratory of Gregory Petsko. This work is evidently long forgotten. More recent work on time resolved x-ray crystallography is reviewed in the Introduction of this manuscript.Therefore, this manuscript has chosen well both the subject for study and the approach of the study. I have no major criticisms that need response.It is interesting that when one actually uses the true substrate, as opposed to a stable analog, organized electron density can be seen, contrasting with the disorder that was seen with the substrate analog. This is appropriately mentioned in the Discussion, but I don't know of any other case where this is been seen.Reviewer #3:eLife.This is an outstanding paper that describes a critical step in the origin of life. Using time-resolved X-ray crystallography, the authors follow the progress of a model non-enzymatic primer extension reaction where an activated ribonucleotide is added to the 3' end of an RNA primer. The reaction consists of monomers binding to the template in multiple conformations, followed by the formation of an imidazolium-bridged dinucleotide intermediate, and finally the formation of a new phosphodiester bond between the primer and adjacent monomer of the activated intermediate. The results strongly suggest that non-enzymatic primer extension reactions proceed by a mechanism where the activated imidazolium-ion intermediate is pre-organized on the template, which helps explain the high rate of synthesis of these RNA analogues. The manuscript was well written, and the experiments are clearly described in the main text and methods sections. Overall, I have no minor or major concerns and strongly feel that this paper would be excellent contribution to  Reviewer #1:[…] 1) It would be helpful to include in Figure 1 the paired structure of the template-primer complex, shown as a homodimer and indicating the sites on both strands for binding the two guanylate residues.As requested, we have added a diagram to Figure 1 to illustrate the RNA sequence for all of the structural studies in our paper.2) Clearly it would have been preferable if the template were all-RNA, rather than containing LNA residues. The authors state that this was done "to rigidify the backbone and facilitate crystallization". I presume that attempts were made to use all-RNA molecules, in which case this should be stated explicitly. It also should be noted that the LNA residues are "locked" into the C3'-endo conformation, which helps enforce an A-form duplex structure. Including this information at the outset will prepare the reader for the discussion in the following paragraph.The reviewer is correct, in that we had indeed tried to co-crystallize the unmodified RNA template with monomers. Although we did obtain crystals, the solved structure only showed the central RNA duplex. The flanking template region was highly disordered, without any interpretable evidence for bound monomer. As a result we chose to use LNA in the template region to rigidify the structure. We have added a brief explanation in the first paragraph of the subsection “Structure of the primer-template duplex co-crystallized with dGMP”.2+ was included in the crystallization or soak buffer for these experiments, so at least with regard to metal ions it is not surprising they were not observed.3) The authors state: "Surprisingly, no metal ion or water molecules are observed close to the N7 positions". My understanding is that no Mg2+ was present in the crystallization buffer. We have deleted the word “surprisingly”, and modified the sentence to clarify that no monovalent ions or water molecules were observed close to N7.The reviewer is correct in that no Mg2+, attaining the proper solvation state, or other factors. The fact that the reaction is slower in the crystal does not diminish the significance of the results, and indeed was helpful for visualizing the reaction trajectory. Clearly that trajectory is qualitatively the same as for the reaction in solution.4) Figure 4 is simply "Wow!"; the payoff of decades of research. To gild the picture just a bit, it should be possible to make a quantitative estimate of the fraction of imidazolium-bridged diguanylate vs. extension product for the 1, 1.5, 2, and 3 hour time points. This assumes one can fit the electron density to a two-state model that corresponds to the fraction reacted at each time. These data in turn could be used to estimate the reaction rate within the crystal. As the authors note, this rate is much slower than in solution, which may be due to constraints of crystal packing, but could also be due to access to the catalytic MgThis is a point that we discussed at great length during the preparation of our original manuscript. Ultimately we decided against including such a plot, for fear that this would be an over-interpretation of the available data. The problem is that our refined models of the imidazolium-bridged dimer and the extension product do not account for 100% of the molecules in the crystal. It is likely that hydrolysis products such as GMP and 2-AIpG occupy some fraction of the template molecules. As a result, we do not feel able to quantitatively estimate the fraction of imidazolium-bridge vs. phosphodiester bond based on our observed electron densities. An additional confounding factor is that the rate of molecular diffusion into the crystal depends on the size the crystal, so quantitative conclusions based on structures from different crystals might be misleading. We certainly hope to overcome these limitations in future work, but believe that a simple qualitative characterization is most appropriate at present.Reviewer #2:The problem that this manuscript addresses, the uncatalyzed template-directed synthesis of RNA, is one of the six or seven central problems in the origins of life.Further, the result that the research reported in this manuscript has achieved, allowing crystallography to follow dynamic processes, here chemical reactions, has long been a "Holy Grail" goal of crystallography, dating back to the early 1980s when cryo-enzymology achieve this for a few cases, notably ribonuclease in the laboratory of Gregory Petsko. This work is evidently long forgotten. More recent work on time resolved x-ray crystallography is reviewed in the Introduction of this manuscript.Therefore, this manuscript has chosen well both the subject for study and the approach of the study. I have no major criticisms that need response.It is interesting that when one actually uses the true substrate, as opposed to a stable analog, organized electron density can be seen, contrasting with the disorder that was seen with the substrate analog. This is appropriately mentioned in the Discussion, but I don't know of any other case where this is been seen.We thank the reviewer for this appreciation of our work. We note that, as stated in the text , the 2-aminoimidazole leaving groups of the bound monomers are disordered – this is similar to what we previously observed with non-hydrolyzable analogs.Reviewer #3:This is an outstanding paper that describes a critical step in the origin of life. Using time-resolved X-ray crystallography, the authors follow the progress of a model non-enzymatic primer extension reaction where an activated ribonucleotide is added to the 3' end of an RNA primer. The reaction consists of monomers binding to the template in multiple conformations, followed by the formation of an imidazolium-bridged dinucleotide intermediate, and finally the formation of a new phosphodiester bond between the primer and adjacent monomer of the activated intermediate. The results strongly suggest that non-enzymatic primer extension reactions proceed by a mechanism where the activated imidazolium-ion intermediate is pre-organized on the template, which helps explain the high rate of synthesis of these RNA analogues. The manuscript was well written, and the experiments are clearly described in the main text and methods sections. Overall, I have no minor or major concerns and strongly feel that this paper would be excellent contribution to eLife.We thank the reviewer for this appreciation of our work.


The federal channel at Port of Miami, Florida, USA, was dredged between late 2013 and early 2015 to widen and deepen the channel. Due to the limited spatial extent of impact-assessment monitoring associated with the project, the extent of the dredging impacts on surrounding coral reefs has not been well quantified. Previously published remote sensing analyses, as well as agency and anecdotal reports suggest the most severe and largest area of sedimentation occurred on a coral reef feature referred to as the Inner Reef, particularly in the sector north of the channel. A confounding regional warm-water mass bleaching event followed by a coral disease outbreak during this same time frame made the assessment of dredging-related impacts to coral reefs adjacent to the federal channel difficult but still feasible. The current study sought to better understand the sedimentation impacts that occurred in the coral reef environment surrounding Port of Miami, to distinguish those impacts from other regional events or disturbances, and provide supplemental information on impact assessment that will inform discussions on compensatory mitigation requirements. To this end, in-water field assessments conducted after the completion of dredging and a time series analysis of tagged corals photographed pre-, during, and post-dredging, are used to discern dredging-related sedimentation impacts for the Inner Reef north. Results indicate increased sediment accumulation, severe in certain times and places, and an associated biological response  extended up to 700 m from the channel, whereas project-associated monitoring was limited to 50 m from the channel. These results can contribute to more realistic prediction of areas of indirect effect from dredging projects needed to accurately evaluate proposed projects and design appropriate compliance monitoring. Dredging projects near valuable and sensitive habitats subject to local and global stressors require monitoring methods capable of discerning non-dredging related impacts and adaptive management to ensure predicted and unpredicted project-related impacts are quantified. Anticipated increasing frequency and intensity of seasonal warming stress also suggests that manageable- but- unavoidable local stressors such as dredging should be partitioned from such seasonal thermal stress events. Numerous dredging projects have resulted in widespread environmental effects on coral reef communities e.g., . Coastal1is composed of two reef habitat types, including a Ridge-shallow (western portion) and Linear Reef (eastern portion). The current direction in the outer sections is dominated by the Florida Current with strong north-northeasterly flows, but the flow environment is complex with common current reversals and multiscale vortices capable of transporting suspended material in complex patterns  of direct impacts  to Outer Reef north and Outer Reef south. The EIS concluded impacts may also include the resuspension and deposition of sediments on nearby coral reef assemblages, but the area of anticipated sedimentation impact was not quantified  via pipeline, backhoe, and clamshell dredges and deposited to a permitted offshore disposal site 2.4 km east-southeast of the project site in 120–240 m depth  prepared by the Army Corps of Engineers (USACE) concluded the dredging would result in 13,355 mantified . Nine yeantified . Additio m depth .2ftp://ftp.dep.state.fl.us/pub/ENV-PRMT/dade/issued/0305721_Miami_Harbor_Phase_III_Federal_Dredging/001-BI/Final%20Order/Miami%20Harbor%20Final%20Order%20052212.pdf.Florida Department of Environmental Protection Permit #0305721-001-BI; was issued in 2012 to the USACE by the Florida Department of Environmental Protection and included conditions for biological monitoring areas adjacent to the channel along each of six coral reef or hardbottom features  and 23–84% greater frequency than a baseline period prior to the start of dredging. This study also documented the greatest frequency and intensity of dredging-associated sediment plumes over the Inner Reef north reef sector. For this reason, we focused post-hoc sediment impact assessment effort in this reef sector, recognizing that additional reef area was likely impacted in the other sectors, with perhaps lesser intensity over a smaller extent.The purpose of the current study was to better understand the sedimentation impacts that occurred in the coral reef environment surrounding Port of Miami, to distinguish those impacts from other regional events or disturbances, and provide supplemental information on impact assessment to inform discussions on compensatory mitigation requirements. This paper reports results from post-hoc field sampling focused on coral condition and standing sediment on reef substrates. Sampling was conducted at five sedimentation assessment locations within the Inner Reef north sector. Parallel sampling was conducted in the associated reference area, chosen and followed as part of the compliance monitoring program, approximately 9 km north of the channel and composed of similar reef habitat types . In addiIn December 2015, field sampling was conducted to quantify coral condition and standing sediment on reef substrates at locations spanning increasing distance from the channel in the Inner Reef north sector, in addition to the reference location  that were selected to correspond with dive sites surveyed in a pre-construction assessment the USACE completed in 2010. Overall, this results in five sedimentation assessment locations , two dive sites per location—one in the Ridge-shallow and the other in the Linear Reef—with the exception of 700 m, where only the Linear Reef was surveyed. A temporary marker buoy was deployed from the boat at the pre-determined coordinates. At each dive site, two 50-m long transects were run in opposite directions from the buoy and sampled at 1.0-m intervals . Observers recorded the occurrence of standing sediment along the line-intercept transects, where present, in two categories. ‘Sediment-over-hardbottom’ (SOHB) was designated if there was a visible accumulation of sediment. For example, algal turfs normally have some sediment embedded within them, but if the turfs were engulfed by sediment, this would be labelled as SOHB. If the sediment was qualitatively observed to be deeper (estimated >4.0 cm), it was labelled as “deep sediment over hardbottom” (DSOHB). In addition, at every 5.0 m along each transect, the depth of the sediment (cm) over hardbottom was measured with a ruler and the deepest of several measurements within one meter of the sample point was recorded.2 belt transects were also sampled along or parallel (within 10 m distance) with the two line-intercept transects at each dive site to quantify the condition of coral colonies within each area. Each scleractinian colony was recorded by species and condition; namely if the coral displayed recent partial mortality . Thus, transects of both habitat types were pooled at each location  to increase replication and power to detect differences among the locations via one-way ANOVAs (on ranks when parametric assumptions were violated).One-way ANOVAs followed by post-hoc tests between the assessment and reference locations were used to determine statistically significant differences in each survey parameter. For each of six parameters, , preliminary one-way ANOVAs  showed no significant differences between the two habitat types  and a four-week post-construction phase (July 2015). Intervals between images ranged from 1to 121 (mean 14–18) days for the channel-side colonies and from 1 to 145 (mean 12–13) days at the reference site. The frequency of images varied according to permit requirements; photographs were to be taken at greater frequency when dredging was occurring in close proximity (<750 m) to the monitoring site. We thus made the conservative assumption that long intervals between photos corresponded to times when dredging activities were relatively distant and less influential, and that relevant instances of sediment interaction with the colonies are appropriately captured in the available time series for each colony. The time series of each colony was examined and the temporal sequence of conditions affecting each colony was noted. Specifically, the presence of sediment accumulation on live tissue, partial sediment burial generally of colony edges, complete colony burial by sediment, the presence of active White Plague disease signs , and ‘sudden death’  were recorded in sequence. We included ‘sudden death’ in a category of disease impact given consistency with described patterns of mortality  associated with a regional outbreak of ‘White Plague’ disease affecting most species of mounding corals during this time frame (2) was calculated by outlining the live tissue area for each colony. Proportional change in live tissue area was calculated for each colony /pre). Colonies which went missing prior to the post-construction phase were excluded from this analysis. The colonies from the two habitat types in each location were pooled and the change in colony area between locations was compared by a Mann–Whitney U-test.Public records of time series photographs of tagged coral colonies taken as part of the compliance monitoring were obtained from the USACE. Tagged colonies were distributed in both the Ridge-shallow and Linear Reef habitat types at the channel-side and the reference . At the reference location, 1% of the survey points exhibited reef substrate characterized as SOHB. The mean percent cover of DSOHB showed significant variation among sampled locations , ranging from 0.8 to 10.8% at distances 100, 200, 500, and 700 m from the channel (The mean percent cover of reef substrate characterized as “sediment over hardbottom” (SOHB) and “deep sediment over hardbottom” (DSOHB) was higher along the Inner Reef north transects , than the reference location transects . Specifically, along the transects located 200 m north of the channel, the mean sediment depth was 3.0 cm compared to 0.3 cm at the reference location . Specifically, the 100, 300, and 700 m locations were significantly different than the reference . The occurrence of sediment accumulation (SA) on live coral tissue ranged from 4.8 to 21.3× higher at sedimentation assessment locations when compared to the reference location with the 100 m and 200 m locations being statistically higher .There was up to a 3.1 to 5.1× increase in the prevalence of corals with recent partial mortality at sediment assessment locations when compared to reference  displayed complete or almost complete colony mortality directly associated with sediment burial , without regard to particular conditions or attributions of coral loss, showed over 4× greater tissue loss on average than the reference colonies  and prevalence of recent partial mortality , significant contrast is evident with the reference location. This pattern also is consistent with the less-objective or ephemeral parameters in our survey, such as the attribution of partial mortality patterns as ‘halos’ or the presence of sediment on live coral tissue. Unfortunately, there are no directly comparable baseline data for these parameters, raising a potential concern that high sediment impact, relative to the reference area, may be characteristic of a reef area adjacent to a high-traffic channel, rather than a direct effect of dredging. Baseline sampling was conducted by USACE at a gradient of sites out to 450 m in the Inner Reef North sector in 2010 and recorded a binary score (yes/no) for the presence of an unspecified ‘sedimentation’ condition and the overall prevalence of coral partial mortality (not specified whether recent or not). The ‘sedimentation’ score was reported as ‘yes’ at all sampled sites except the Inner Reef North site at 200 m where a ‘no’ was recorded for ‘sedimentation’. The dive site for one of the two sampled in the current study at 200 m distance 200-RR,  was overNeither the dredging process nor the Port of Miami entrance channel environment was conducive to a simple sedimentation gradient leading away from the channel and into the coral reef habitat. While the material dredged from the federal channel was predominantly limestone and sand below a thin layer of silt, the proportion of silt within material from the inner harbor was generally higher than from the entrance channel. Three types of dredges  operated at various times and locations, and the amount of sediment suspended from these dredges can differ substantially under normal operation . In addiWhile sediment movement and deposition is a normal process in a coral reef ecosystem, offshore coral reefs are not capable of developing or sustaining ecological functions when substrates are covered by sediment over prolonged periods. The presence of deep sediment pockets within patchy reef habitats may also be a normal reef habitat feature. However, the presence of emergent sessile invertebrates (particularly soft corals, but also scleractinian corals and sponges, Although our replication was not adequate to discern habitat effects, the post-hoc survey results suggest differences in the severity of sedimentation impact between the habitat types. Most survey locations in the vicinity of the channel showed a trend for higher sediment cover and depth in the Linear Reef than the Ridge-shallow habitat . The LinSedimentation on reefs can reduce coral recruitment, survival, and settlement of coral larvae  and suppThe implementation of seasonal shutdowns for dredging projects near coral reefs has largely been based on protecting corals during major spawning events. Unfavorable conditions during a coral spawning period could negate the entire reproductive output for the year . Conduct3 of material. However, recent thermal stress and disease impacts have rendered the baseline reef condition as further impaired and less able to tolerate increments of ‘standard’ sedimentation stress associated with dredging activities in the past (e.g., Another port expansion at Port Everglades, located approximately 37 km north of Port of Miami, is on the horizon for southeast Florida. The construction plans at Port Everglades are similar in scale with USACE proposing to remove 4.21 million mst e.g., . The prost e.g., , though st e.g.,  with neast e.g., , and locst e.g., . Such an10.7717/peerj.2711/supp-1Data S1Click here for additional data file.10.7717/peerj.2711/supp-2Table S1Percentage of scleractinian corals at each site with recent partial mortality, sediment accumulation, or sediment halo and number of corals from the belt transects partitioned by habitat type. Percentage of survey points with sediment over hardbottom and deep sediment from line-intercept transects.Click here for additional data file.10.7717/peerj.2711/supp-3Supplemental Figures 1–3Click here for additional data file.


O stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (<ΦF> = 0.22) that is virtually unaffected by the neighbouring bases (ΦF = 0.20–0.25), resulting in an average brightness of 1900 M−1 cm−1. The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (<ΦF > = 0.24) compared to dsRNA, with a broader distribution (ΦF = 0.17–0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (<ΔTm> = + 2.3 °C). These properties make tCO a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.The bright fluorescent cytosine analogue tC The discovery two decades ago that short RNA sequences can up- or downregulate gene expression through the RNA interference (RNAi)i.e. supertertiary structure6, for their function, and underline the need for tools that allow a better understanding of these parameters8. Traditionally, high-resolution structural insight into nucleic acids has been achieved using nuclear magnetic resonance (NMR) spectroscopy9 or X-ray crystallography10, often complemented by lower-resolution techniques such as Förster resonance energy transfer (FRET)12. Single-molecule FRET opens up new possibilities when it comes to observing biomolecular structure and dynamics in live cells14. For in-cell single-molecule FRET measurements, external fluorophores such as Cy-dyes, Alexa-dyes and Atto-dyes have been employed because of their high brightness and photostability17. However, these probes are less useful for dynamics (anisotropy) and detailed, smaller structural features. If an internal, non-perturbing fluorescent probe is employed, real-time information on the structure and intrinsic dynamics of nucleic acids may also be obtained18, which could pave the way towards a deeper understanding of important cellular processes such as the genome editing CRISPR systems23. It may also support the development of novel oligonucleotide-based therapeutics, such as ASOs, through visualisation of their intercellular transport and subcellular localisation2.The broad range of conformation and functions observed for RNA reflects the importance of the secondary and tertiary structure as well as dynamics, 25, and the search for new probes with improved photophysical properties is continuing. However, most of the probes reported to date are not synthesized for or characterized in RNA systems, and the vast majority of these probes are quenched considerably upon incorporation into nucleic acids, the effect being dependent on base sequence, position and whether the nucleic acid is single- or double-stranded26. This feature has been extremely useful in studies that probe e.g. RNA translation and catalysis28 and DNA dynamics29. However, for structure and dynamics studies employing FRET or fluorescence anisotropy, or cellular tracking, a bright and stable fluorescent probe would be preferred.The number of internal probes for nucleic acids, especially fluorescent base analogues, has increased considerably over the last decadesO31. These probes are unique in that they retain their high fluorescence quantum yield (ΦF ~ 0.20) when incorporated into duplex DNA regardless of base sequence or position, positioning them among the brightest internal DNA probes reported to date32. We have shown that both probes are excellent cytosine analogues that are rigidly stacked within the duplex, where they have only limited effects on the native conformation of DNA31. Both compounds are good substrates for human DNA and RNA polymerases34, that have been used to investigate DNA-protein interactions36 and to monitor the i-motif transition38. More recently, we developed the non-emissive FRET acceptor tCnitro that can be used with tCO to accurately distinguish distance- from orientation-changes through internucleobase FRET40 and thereby quantitatively resolve solution structures41. Even small modifications of the DNA conformation may result in significant changes in the FRET efficiency of these probes, which have been used to gain insight into mammalian mitochondrial transcription43, the structure and dynamics of the DNA binding RAD4/XPC complex44, and the mechanism and kinetics of the B- to Z-DNA transition45.We have previously reported on two internal fluorescent probes based on a tricyclic cytosine scaffold, tC and tCO ribonucleoside, and characterize its base-mimicking and fluorescence properties as a probe for RNA systems. Two series of 10-mer RNA-sequences were studied  in detail. Importantly, tCO has a high fluorescence quantum yield inside RNA duplexes that is virtually unaffected by the nature of the surrounding base pairs. These unique properties should make tCO a useful internal RNA probe for detailed FRET structure measurements, in fluorescence microscopy, and for probing the dynamics of subdomains of complex RNA 3D-structures with fluorescence anisotropy.Herein, we report the synthesis, RNA-incorporation and characterization of the tCO-ribonucleoside was synthesized for the first time in a two-step protocol, followed by three protection steps, to afford the tCO phosphoramidite monomer (4)  with 2-mesitylenesulfonyl chloride produced an active O-4-sulphonate ester, which was reacted with 2-aminophenol to produce 2. Deprotection and cyclization was achieved in one pot, using KF to facilitate the cyclization, to produce 3. Finally, the tC° ribonucleoside was protected with 5′-O- (DMT), followed by 2′-O-tert-butyldimethylsilyl (TBDMS) and a 3′-phosphoramidite for incorporation into RNA oligonucleotides using solid-phase synthesis (see Supplementary Table The tC(4) Fig. . This apO  as well as melting temperature was measured on both the modified RNA duplexes and the corresponding reference duplexes.In a preliminary study, a small-scale batch of four modified 10-mer RNA sequences was synthesized, differing only in the bases neighbouring tCO-modified sequences exhibit the general characteristics of A-form RNA, namely a positive band at 265 nm and a strong negative band at 210 nm, indicating that when a cytosine is replaced with tCO in a RNA duplex, the RNA adopts A-form geometry. In a similar manner to tCO in DNA, no significant CD signal was found for the long wavelength absorption band of tCO31. The thermal stability of the modified and the corresponding unmodified RNA duplexes is summarized in Table O increases the melting temperature by 2.3 °C. A similar increase in stability (2.7 °C) was observed for tC- and tCO-incorporation into DNA, and presumably reflects that the increased base stacking interactions act to stabilize the duplex structure31. In the few reports on fluorescent base analogues in RNA, a slightly stabilizing effect was also reported for the emissive guanosine isomorph, thG47, whereas a negligible effect was reported for the uridine analogue UDz 48, and a destabilizing effect was reported for benzo[b]thiophene-49, benzofuran-50 and naphthalimide-conjugated51 uridine analogues.Circular dichroism spectra . Three of the sequences  were chosen to investigate possible effects from guanines positioned two bases away from tCO, as a slight quenching effect of tCO by guanines have been observed in DNA31.Having established that RNA duplexes retains their native A-helix conformation and are slightly stabilized after incorporating tCO in single- and double-stranded RNA together with the corresponding spectra of tCO as a monomer. As is the case for DNA, the long-wavelength absorption maximum is slightly red-shifted upon incorporation in ssRNA (362–368 nm) compared to the tCO monomer (359 nm)31. The emission spectrum of tCO in ssRNA shows slightly more vibrational fine structure than that of the monomer, with an emission maximum (453–458 nm) very close to that of the monomer (457 nm). Similar spectral effects were observed for tCO in DNA and is common for fluorescent base analogues incorporated into nucleic acids25.Figure F, mean fluorescence lifetimes, <τ>, and calculated radiative rate constants, kr, and non-radiative rate constants, knr, of tCO in ssRNA. The fluorescence decays could all be fitted with two fluorescence lifetimes  than both the free monomer (3.4 ns) and the other three single-strands, GG, AA and CC (4.7–5.4 ns). These six sequences also have a reduced fluorescence quantum yield and a higher non-radiative rate constant  compared to the free monomer  as well as the other three sequences . Interestingly, this quenching effect appears to be similar regardless of whether the uracil is positioned on the 3′ side or the 5′ side (cf. sequences UG and GU). The effect is not increased significantly in the two sequences where tCO is positioned between two uracils (GUUU and UUUG), and the differences observed in quantum yield and mean fluorescence lifetime between them is too small for us to conclude whether the quenching is dependent on the relative orientation and interaction between tCO and its flanking uracils. Comparing sequences AU with GAUU and GUUU with UUUG, we find that replacing a uracil with guanine two steps away from tCO only has a small or negligible effect on the fluorescence quantum yield and average fluorescence lifetime. In ssDNA, sequences containing a guanine on the 5′ side to tCO were shown to have shorter lifetimes (2.8–3.0 ns) and quantum yield (ΦF = 0.14–0.17) compared to other sequences (<τ> = 4.4–5.8 ns and ΦF = 0.29–0.41), whereas sequences with guanine on the 3′ side were among the brightest, suggesting that subtle structural differences such as relative orientation and stacking plays an important role31.Table O in double-stranded RNA is included in Fig. O in dsRNA was also observed for tCO in dsDNA, indicating that tCO is firmly stacked inside the RNA helix as has previously been found in DNA31.Representative absorbance and emission spectra of tCO-modified RNA duplexes show the general characteristics of A-form RNA, namely a positive band at 265 nm and a strong negative band at 210 nm. The long-wavelength band of tCO is not observed in any of the CD spectra . A similar trend has been observed for tCO in dsDNA (ΦF = 0.22 ± 0.05)31. Inside dsDNA, tC and tCO are one of the few internal base analogues that have a single lifetime (<τ > = 3.4–4.8 ns for tCO), which is a great advantage in techniques such as fluorescence anisotropy and FRET. However, while the fluorescence decays of tCO in dsRNA can be fitted using a single lifetime, slightly better fits are obtained when using two lifetimes: a large component of 4.1–4.9 ns and a smaller component of ~2 ns, resulting in mean fluorescence lifetimes ranging between 3.8 and 4.7 ns . Much like in dsDNA, the radiative rate constant is reduced by ~40% compared to the tCO monomer (kr = 8.8·107 s−1), most likely a consequence of the electronic interaction between tCO and other bases in the DNA, leading to a hypochromic effect31. This would result in a reduction of the radiative rate constant, since it is proportional to the extinction coefficient according to the Strickler-Berg relation52. The observed change in the radiative rate constant is similar to the hypochromicity of dsDNA as compared to a free nucleoside, which is typically <40%53. Altogether, our investigations of the structural and photophysical properties in double-stranded RNA suggest that tCO is a very promising non-perturbing internal RNA fluorophore.Table O can be incorporated into RNA, where it preserves the A-form duplex and slightly stabilizes it. The tCO ribonucleoside can be synthesized through a simple and straight-forward synthetic protocol that affords the phosphoramidite-protected tCO ribonucleoside in high yield. As is the case for DNA, tCO retains its high fluorescence quantum yield inside RNA duplexes, and is virtually unaffected by the nature of the surrounding base pairs. Because of these properties, the tCO-ribonucleoside is potentially a valuable fluorescent RNA base analogue whose average brightness (ε·ΦF) of 1900 M−1 cm−1 in dsRNA is the greatest for any internal RNA analogue reported to date. As we have previously demonstrated for DNA-systems, we envisage its main advantages in RNA to be in detailed FRET-based structural measurements, where there is a current lack of internal FRET-probes, as a bright label in microscopy, and in fluorescence anisotropy probing the dynamics of subdomains of complex RNA 3D-structures.We have, for the first time, shown that the base analogue tC1H (400 MHz) and 13C (100.6 MHz) NMR spectra were recorded at room temperature using a Jeol Eclipse 400 NMR spectrometer. All shifts are recorded in ppm relative to the deuterated solvent . Positive FAB high resolution mass spectra were obtained on a JEOL SX102 mass spectrometer at Instrumentstationen, Lund University, Sweden. Samples were desorbed from a 3-NBA matrix using 6 kV xenon atoms. Synthesis and characterization data of the synthesized compounds can be found in the Supplementary Information online.Commercially available reagents were used without further purification. Deoxygenation of reaction mixtures was achieved by bubbling argon through the solution for 30 min. Column chromatography were performed using silica gel . O-tert-butyldimethylsilyl (TBDMS) protection implemented on an Applied Biosystems 394 synthesizer1. Oligoribonucleotides were cleaved from the support and base deprotected in 25% ethanol/ammonia solution at 20 °C for 3 h, and evaporated to dryness. Removal of TBDMS protecting groups was achieved by redissolving oligoribonucleotides in 115 μL dimethyl sulfoxide to which was added 125 μL 1 M triethylamine trihydrofluoride (Sigma-Aldrich) and incubated at 65 °C for 2.5 h prior to butanol precipitation. All oligonucleotides were purified by gel electrophoresis in 20% polyacrylamide under denaturing conditions (7 M urea) in 90 mM Tris-borate (pH 8.3), 10 mM EDTA (TBE buffer). The full-length RNA product was visualized by brief ultraviolet shadowing. The band was excised and electroeluted using an Elutrap (Whatman) into 45 mM Tris-borate (pH 8.5), 5 mM EDTA buffer, 8 M ammonium chloride at 200 V. The RNA was precipitated with ethanol, washed with 70% ethanol, dried and resuspended in water. Oligoribonucleotides were subjected to further purification by reversed-phase HPLC , using an acetonitrile gradient with an aqueous phase of 100 mM triethylammonium acetate (pH 7.0).Oligoribonucleotides were synthesized using UltraMILD ribonucleotide phosphoramidites (Link Technologies) with 2′-54. Molar absorptivities of modified strands were calculated in the same way, with the modified base replaced by cytosine, and correcting for the molar absorptivity difference between tCO (εtCO = 11000 M−1 cm−1) and cytosine (εC = 7400 M−1cm−1) at 260 nm. The total extinction coefficient of each sequence can be found in Supplementary Table All samples used in this study, unless stated otherwise, were prepared in a sodium phosphate buffer, pH 7.5 with 100 mM added NaCl and 1 mM EDTA. All samples were mixed and handled in sterile, RNase-free environments. The oligonucleotide concentration was determined by measuring the absorption at 260 nm. The molar absorptivities of the unmodified oligonucleotide single strands at 260 nm were calculated in IDT’s online oligonucleotide analyzerMelting curves were recorded on a Cary 4000 spectrophotometer (Varian Technologies) with a programmable multi-cell temperature block, by heating the samples to 85 °C at 0.5 °C/min, followed by cooling to 5 °C at a rate of 0.5 °C/min. The temperature was kept at 85 °C for 5 min between heating and cooling. For unmodified duplexes a temperature range of 15 °C to 92 °C was used. The absorption at 260 nm was recorded every 0.5 °C. Melting temperatures presented in this article are averages of the temperature values at the maximum of the first derivative and at half maximum of the melting curves, and were measured at least twice.Circular dichroism (CD) spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics) at 25 °C. Spectra of solutions containing 7 mM RNA duplexes, prepared as described above, were recorded between 200 and 450 nm at a scan rate of 1 nm/s. All spectra were corrected for background contributions, and smoothed (3-point adjacent-averaging).F) of the different tCO-modified RNA oligonucleotides were determined relative to the quantum yield of the potassium salt of a tCO-monomer in H2O (ΦF = 0.30)31. Samples containing duplex RNA were prepared as described above. The single-stranded RNA oligonucleotides were set to have an absorption around 0.05 at the excitation wavelength. Spectra were recorded on a SPEX fluorolog 3 spectrofluorimeter (JY Horiba). The samples were excited at 358 nm, and emission spectra were recorded between 385 and 710 nm.Quantum yields . The excitation pulse was generated by a PicoQuant pulsed (10 MHz) laser diode, emitting at 377 nm. The emission was monitored at 470 nm. Photons were collected by a microchannel-plate photomultiplier tube  and fed into a multichannel analyzer  with 4096 channels. 10000 counts were recorded in the top channel. The intensity data were convoluted with the instrument response function and fitted to mono-, bi- or tri-exponential expressions using Fluofit Pro v.4 software (PicoQuant GmbH). The average lifetimes were amplitude-weighed: <τ> = ∑αSupplementary Information