The association between self-rated health (SRH) and mortality is well documented in the literature, but studies on the subject among young adults in Latin America are rare, as are those evaluating this association using repeated SRH measures, beyond the baseline measurement. This study aims to evaluate the association between SRH evaluated at three data collection stages and mortality.Pró-Saúde Study, n = 4009, men = 44.4%).Cox regression models were used to examine the association between SRH  varying over time and mortality, over a 10 year period, in a cohort of non-faculty civil servants at a public university in Rio de Janeiro, Brazil  and women, 3.43 (CI95% 1.23-9.59), as compared with those who reported “Very good” SRH.In a population of young adults, our findings reinforce the role of SRH as a predictor of mortality, even controlling for objective measures of health. Self-rated health (SRH) is a subjective measure that is being used to monitor the health of various populations,2. AlthoThe single item used to gauge SRH – commonly implemented by the question “In general, how would you rate your health?” – has attracted health researchers' attention because it is easy to answer and low-A number of studies evaluating population groups in various countries for varying follow-up periods have found that respondents who perceived themselves to be in poor health display hazard of mortality 2 to 7 times greater than those who perceived their health to be excellent, even after adjustment for diseases and other important health conditions,9. A dosAlthough the relationship between SRH and mortality is well documented in the literature, most studies have evaluated SRH at only one point in time, generally at the study baseline-13. HoweThere are many issues that give good reason for studying the association between SRH and mortality in our specific cultural context. First, there are few studies on such relationship arising from Brazil, a country with a different socioeconomic profile as compared to most developed countries where the majority of reports on the relationship between SRH and mortality comes from. The three existing studies have investigated the association only in populations over 60 years of age and used only one measure of SRH, taken at the baseline-19. SecoPró-Saúde study), directed mainly to investigating social determinants of health outcomes.This study draws on a longitudinal prospective study of non-faculty civil servants at a university in Rio de Rio de Janeiro, Brazil (the At stage 1 of the study (1999), all regular employees in technical administrative positions were considered eligible, except those who had retired, were on leave of absence, had been dismissed or seconded to other institutions . The analyses presented here include all employees participating in stage 1 with valid responses to the question on SRH (N = 4009). Of these, 80.7% (N = 3237) were followed up at stage 2 of the study (in 2001) and 81.2% (N = 3255), at stage 3 (in 2006). These participants’ life status was monitored until May 2009. The responses for SRH at stage 2 and stage 3, whenever available, were incorporated into the analyses.Participants filled out a self-administered questionnaire. Self-rated health was evaluated on an ordinal basis, as measured by the question “In comparison with people of your age, how do you rate your own overall health status?”. The response options were: “Very good”, “Good”, “Fair” or “Poor”. For the analyses, the levels “Fair” and “Poor” were grouped into a single category, because the category “Poor” was mentioned by only a small number of employees at stage 1 (N = 63). A test-retest reliability study with a two-week interval between responses was performed among individuals not enrolled in the study cohort, but whose sex, age and literacy profile was similar to that of the cohort. Reliability for the SRH reported at stage 1 of the study (1999) was estimated using weighted kappa (square weighting), returning a value of 0.65 (CI95% 0.54-0.72).Deaths occurring in the cohort between 1999 and 2009 were identified by consulting the university human resources department. In order to investigate their causes, these deaths were located in the national Mortality Information System (SIM) database up to 2006 – the most recent data available – by means of probabilistic record linkage technique. Reclink was used2, in kg/m2): calculated from weight and height measured and classified according to World Health Organization recommendations – categorised as underweight or normal weight (<25 kg/m2); overweight (25 to 29.9); or obesity (≥30)[The covariates included in the study were defined as: a) age (years): < 35, 35 to 44, 45 to 54 and >54; b) sex: male or female; c) income: per capita family income terciles in U.S. dollars (US$), calculated by dividing the median of the net income bracket by the number of dependents on that income, and converting to August 1999 dollar values (median date of entry into the study in 1999) of US$1.00 = R$1.70; d) schooling: fundamental (up to incomplete high school), middle (complete high school and incomplete university) or university (complete university or more); e) chronic diseases: self-reported lifetime medical diagnosis of arterial hypertension and/or diabetes mellitus and/or myocardial infarction and/or cerebral vascular accident and/or pulmonary emphysema/chronic bronchitis – categorised as none, or at least one, of these conditions; f) presence of common mental disorders: evaluated on the General Health Questionnaire (GHQ-12) scale – categorised as present  or absent; g) body weight < kg/m2; oAll variables analyzed were collected at stage 1 only, except the main exposure variable (SRH) and age, updated in 2001 (stage 2) and 2006 (stage 3), and the covariate “chronic diseases”, updated in 2001 but not available in 2006. The pattern of change in the SRH over time was investigated.All participants signed a declaration of informed consent, and the research protocols were approved by the Research Ethics Committee of the Institute of Social Medicine of the State University of Rio de Janeiro.The association between SRH and mortality was estimated using extended Cox proportional hazard models with both SRH and chronic diseases varying over time. Follow-up started on study entry date, and the variable was updated at each new interview. The Kaplan-Meier estimator and the log-rank test (p < 0.15) were used to select the variables to be evaluated in bivariate Cox models, where hazard ratios (HR) were estimated. The variables that proved statistically significant in the bivariate models (p < 0.05) were then included in the multivariate analysis.a priori that “chronic diseases” would be retained in the final model independently of these criteria, due to its importance as an alternative explanation of the causal chain. The Cox model’s assumption of proportional hazard for each variable over time was checked by analysis of Shoenfeld’s residual analysis and the functional form of the continuous variables age and income was checked by analysis of Martingale’s residual analysis (p < 0.05). All analyses were performed using the R statistical package, version 2.10.0[Variables' entry into the multivariate models was determined on the hierarchical causality theoretical model, including first the distal characteristics  and then the intermediate characteristics . In order to be maintained as a confounder in the multivariate model, each variable had to be associated with the outcome at a 5% level of significance (p < 0.05) and/or alter the effect of SRH on mortality by at least 20%. It was decided n 2.10.0 with then 2.10.0.Due to the difference in hazard of death encountered among men and women, by SRH, the analyses were performed separately by sex Table. The groAt stage 1, health was self-rated as “Very good” by 28.2% of participants, “Good” by 53.2%, “Fair” by 17% and “Poor” by 1.6%. Self-perceived health was worse among women than among men, with 21.3% reporting SRH as “Fair” or “Poor”, compared with 13.2% of the men (p < 0.001). In both sexes, worse SRH was also observed among the older individuals, those with less income, less schooling, who were widowed, who reported some disease, had higher BMI, were classified as positive for common mental disorders or as smokers, for both sexes (p < 0.05) (data not presented). At stages 2 and 3, SRH prevalences were, respectively, 27.8% and 26.3% (Very good), 51.5% and 50.7% (Good), 17.8% and 20.2% (Fair), and 1.6% and 2.2% (Poor).As regards changes in SRH over the course of follow-up Figure, 36.2% oOf the 4009 participants with complete SRH data at stage 1 (99.5% of participants at that study stage), 117 had died by May 2009  were distributed as follows: diseases of the circulatory system , neoplasms , diseases of the respiratory system , external causes , infectious and parasitic diseases , endocrine, nutritional and metabolic diseases , diseases of the digestive system , symptoms, signs and abnormal clinical and laboratory findings, not elsewhere classified , and others . Distribution of causes of mortality was similar for men and women, with the exception of external causes, which constituted the second cause of death among men and did not figure among causes of death for women.The crude survival functions indicated that hazard of death for women with “Fair/Poor” SRH was seven times greater than for those with “Very good” SRH Table. Among mAs far as we were able to discover, this study, which involved follow-up over a 10-year period, is the first in Brazil to investigate the association between SRH and mortality in a population of young adults. Men and women with “Fair/Poor” SRH had 2.1 and 3.4 times greater hazard of mortality, respectively, than those with “Very good” SRH, independently of reporting diagnoses of chronic diseases and other covariates.A number of studies,10, inclPró-Saúde study cohort and the population of Rio de Janeiro State have similar profiles in terms of causes of mortality, except for external causes, which had more influence on mortality in the overall population than in the cohort, and diseases of the respiratory system, which occurred more often in the Pró-Saúde study population[The prevalences of the worst category of SRH estimated in our population were similar to those found among industrial workers in Brazil and lowepulation.The results of previous studies have varied as regards whether hazard of death, comparing the worst and best SRH categories, is greater among men or women. Some estimated greater relative hazard of mortality among women than among men associated with “Poor” SRH,16,31; oThe presence of diseases is identified as the main potential confounder of the relationship between SRH and mortality. In our Idler & Benyamini suggest Many studies have indicated that the ability of SRH to predict mortality diminishes with increasing cohort follow-up time,31,35. TIn the study by Strawbridge & Wallhagen, time-deSome limitations of this study deserve mention. Our results might have been biased due to lack of complete information on changes of SRH over time for the participants. About 14% of them had only the first baseline SRH measure, and other 10% had only two SRH measures (baseline plus SRH recorded on stages 2 or 3). The potential effect of such problem on the results is unknown, but one might suppose that those who drop out would probably have worse health as compared to their earliest SRH evaluation and higher probability of death. Including only the first SRH assessment for these participants would probably lead to underestimation of the strength of the association between SRH and mortality. In addition, we didn’t have the measure of chronic conditions on stage 3, thus possibly slightly overestimating the independent effect of SRH. Last, it was not possible to update the status of some covariates in our analyses. However, considering that the population is made up of staff at a single public institution, changes in income and schooling are uncommon.Moreover, deaths occurring after 2006 could not be identified, as they were not available in the Mortality Information System (SIM). However, the high proportion (96%) of deaths recorded in the university human resource system that were also found in the SIM database between 1999 and 2006 warrants our belief that the university records for deaths occurring from 2007 to 2009 are valid. Besides, the university records system is extremely reliable, as the family must notify the institution of any death in order to secure their right to a regular pension and funeral costs. Lastly, it cannot be guaranteed that residual confounding is absent, given that only some medical diagnoses were included as self-reported chronic diseases, and objective measures of health  were not used. Nonetheless, we believe that this potential residual confounding is not considerable, because a number of studies using objective measures have arrived at results similar to ours,12,38.Analysis of SRH in three categories (instead of the “SRH positive”/“SRH negative” dichotomy found in most research) and also the use of both SRH and presence of diseases as time-dependent covariates, make the results of this study more robust and comparable to those of the few studies that use a similar strategy. We consider that analysis of SRH that changes over time is the most appropriate analytical method for investigating the relationship between this variable and mortality, given that these alterations are frequent. Failure to incorporate such information can result in misclassification, which in our study would affect about 40% of the participants.Pró-Saúde study cohort may represent an approximation to what is occurring in the middle strata of the economically active population of Brazil's major metropolises. Subsequent studies could investigate the role of SRH in predicting specific causes of mortality, which was not possible in the Pró-Saúde study given the small number of deaths. It would also be interesting to ascertain whether specific causes of mortality can explain the differences observed between men and women in the SRH-mortality relationship. It is also suggested that studies investigate different SRH trajectories over time, and their association with mortality.As regards external validity, the results obtained in the Our findings reinforce the importance of using SRH in epidemiological research as a simple, low-cost and complementary measure for population health monitoring.The authors declare that they have no competing interests.JMNG wrote the first draft of the manuscript. JMNG and MSC carried out the data analyses. JMNG, DC, GLW, CMC, CSL and EF were responsible for the conception and design of the study. All authors significantly contributed to the interpretation of the results, commented extensively on subsequent revisions and have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/676/prepub


Barley yellow dwarf virus (BYDV) – like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5′ proximal end of the TNV-AC coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5′ or 3′ sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (−3 to +6 of the CP initiation site) within TNV-AC RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-AC, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the  Tomato bushy stunt virus (TBSV) Red clover necrotic mosaic virus (Dianthovirus) Cucumber leaf spot virus (Aureusvirus) Potato virus X (Potexvirus) Necrovirus gRNAs, cap-independent functional elements related to the BYDV–like translational enhancer (BTE) in the 3′ UTR are involved in long-distance RNA-based interactions during translation and replication. These interactions have been well studied to understand the mechanistic features of virus reproduction in vitro translation Positive-stranded RNA viruses often harbour RNA elements within their genomic (g) RNAs that mediate a variety of fundamental viral processes. Viral RNA elements were conventionally viewed as localised sequences or structures, such as poly(A) tails, pseudoknots, tRNA-like structures or RNA hairpins Necrovirus genus in the Tombusviridae family C that was obtained from soybean (Glycine max) in China, causes a localised infection in Chenopodium amaranticolor leaves and systemic infections in soybean and Nicotiana benthamianaC has a 3682 nucleotide (nt) linear monopartite gRNA TNV-A is the type member of the C coat protein (CP) is dispensable for infection of C. amaranticolor, and that the 5′ terminal nucleotides (NT) of the CP coding region affects local symptoms and viral RNA accumulation C element, we have now precisely identified functional nucleotides flanking the CP initiation site by site-directed mutagenesis. We also demonstrated pronounced positional effects of an internal element on viral RNA synthesis and translational enhancement of a reporter gene fused to the element.We reported previously that the intact TNV-AC CP by either deleting the entire coding region or by prematurely terminating the translational start codon with a CAC triplet resulted in attenuated local symptoms and reduced levels of viral RNA accumulation in C. amaranticolor plants C. amaranticolor. At 4 dpi, the results revealed that deletions in the 5′ end of the coding region  eliminated symptoms and greatly reduced RNA synthesis, compared to the internal or the 3′ end deletions TNV-AC gRNA , which had elevated RNA levels similar to those of wild type (wt) TNV-AC (C CP (data not shown). These results suggest the existence of a gRNA element near the 5′-proximal end of the CP gene that is responsible for viral RNA synthesis. A mutant containing this element is represented by pTCPΔ814, which contains the 17 nt at the 5′ end of the CP gene, but lacks the remainder of the CP ORF and the stop codon  reporter was substituted into the TNV-AC genome to replace the CP gene (nt 2612–3442) to produce pTGUS-I, or was fused in-frame with the 5′ proximal 18 nucleotides (nt 2612–2629) of the CP gene to generate pTGUS-II (C. amaranticolor at 4 dpi. In contrast to the necrotic lesions caused by wt TNV-AC (pMTC27), attenuated symptoms consisting of a few chlorotic spots were observed in leaves inoculated with pTGUS-II and pTGUS-I 294, whereas lesions failed to appear on leaves inoculated with pTGUS-I, pTGUS-I 17 and pTGUS-I 81 , but much lower levels were present in leaves inoculated with pTCPΔ826 to pTCPΔ830 RNAs . The latter results possibly reflect negative regulation of polymerase translation by sequences within the 2609 to 2617 nt region . First, the viral fragments were fused with a firefly luciferase (F-Luc) gene in the Dual-luciferase assay system to quantitatively evaluate translational efficiency. Considering the potential interference from the upstream sequence as shown in Renilla luciferase (R-Luc) from pRL-TK were co-transfected into BY-2 protoplasts. The relative luciferase activities of each construct were analysed at 16 hpt, and normalised to values for the pGL-Luc control vector, which was defined as 100%. To preclude an effect from the sequence insertion, pGL-RN-Luc, a construct that contained 81 nucleotides (RN) from the plasmid pGL3-Basic was used as a negative control. As shown in As shown in C UTR constructs were generated  were created as controls  that is essential for virus replication and high efficiency expression of reporter genes in plant leaves or in tobacco cells. The element is associated with viral UTRs, mainly with the 3′ UTR and these associations are required for synergistic enhancement.In summary, we have identified an RNA regulatory element in the internal region of TNV-ATombusviridae, reduced levels of Turnip crinkle virus (TCV) CP are responsible for sat-RNA mediated symptom attenuation ArabidopsisCymbidium ringspot tombusvirus (CymRSV) CP gene triggers an HR-like resistance response in Datura stramoniumCBeet black scorch virus (BBSV) C. amaranticolor were attenuated or delayed when the CP initiation codons were mutated or intact genes were deleted. In contrast, a reverse mutation of the CP start codon from AG2613G back to AUG in the construct pTCPM-I lead to a restoration of severe lesion phenotype similar to that of wt TNV-ACC CP functions as a silencing suppressor or an HR elicitor. Comparative analysis revealed that a 9-nt key oligonucleotide adjacent to the CP initiation site (nt 2609–2617) in TNV-AC also occupies the same position in other TNV-A isolates Tombusviridae family members, including BBSV Carnation Italian ringspot virusTombusviridae contain a highly-conserved sequence in the vicinity of the CP start codon that is remarkably resistant to spontaneous mutation during evolution. As shown in 2460 reported previously C sgRNA2 transcription. Moreover, although alignments of the homologous RNA regions among the necroviruses and tombusviruses showed no significant conservation throughout the 87-nt region, the nucleotides 2593UUUC2596 in the 87-nt region that exist in some other necroviruses could base-pair with the 3512GAAA3515 sequences of the BTE stem-loop I motif (3503GGAUCCUGAGAAACAGG3519) and 3623GAAA3626 of the 3′ end stem-loop II motif in the 3′UTR. This possible terminal bulge base-paring may be important for formation of the conserved 3′ UTR secondary structure predicted by Na 3512GAAA3515 loop is responsible for the high level CP accumulation and the severer symptoms of BBSV, TNV-A and TNV-D by Xu 2612AUGGCA2617) is highly conserved amongst necroviruses and tombusviruses. This sequence is partially complementary to the 3466UGCC3469 or 3609GCCA3612 sequences in the 3′UTRs, but appear not to be involved in the 5′ and 3′ long-distance interactions described by Shen The CP's of plant viruses frequently have multiple functions during infection Tobacco etch virus (TEV) Potato virus Y (PVY) Turnip mosaic potyvirus (TuMV) Blackcurrant reversion virus (BRV) RNA2 Tobacco mosaic virus (TMV) C genome, which is composed of the 9 nt key region. An additional sequence upstream of the sgRNA2 leader is also indispensable for viral RNA replication and functions in translational enhancement. Intriguingly, when present in reporter vectors lacking a TNV-AC trailer sequence, the element can also increase the efficiency of protein expression significantly. These results demonstrate that the sequence motif can function independently of a viral context to facilitate translation of heterologous mRNAs.Various regulatory elements mostly located at the termini of viral genomes are employed by RNA viruses to mediate appropriate timing of gene expression and levels of accumulation. For instance, the internal ribosome entry sites (IRES) in the 5′ UTR of 2602CAAAAUAGAC2611 present in the TNV-AC enhancer element shares very high similarity with the upstream region 28CAAAACAAAC37 flanking pseudoknot 1 (PK1) in the TEV 5′-leader, which is necessary for promotion of cap-independent translation C genome may be involved in translational enhancement by generating functional structure with other viral sequences.In addition, the sequence C gRNA that may provide an alternative mechanism to stimulate protein translation. Distinct functional features of this element still remain to be elucidated and will be a subject for future investigations in our lab.In addition to the 3′-BTE, we have identified a new element for cap-independent translation in the centre of the TNV-AC (pMTC27) Aor51HI and EcoRV to construct the mutant pTCPΔ488  was amplified by PCR from pBGUS DraIII and EcoRI before cloning into pMTC27 to create pTGUS-I  leader sequence from plasmid pRTL2 C sequences for translational enhancement activities  reporter was fused upstream or downstream of TNV-A plasmid  was used plasmid . Six of  plasmid . In cate plasmid . In a setivities . To test control .Renilla luciferase (R-LUC) gene from pRL-TK (Promega) driven by T7 or 35S promoter was used as a control throughout the experiments.For transient expression in tobacco cells, the 35S promoter-driven constructs were created by transferring expression fragments from pGL-T142-Luc, pGL-T87-Luc or pGL-T21-Luc into the plant expression vector pRTL2 C. amaranticolor plants used in this experiment were grown in a growth chamber at 24°C with a 14-hour-light/10-hour-dark cycle. Tobacco BY-2 (N. tabacum L. var Bright Yellow 2) suspension cells in vitro with T7 RNA polymerase (Promega) and purified with HiBind® spin cartridges (OMEGA) according to the manufacturer's instructions. RNA concentrations were determined by spectrophotometry , and RNA integrity was assessed by 1% agarose gel electrophoresis. One to two μg of in vitro synthesised RNAs were mixed with an equal volume of inoculation buffer  for mechanical inoculation of C. amaranticolor leaves. Transfection of protoplasts was carried out as described previously 6 cells/ml) isolated from BY-2 suspension cells were electroporated with 10 μg of the viral RNA transcripts and incubated in the dark at 24°C for 18 hours. To eliminate background interference, a mock treatment was performed by mixing the protoplasts with RNA transcripts without electroporation.Viral RNAs were synthesised C. amaranticolor at 4 days post-inoculation (dpi) as previously described C, prepared as in previous reports C CP Total RNA was isolated from leaves of 6 cells/ml) were co-electroporated with 15 μg of uncapped RNA transcripts, or 20 μg of DNA containing a F-Luc gene or 1.5–2 μg of the R-Luc internal control and then were incubated at 24°C for 16 h. Cells were harvested by centrifugation at 150 g for 3 min at room temperature, washed with phosphate buffered saline (PBS), and centrifuged at 10,000 g for 3 min at 4°C. The pellet was lysed in 250 μl of passive lysis buffer (Promega) before being subjected to one or two freeze-thaw cycles to completely disrupt the cells. Aliquots (20 μl) of cell lysate were measured for firefly and Renilla luciferases activities with a GloMax® 20/20 Luminometer (Promega), in which light readings of extracts from mock-inoculated protoplasts were used as background controls. The relative luciferase activities of the constructs were normalised with Renilla luciferase standard and expressed as a percentage relative to the pGL-Luc or p35S:Luc controls, which were set as 100%.BY-2 protoplasts  The organisation of wtTNV-AC (pMTC27) ORFs are illustrated by gray boxes. The region at the 5′ end of the CP ORF is indicated by solid lines to illustrate the sequence used for the deletions, and the deleted sequences are indicated by dashed lines with numbers identifying the deleted sequences appearing above the dotted regions. Designations of the eight deletion mutants and the nucleotide deletions are shown on the left side of the figure. (B) The inoculated C. amaranticolor leaf phenotype was photographed at 4 dpi, and the mutant viral RNAs used for inoculations are shown above each photo. (C) Viral RNA accumulation in C. amaranticolor plants inoculated by the respective mutants was assessed by Northern blots with the same probe used in C RNA species are indicated on the left side of the gel photographs and plant rRNAs are shown as loading controls in the bottom panel.(TIF)Click here for additional data file.Figure S2Viral RNA accumulations in inoculated tobacco BY-2 protoplasts and in vitroC RNAs containing deletions of the internal region. translation assay of the P23 replicase protein with mutant TNV-A (A) Schematic representation of pTCPΔ823 (see C RNA species are indicated on the left side of the gel photographs and plant rRNAs used as loading controls are shown in the bottom panel. (C) Western blot analysis of P23 replicase proteins synthesized during in vitro translation with wheat germ extract (Promega). Viral constructs used for in vitro mRNA synthesis are indicated above each lane in panels B and C Click here for additional data file.Table S1Primers used in this study.(PDF)Click here for additional data file.


The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex , which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) immunity has been exploited widely over the past few months  (precursor CRISPR RNA (pre-crRNA) forming a dual-RNA  ,16 bindsdual-RNA  that is dual-RNA , and invdual-RNA ,17–19. Ang crRNA  introducng crRNA ,17–19. Cng crRNA ,20,21 thng crRNA ,22,23, eng crRNA ,14,24,25ng crRNA ,23,26–28Recent studies have demonstrated that RNA-guided Cas9 can be employed as an efficient genome editing tool in human cells ,2,8,11, S. pyogenes, S. thermophilus** and Neisseria meningitidis have been developed into tools for genome manipulation  using a microplate reader (BioTek PowerWave).escribed ,16. BraiEscherichia coli was transformed with plasmid DNA according to standard protocols , finally suspended in the same buffer and equalized to the same OD620. For electroporation, 1 µg of plasmid was incubated with the competent cells on ice for 10 min. The conditions were 25 µF, 600 Ω and 1.5 V using 1 mm electroporation cuvettes (Biorad). After a regeneration time of 3 h, bacteria were spread on agar medium supplemented with kanamycin (300 µg/ml). Transformation assays were performed at least three times independently with technical triplicates. The efficiencies were calculated as colony-forming units (CFU) per µg of plasmid DNA. Positive and negative control transformations were done with backbone plasmid pEC85 and sterile H2O, respectively.rotocols . Transfoescribed  with som® High-Fidelity DNA Polymerase, Finnzyme), DNA digestion , DNA ligation , DNA purification  and agarose gel electrophoresis were performed according to the standard techniques or manufacturers’ protocols with some modifications , polymerase chain reaction (PCR) (Phusionications . Site-diications . Synthetrnc genes) of S. pyogenes, S. mutans, S. thermophilus, C. jejuni, N. meningitidis, P. multocida, F. novicida, E. coli and S. aureus, and the genes encoding truncated and inactive RNase III variants (truncated and inactive (D51A) rnc mutants) of S. pyogenes were cloned in pEC483 (pEC85 containing the native promoter of S. pyogenes rnc) using NcoI and EcoRI restriction sites . The orthologous and mutant cas9 genes were cloned in pEC342 (pEC85 containing a sequence encoding tracrRNA-171 nt (S. pyogenes cas operon) using SalI and SmaI restriction sites (Supplementary Table S1). Note that in a previous study, we observed low abundance of tracrRNA in the cas9 deletion mutant. For this reason, plasmids used in cas9 complementation studies were designed to encode tracrRNA in addition to cas9 . Plasmid integrity in all complemented strains was checked by plasmid DNA extraction and digestion.The backbone shuttle vector pEC85 was used for complementation study ,39. The A-171 nt  and the  to cas9 . The genspeM protospacer sequence was cloned in the PstI site of plasmids containing coding sequences of WT or mutated cas9 from S. pyogenes (Supplementary Table S1).Plasmid pEC85 was used as backbone vector for transformation studies. A DNA fragment containing WT Supplementary Table S1). The S. pyogenes cas9 mutant genes were PCR amplified from the complementation plasmids mentioned above. All orthologous and mutant cas9 genes were cloned into the SalI and NotI sites of pEC621.The overexpression vector pET16b (Novagen) was modified by inserting three additional restriction sites  into the NdeI restriction site, generating pEC621. The genes coding for the orthologous Cas9 proteins were PCR amplified from genomic DNA of the corresponding strains using primers containing a SalI and a NotI restriction site  was modified by PCR-mediated site-directed mutagenesis (Supplementary Table S1).Plasmid pEC287 that contains the agenesis  using onS. pyogenes SF370 WT, deletion mutants and complemented strains was prepared from culture samples collected at the mid-logarithmic phase of growth using TRIzol (Sigma-Aldrich). The total RNA samples were treated with DNase I (Fermentas) according to the manufacturer’s instructions. The concentration of RNA in each sample was measured using NanoDrop.Total RNA from 32P (20 μCi) using the T4-polynucleotide kinase  and purified using G-25 columns  prior use. Visualization of the radioactive signal was done using a phosphorimager. 5S rRNA served as loading control.Northern blot analysis was carried out essentially as described previously . Total REscherichia coli Rosetta2(DE3) and E. coli NiCo21(DE3) (New England Biolabs) were transformed with overexpression plasmids coding for S. pyogenes WT and mutant or orthologous Cas9, respectively. Cells were grown at 37°C to reach an OD600 of 0.7–0.8, protein expression was induced by adding IPTG to a final concentration of 0.5 mM and cultures were further grown at 13°C overnight. The cells were harvested by centrifugation and the pellet was resuspended in lysis buffer  and lysed by sonication. The lysate was cleared by centrifugation (>20 000 × g) and incubated with Ni-NTA (Qiagen) for 1 h at 4°C. After washing the Ni-NTA with lysis buffer and wash buffer , the recombinant protein was eluted with elution buffer ) and the fractions were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). In the case of S. pyogenes Cas9 WT and mutants, the protein containing eluates were pooled and further purified via HiTrap SP FF  cation-exchange chromatography. Briefly, the protein was loaded on the column equilibrated with buffer A  using an FPLC system . Cas9 was eluted with a gradient of buffer B  over 12 ml. 1 ml fractions were collected and analyzed by (SDS–PAGE). The protein containing fractions were pooled and dialyzed overnight . For Cas9 orthologs, the eluates from Ni-NTA purification were checked for purity by SDS–PAGE. In case of contaminants, a second purification over chitin beads was performed as described in the manual for NiCo21(DE3) cells from New England Biolabs. Briefly, 1 ml chitin beads (New England Biolabs) equilibrated with buffer A was incubated with the Ni2+-IMAC eluates for 1 h at 4°C. Afterwards, the beads were added onto a column and the Cas9 containing flowthroughs were collected and again checked for purity by SDS–PAGE . The purified proteins were dialyzed overnight. The protein concentration was calculated by measuring the OD280 using the extinction coefficient. The detailed characteristics of purified proteins are summarized in Supplementary Figure S1A.in vitro DNA cleavage assays was generated by in vitro transcription using the AmpliScribe™ T7-Flash™ Transcription Kit (Epicentre) according to the manufacturer’s instructions. PCR products or synthetic oligonucleotides used as templates are listed in Supplementary Table S1. The synthesized tracrRNA and repeat region of crRNA from each bacterial species correspond to the mature forms of RNAs as determined by deep RNA sequencing , heated up to 95°C for 5 min and slowly cooled to room temperature before use.RNA for quencing  or bioinyo1h_002 ). TranscS. pyogenes dual-RNA in cleavage buffer  for 15 min at 37°C. Plasmid DNA (5 nM) containing speM (NGG PAM) was added and further incubated for 1 h at 37°C. The reaction was stopped by addition of 5X loading buffer  bromophenol blue) and analyzed by 1% agarose gel electrophoresis in 1X TAE. Cleavage products were visualized by ethidium bromide staining. All other cleavage assays were carried out using the same conditions with the following modifications: KGB  and used to find cognate protospacer candidates using megaBLAST (http://blast.ncbi.nih.gov/Blast). Protospacer candidates were defined as containing a sequence with ≥90% similarity to the crRNA spacer sequence and originating from phage, plasmid or genomic DNA related to the bacterial species of the targeting CRISPR-Cas. For the investigated CRISPR-Cas loci, the orientation of transcription was determined previously by RNA sequencing or northern blot analysis (http://weblogo.berkeley.edu/) showing the most abundant nucleotides was created and PAM sequences were predicted. In the cases of CRISPR-Cas loci for which no suitable protospacer sequences could be identified , closely related strains of the same species were selected (Supplementary Table S2). The spacer contents of the type II CRISPR arrays in selected strains were analyzed (http://crispr.u-psud.fr/Server/). The spacer sequences were then used to select cognate protospacer sequences as described above.Spacer sequences of the selected bacterial species were extracted from the CRISPRdatabase . This procedure produced 82 clusters. In the case of sequences reported in this study, one or several representatives from each cluster were selected and aligned using the MUSCLE program  with 285 informative positions were used for maximum likelihood tree reconstruction using the FastTree program (cas operons (Supplementary Table S2). A few incomplete sequences were substituted by other Cas1 sequences from the same Cas9 cluster (Supplementary Table S2). Several Cas1 proteins from subtypes I-A, B, C and E were included as an outgroup. Cas1 sequences were aligned using the same approach described above and 252 informative positions  were used for maximum likelihood tree reconstruction using the FastTree program. RNase III multiple sequence alignment was prepared using the MUSCLE program.Position-Specific Iterated (PSI)-BLAST program  was used program  with def program  and HHpr program . The con program  with thehttp://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). The structure predictions were then visualized using VARNA . After removing incomplete or highly similar sequences, we selected 83 diverse, representative Cas9 orthologs for multiple sequence alignment and phylogenetic tree reconstruction . The Cas9 tree topology largely agrees with the phylogeny of the corresponding Cas1 proteins  and fully supports the previously described classification of type II CRISPR-Cas into three subtypes, II-A (specified by csn2), II-B (characterized by long and most diverged cas9 variants (formerly csx12) and cas4) and II-C (three-cas gene operon)  .Figure cas genes, transcription direction of the CRISPR arrays with respect to that of the cas operon, and location and orientation of tracrRNAs resulted in the division of subtypes into groups with distinct locus characteristics, especially within the subtype II-A  and S. mutans were chosen for type II-A systems associated with shorter, ∼220 amino acid Csn2 variants (Csn2a). Cas9 of S. thermophilus** (CRISPR1) represents a distinct group of type II-A sequences associated with longer, ∼350 amino acid version of Csn2 orthologs (Csn2b). Cas9 of F. novicida was selected for type II-B. The closely related Cas9 orthologs of P. multocida and N. meningitidis and the distinct, short Cas9 of C. jejuni were chosen for type II-C .Analysis of the composition of ype II-A . We seleype II-C B. Expresquencing ,16. The S. pyogenes and S. thermophilus, RNase III plays an essential role in the biogenesis of dual-RNA:Cas9 systems by coprocessing tracrRNA and pre-crRNA at the level of antirepeat:repeat duplexes . Northern blot analysis shows that all RNases III studied here can coprocess the RNA duplex , indicating that there is no species-specificity for tracrRNA:pre-crRNA cleavage by RNase III. Multiple sequence alignment of RNase III orthologs demonstrates conservation of the catalytic aspartate residue and the dsRNA binding domain  that are both required for RNA coprocessing . These data imply that the conservation of tracrRNA:pre-crRNA coprocessing by bacterial RNase III provides a degree of flexibility allowing the functionality of dual-RNA:Cas9 systems in multiple species upon horizontal transfer.As described in duplexes ,17. We aC. jejuni to 1648 amino acids for F. novicida), especially in the linker sequence between the highly conserved N-terminal RuvC and central RuvC-HNH-RuvC regions and in the C-terminal extension . Several studies demonstrated the importance of the nuclease motifs for dsDNA cleavage activity by mutating one aspartate in the N-terminal motif of the RuvC domain and one or several residues in the predicted catalytic motif of the HNH domain of the Cas9 enzyme . Northern blot analysis of S. pyogenes cas9 deletion mutant complemented with each of the cas9 point mutants revealed the presence of mature tracrRNA and crRNA forms, demonstrating that none of the catalytic motifs is involved in dual-RNA maturation by RNase III. This is in agreement with previous data showing that RNase III is the enzyme that specifically cleaves tracrRNA:pre-crRNA duplex .Comparison of Cas9 sequences revealed high diversity in amino acid composition and length  and WT ay . Hence, we investigated whether this variability plays a role in the specificity of Cas9 with regard to tracrRNA:pre-crRNA duplex and mature crRNA stabilization. We complemented S. pyogenes cas9 deletion mutant with Cas9 from selected bacterial species representative of the various type II groups and analyzed tracrRNA:pre-crRNA processing by northern blot. Cas9 proteins from S. mutans and S. thermophilus* can substitute for the stabilizing role of S. pyogenes Cas9 in RNA processing by RNase III . In contrast, Cas9 from S. thermophilus**, C. jejuni, N. meningitidis, P. multocida and F. novicida could not complement the lack of RNA processing in the cas9 mutant of S. pyogenes. In these strains, the 75-nt processed form of tracrRNA is observed as a very weak signal of background level of dual-RNA processed by RNase III in the absence of Cas9. Overall, only Cas9 from closely related systems of S. pyogenes in the type II-A cluster can substitute endogenous Cas9 role in dual-RNA stabilization and subsequent maturation by RNase III.Beside the conservation of the HNH and split RuvC domains involved in DNA cleavage ,15, the S. pyogenes and S. thermophilus* types II-A, PAMs were identified as NGG and NGGNG, respectively. In these two species, mutating the PAM abrogates DNA interference by dual-RNA:Cas9  and analyzed their spacer sequences following the same approach (Supplementary Table S3). We aligned the identified 10 nt sequences located directly downstream of the protospacer sequence and delineated the most common nucleotides that could represent PAM sequences. Based on the data visualized as a logo plot  and dual-RNA orthologs were designed based on deep RNA sequencing data . The previously published PAMs for Cas9 from S. pyogenes (NGG), S. mutans (NGG), S. thermophilus* (NGGNG) and N. meningitidis (NNNNGATT) . This is in contrast to data obtained in vivo, where mutation of the third G abrogates interference by Cas9 of S. thermophilus* . Using the same approach, we also validated the PAM activity of the most common protospacer-downstream sequences for C. jejuni, F. novicida and P. multocida by in vitro cleavage assays, resulting in the most probable PAM sequences being NNNNACA (C. jejuni), GNNNCNNA (P. multocida) and NG (F. novicida) . Analysis of the protospacer-adjacent sequence from C. jejuni shows the same frequency of C and A (‘NNNNCCA’ or ‘NNNNACA’) at position 5 downstream of the protospacer (Supplementary Table S3). Hence, we tested both substrates for cleavage activity by C. jejuni dual-RNA:Cas9. Only the DNA target containing A at this position was cleaved efficiently . This result could be explained by the origin of the protospacer, with the ‘NNNNCCA’ PAM being mostly found in genomic DNA or prophages of Campylobacter strains. In this case, the mutated PAM sequence on the chromosomally located protospacer prevents self-targeting. The P. multocida PAM requires further verification given that the multiple sequence alignment was derived from only two protospacer sequences. Thus, we identified a series of specific PAMs that enable dsDNA cleavage by dual-RNA:Cas9 complexes from different bacterial species in vitro. For gene editing purposes, it would be advisable to test a range of potential motifs to select those PAMs that would allow efficient targeting with limited off-site effect.In RNA:Cas9 ,22,23. Togo plot A, we desM or not B. The CaNNNGATT) ,28,53,54 NNAGAAW , which dS. pyogenes Cas9 in tracrRNA:pre-crRNA processing by RNase III in vivo , but not from any other tested species. The same result was observed when the dual-RNA from S. pyogenes was incubated with Cas9 orthologs from different bacteria . More striking was the gradient of activity dependent on how closely related the species are in the corresponding type II group. This effect can be observed for C. jejuni Cas9 that is able to cleave DNA in the presence of dual-RNA from P. multocida and N. meningitidis, but not as efficient as with its own RNA . This finding is in good agreement with the phylogenetic tree of Cas9 . Most of the strains that harbor type II CRISPR-Cas systems (and accordingly Cas9) are pathogens and commensals of vertebrates. A majority of these strains were isolated from gastrointestinal tracts and feces of mammals, fish and birds, but also from wounds, abscesses and spinocereberal fluid of septicaemia patients. We also identified strains isolated from invertebrates and environmental samples, including fresh and sea water, plant material, soil and food, the latter comprising species used in fermentation processes. Cas9 is also present in species from extreme environments such as deep sea sediments, hot springs and Antarctic ice, further demonstrating the wide spread of type II CRISPR-Cas systems in bacteria. A comparison of the taxonomy and habitats of representative strains with the phylogenetic clustering of Cas9 sequences shows little correlation . In particular, we identified clusters of Cas9 genes from taxonomically distant bacteria that were isolated from similar habitats. Examples include diverse Firmicutes, Molicutes, Spirochaete and Fusobacteria, that were all isolated from gastrointestinal tracts of mammals, and members of different Proteobacteria, Firmicutes and Fusobacteria families mostly found in environmental samples . A few exceptions involve grouping of Cas9 genes from closely related species isolated from diverse habitats such as Actinobacteria isolated from human and dog specimens but also from hot springs . This complex distribution of Cas9 across bacterial genomes indicates that evolution of dual-RNA:Cas9 systems in bacteria occurs both vertically and horizontally  results in nicking activity of Cas9 originating from the other motif , F. novicida (II-B) and C. jejuni, N. meningitidis and P. multocida (II-C). Substitution of orthologs from the selected species for the endogenous S. pyogenes Cas9 shows that only Cas9 proteins from the S. pyogenes subcluster are capable of assisting tracrRNA:pre-crRNA processing by RNase III. This result indicates that the less-conserved inter-motif regions, which are the basis for the Cas9 subgrouping, could be responsible for Cas9 specificity for certain dual-RNAs.Dual-RNA and Cas9 sequences have widely evolved in bacteria . Howeverer motif ,8,24,25.in vitro. The interchangeability between dual-RNA and Cas9 from different subclusters was tested using plasmid cleavage assays. Only closely related Cas9 proteins can exchange their cognate dual-RNAs and still exert cleavage activity when using the Cas9 specific PAM. The specificity of Cas9 towards dual-RNAs is highly sensitive to the Cas9 sequence relatedness. This sensitivity is observed with Cas9 from C. jejuni that displays full cleavage activity with its cognate dual-RNA but reduced activity with dual-RNAs from N. meningitidis or P. multocida which belong to different subclusters of type II-C. We hypothesize that Cas9 possesses specificity for the secondary structure of dual-RNAs, given that bioinformatics predictions suggest similar structures of repeat:antirepeat duplexes in closely related CRISPR-Cas systems .To investigate the interchangeability between type II subgroups at the level of DNA interference, we first determined the PAMs specific for each of the eight selected Cas9 orthologs . By aligS. thermophilus , each set al. . K.C. was a fellow of the Austrian Doctoral Program in RNA Biology. Funding for open access charge: Helmholtz Centre for Infection Research.Conflict of interest statement. None declared.


SeU) phosphoramidite and Se-nucleobase-modified RNAs (SeU-RNAs), where the exo-4-oxygen of uracil is replaced by selenium. Our crystal structure studies of U-A and U•U pairs reveal that the native and Se-derivatized structures are virtually identical, and both U-A and U•U pairs can accommodate large Se atoms. Our thermostability and crystal structure studies indicate that the weakened H-bonding in U-A pair may be compensated by the base stacking, and that the stacking of the trans-Hoogsteen U•U pairs may stabilize RNA duplex and its junction. Our result confirms that the hydrogen bond (O4…H-C5) of the Hoogsteen pair is weak. Using the Se atom probe, our Se-functionalization studies reveal more insights into the U•U interaction and U-participation in structure and function diversification of nucleic acids.Unlike DNA, in addition to the 2′-OH group, uracil nucleobase and its modifications play essential roles in structure and function diversities of non-coding RNAs. Non-canonical U•U base pair is ubiquitous in non-coding RNAs, which are highly diversified. However, it is not completely clear how uracil plays the diversifing roles. To investigate and compare the uracil in U-A and U•U base pairs, we have decided to probe them with a selenium atom by synthesizing the novel 4-Se-uridine ( Unlike natural DNA, which merely stores genetic information in cells , naturalHowever, considering the similar chemical structures of nuclei acid building blocks , it is striking that RNA with the extra 2′-OH is able to establish much more diversified structures and functions than DNA ,21. In aSeU-RNA synthesis challenge, meet the urgent needs in ncRNA investigation and probe U-A and U•U pairs by a Se atom. Herein, we report the first synthesis of the 4-Se-uridine phosphoramidite (SeU) and the corresponding SeU-RNAs by replacing 4-oxygen with selenium. We have found that this Se-modification does not cause significant perturbation and that the native and modified structures are virtually identical. We also found that via the stacking and hydrogen bonding, the uracil nucleobase interacts differently in RNA duplex and duplex junction. Moreover, the accommodation of the larger selenium atom by both U-A and U•U pairs implies the RNA flexibility. Our studies suggest that by presenting their different faces and edges, uracil and uridine are capable of diversifing structure and function of ncRNAs. Furthermore, this Se-modified uridine offers the Se-RNAs with additional UV absorption . Excitingly, after a single-oxygen atom replacement with selenium, we have observed for the first time the color RNAs (light yellow) as well as color RNA crystals (dark yellow). The color property of the SeU-RNAs is unique and has great potentials in RNA visualization, detection, spectroscopic study and crystallography of RNAs and protein-RNA complexes and interactions, demonstrating the usefulness of selenium-derivatized nucleic acids (SeNA)  ,37 in st4 suspension (250 mg of NaBH4 in 3 ml of EtOH) was injected into a flask containing di(2-cyanoethyl) diselenide  and THF (10 ml) in an ice bath with argon. The yellow color of the diselenide disappeared in ∼15 min, giving an almost colorless suspension of sodium selenide (NCCH2CH2SeNa). Then, the reacted solution of compound 1 was slowly injected into this selenide solution. After the selenium incorporation was completed in 45 min , water (100 ml) was added to the reaction flask. The solution was adjusted to pH 7–8 using CH3COOH (10%) and was then extracted with ethyl acetate (3 × 100 ml). The organic phases were combined, washed with NaCl , dried over MgSO4 (s) for 30 min and evaporated to minimum volume under reduced pressure. The crude product was then dissolved in methylene chloride (5 ml) and purified on a silica gel column equilibrated with hexanes/methylene chloride (1:1). The column was eluded with a gradient of methylene chloride . After the collected fraction evaporation and dry under high vacuum, pure compound 2 was obtained as a slightly yellow foam product . 1H-NMR  δ: 0.21 , 0.38 , 0.95 , 2.31-2.37 , 3.00 , 3.37-3.41 , 3.50-3.52 , 3.81 , 4.17–4.22 , 4.31 , 4.40–4.50 , 5.78 , 5.90 , 6.8–6.90 , 7.20–7.46 , 8.31 . 13C-NMR  δ: −4.30, −4.40 (CH3), 18.1 (CH2-CN), 19.0 (CH2-CH2-CN), 20.5 [(CH3)2C(t-Bu)], 25.9 (CH3), 55.3 (OCH3), 68.7 (C-3′), 76.4 (C-2′), 83.1 (C-4′), 91.0 (C-1′), 106.0 (C-5), 118.8 (CN), 113.3, 127.1, 128.0, 128.2, 130.1, 135.0, 135.3, 144.2, 158.7 (Ar-C), 140.4 (C-6), 153.3 (C-2), 175.0 (C-4). HRMS (ESI-TOF): molecular formula, C39H49N3O7SeSi; [M+H]+: 778.2413 .To a dry THF solution (10 ml) of the starting material compound , 4,4′-dimethylamino-pyridine  and triethylamine  under argon, the dry tetrahydrofuran (THF) solution (10 ml) of 2,4,6-trisopropylbenzenessulfonyl chloride  was added dropwisely. The reaction was stirred for 1 h before it is finished (monitor by thin layer chromatography (TLC), 5% methanol in dichloromethane). At the same time, the NaBH2  under argon, dry methylene chloride (2.5 ml), N,N-diisopropylethylamine , and 2-cyanoethyl N,N-diisopropyl-chlorophosphoramidite  were added sequentially . The reaction was then quenched with NaHCO3  and stirred for 5 min, followed by the extraction with CH2Cl2 (3 × 8 ml). The combined organic layer was washed with NaCl  and dried over MgSO4 (s) for 30 min, followed by filtration. The solvent was then evaporated under reduced pressure, and the crude product was re-dissolved in CH2Cl2 (2 ml). This solution was added drop-wise to cold petroleum ether (or hexane) (200 ml) under vigorous stirring, generating a white precipitate. The petroleum ether layer was decanted. The crude product was re-dissolved again in CH2Cl2 (2 ml) and then loaded on Al2O3 column  that was equilibrated with CH2Cl2/Hexanes (1:1). The column was eluded with a gradient of methylene chloride and ethyl acetate [CH2Cl2 to CH2Cl2/EtOAc (7:3)]. After solvent evaporation and dry over high vacuum, the compound 3 (612 mg) was obtained as a white foamy product (92% yield). 1H-NMR  δ: 0.2–0.4 , 0.85–1.20 , 2.30–2.38 and 2.70–2.82 , 2.34 and 2.64 , 3.00–3.04 , 3.32–3.44 , 3.52–3.64 , 3.73–3.84 , 3.82 and 3.83 , 4.12–4.35 , 4.43–4.48 , 5.70–5.90 , 6.83–6.88 , 7.27–7.43 , 8.30 and 8.39 . HRMS (ESI-TOF): molecular formula, C48H64N5O8PSeSi; [M + H]+: 978.3479 .To the flask (25 ml) containing SeU-phosphoramidite was 0.08 M in acetonitrile, compared with the regular ones (0.1 M). Coupling was carried out using 5-(benzylmercapto)-1H-tetrazole solution (0.25 M) in acetonitrile with 12 min coupling time for both native and Se-modified phosphoramidites. Three percent trichloroacetic acid in methylene chloride was used for the 5′-detritylation. Synthesis was performed on control-pore glass (CPG-500) immobilized with the appropriate nucleoside through a succinate linker. All oligonucleotides were prepared in dimethoxy trityl (DMTr)-on form. After synthesis, the RNAs were cleaved from the solid support and fully deprotected by 0.05 M K2CO3 (methanol solution) for 8 h at room temperature, followed by neutralization, evaporation and the treatment of tetrabutylammonium fluoride (TBAF) solution (1 M in THF) for overnight. After desalting and HPLC purification, the 5′-DMTr group was removed by 3% aqueous solution of trichloroacetic acid, and the solution was neutralized to pH 7.0 with a freshly made triethylammonium acetate (TEAAc) buffer and precipitated with NaCl  and ethanol (3 volumes). The ethanol suspension was placed at −80°C for 1 h, followed by centrifugation to collect the RNAs.All the RNA oligonucleotides were chemically synthesized in 1.0 μmol scale on solid phase. The ultra-mild RNA phosphoramidites protected with 2′-TBDMS were used (Glen Research). The concentration of the The RNA oligonucleotides were analyzed and purified by reverse-phase high performance liquid chromatography (RP-HPLC) in DMTr-on form. After the TBAF desilylation and desalting with sephadex G-25, HPLC purification was carried out using a 21.2 × 250 mm Zorbax, RX-C8 column at a flow rate of 6 ml/min. Buffer A consisted of 10 mM TEAAc (pH 7.1), whereas buffer B contained 50% acetonitrile and 10 mM TEAAc (pH 7.1). Similarly, the HPLC analysis was performed on a Zorbax SB-C18 column (4.6 × 250 mm) at a flow of 1.0 ml/min using the same buffer system. The DMTr-on oligonucleotides were eluded in a 20-min linear gradient of 100% buffer A to 100% buffer B. The HPLC analysis for both DMTr-on and DMTr-off oligonucleotides were carried out with up to 60% of buffer B in a linear gradient in the same period of time. The collected fractions were lyophilized, and the purified RNAs were re-dissolved in water for the detritylation and precipitation steps.Solutions of the duplex RNAs (1 or 2 μM) were prepared by dissolving the purified RNAs in sodium phosphate [10 mM (pH 6.5)] buffer containing 100 mM NaCl. The solutions were heated to 75°C for 3 min, then cooled down slowly to room temperature and stored at 4°C overnight before Tm measurement. Before thermal denaturation, the Se-RNA samples were bubbled with argon for 5 min. Each denaturizing curves were acquired at 260 nm by heating and cooling from 5 to 70°C for four times in a rate of 0.5°C/min, using Cary-300 UV-Visible spectrometer equipped with temperature controller system.The purified RNA oligonucleotides (1 mM) were heated to 70°C for 2 min and cooled down slowly to room temperature. Both native buffer and Nucleic Acid Mini Screen Kit (Hampton Research) were applied to screen the crystallization conditions at different temperatures using the hanging drop method by vapor diffusion (1 µl of RNA and 1 µl of buffer). Thirty percent glycerol, PEG 400 or the perfluoropolyether was used as a cryoprotectant during the crystal smounting, and data collection was taken under the liquid nitrogen stream at 99°K. The Se-RNA crystal data were collected at beam line X12B and X12C in NSLS, Brookhaven National Laboratory. A number of crystals were screened to find the ones with strong anomalous scattering at the K-edge absorption of selenium. The distance of the detector to the crystals was set to 150 mm. The radiation wavelength at 0.9795 Å was chosen for diffraction data collection and selenium single-wavelength anomalous dispersion (SAD) phasing. The crystals were exposed for 10 s per image with 1° oscillation, and a total of 180 images were taken for each data set. All data were processed using HKL2000 and DENZO/SCALEPACK .The structures of Se-RNAs were solved by both SAD with HKL2MAP and molecular replacement with Phaser , followe4 in ethanol solution (2CO3 in methanol). Finally, the 4-Se-uridine derivative (2) was converted to the corresponding phosphoramidite (3) in 92% yield. The analysis data are shown in the supporting information .We have developed a facile strategy to synthesize the Se-phosphoramidite. As showed in solution . This pr2CO3 in methanol)  instead of acetic anhydride was used in the capping step to avoid the acetylation of guanosine, which is difficult to remove under the mild deprotecting conditions (K2CO3 in methanol). All Se-RNAs were synthesized in DMTr-on form, followed by cleavage and deprotection with 0.05 M methanol solution of K2CO3. After the deprotection, the solution was carefully neutralized with 1 M HCl and evaporated to dryness. Then the 2′-TBDMS groups were removed by treating with 1 M TBAF solution in THF at room temperature overnight. After desilylation and desalting, a typical HPLC profile of the crude Se-RNAs is shown in Supplementary Figure S8, which indicates a high coupling yield of the Se-uridine phosphoramidite (96%), compared with incorporation of the non-modified phosphoramidites. After desalting with Sephadex-G25 matrix, the pure Se-RNAs were obtained by RP-HPLC purification, followed by the mild detritylation . Excitingly, we observed for the first time that the RNA with the single Se-atom substitution is visible and has yellow color. UV-vis spectroscopic study indicated the Se-RNA with λmax at 260 and 370 nm  ,33,35,43tylation . Severald 370 nm  resultedanalyses A and 2.SeU) by comparing with the native nucleotide, we synthesized and purified the SeUMP and 5′-SeUU-3′. Their HPLC profiles are presented in SeU residue absorbs at both 260 and 370 nm  and 370 nm (only by SeU) is determined as 0.920. Thus, 3 M−1cm−1) is known  calculation indicates that each Se atom contributed additional stabilization  to the stability of the RNA duplexes. This increased RNA duplex stability is attributed to the increased stacking interaction of SeU on the duplex ends; the support from the high-resolution structure data is presented later. Via the Se-atom probe, the UV-melting study of the duplex RNAs containing the UU junction indicates that the uracil stacking contributes significantly to RNA duplex stability.It is reported that a U•U pair is less stable comparing with a U-G or C-A mispair in a RNA duplex ,47. In RSeU-CGCG-3′)2 with overhangs and octamer (5′-rGUG-SeU-ACAC-3′)2 with a perfect duplex]. Crystals of both Se-RNA sequences were formed in 2–5 days at room temperature (25°C) with the Hampton nucleic acid mini-screen kit . Excitingly, all crystals of both Se-RNAs had strong yellow or dark yellow color because of the selenium modification (1). Similarly, the Se-RNA octamer formed crystals in 22 of 24 buffers using the same kit, and these crystals , 12 mM Spermine tetra-HCl, 12 mM NaCl and 80 mM KCl] for the Se-hexamer and No.12  for the Se-octamer. The statistic data of the structural analysis are summarized in To investigate the Se-nucleobase modification and its structural property, we have crystallized two Se-RNA sequences [hexamer (5′-rU-fication  and 6. Tfication  diffractterature . The natSupplementary Table S1) and Hoogsteen base pairs. The structures determined via SAD and molecular replacement approaches are identical. The Se-modified structure  and the corresponding native structure  (SeU•U pairs (Hoogsteen pair) have been observed in the Se-RNA  infinitely stacking and elongating along the 21 screw axis in the crystals with nicks on the 5′-end of each 5′-U(SeU). This 5′-U-SeU sequence forms the two symmetrical SeU•U base pairs, which is virtually identical to the native U•U pair   are virtolution) C with tholution) . Furtherolution) D and E; olution) D-I suggeolution) . These rolution) , two SeUe Se-RNA F and G. U•U pair G. NamelySeU•U pair displays a conventional hydrogen bond between O4 of the native uracil (U1) and N3 of the Se-uracil (U14) and an unusual C-H…Se hydrogen bond between C5 of native U and Se4 of Se-U, through the Hoogsteen edge of native U and the Watson–Crick edge of Se-U. These interactions result in a trans-Hoogsteen U•U pair (…Se (or O) hydrogen bond (bond length: 3.36 Å in the Se case), between C5 of native U and Se4 of Se-U  of the Hoogsteen pair is weak. Thus, the large Se atom probe indicates that the Hoogsteen H-bond is less important in the U•U pairing. This also suggests that the trans-Hoogsteen pair can tolerate a larger substitution and that the Hoogsteen pair is not rigid, which gives the duplex junction sufficient flexibility. Moreover, it is counterintuitive that the distance (3.11 Å) between these two big neighboring 4-Se atoms . Moreover, we have superimposed the structures of SeU-A (or SeU4-A13 pair) and U2-A15 pair  suggests a weak hydrogen bond after the Se-modification. On the other hand, the polarizable and large Se atom with delocalizable electrons may facilitate the base stacking interaction, supported by the narrower base-pair gap and the computational study of the Se-nucleobase-modified DNA , progress in RNA crystallography is limited, especially in the ncRNA structure study. Inspired by the protein Se-derivatization, multi-wavelength anomalous dispersion phasing and SAD phasing , our labtrans-Hoogsteen U•U pairs is the main contributor to the duplex junction stability, whereas the Hoogsteen H-bond is weak. Moreover, the accommodation of larger Se atoms in uracil by both U-A and U•U pairs implies the RNA flexibility. Using the Se atom probe, our studies confirm that uracil is capable of interacting in multiple modes, thereby diversifying U•U and U-A pairs in structure and function. Our thermodynamic and structural studies have also demonstrated that this Se-modification can facilitate the nucleobase stacking interaction and potential crystal growth without significant perturbation. Furthermore, this Se-modification generates color RNA for the first time by single atom replacement, and it shifts the uridine UV spectrum over 100 nm . This color property is useful for RNA-protein co-crystallization, RNA visualization, detection and spectroscopic study. This work provides a new strategy for crystallization, phasing, structure and function studies of ncRNAs and protein-RNA complexes.To probe uracil-mediated interactions and base-pairs with a single selenium atom, we have synthesized the 4-Se-uridine phosphoramidite and Se-RNAs. Our thermostability and structure studies indicate that the modified and native structures are virtually identical, that the H-bonding decrease in U-A pair can be compensated by the base-stacking increase, and that the uracil stacking in duplex junction may increase duplex thermostability. We also found that the stacking interaction of the two 3HGA, 4IQSSupplementary Data are available at NAR Online.


Adapter ligation is a critical first step in many microRNA analysis methods including microarray, qPCR, and sequencing. Previous studies have shown that ligation bias can have dramatic effects on both the fidelity of expression profiles and reproducibility across samples. We have developed a method for high efficiency and low bias microRNA capture by 3′ adapter ligation using T4 RNA ligase that does not require pooled adapters. Using a panel of 20 microRNA, we investigated the effects of ligase type, PEG concentration, ligase amount, adapter concentration, incubation time, incubation temperature, and adapter design on capture efficiency and bias. Of these factors, high PEG% was found to be critical in suppressing ligation bias. We obtained high average capture efficiency and low CV across the 20 microRNA panel, both in idealized buffer conditions (86%±10%) and total RNA spiking conditions (64%±17%). We demonstrate that this method is reliable across microRNA species that previous studies have had difficulty capturing and that our adapter design performs significantly better than the common adapter designs. Further, we demonstrate that the optimization methodology must be specifically designed for minimizing bias in order to obtain the ideal reaction parameters. MicroRNA and other small RNA have added a new dimension to the connection between genotype and phenotype. These new mechanisms for gene expression regulation have led to a wealth of studies detailing the pervasive roles of microRNA in areas such as developmental biology de novo and due to their ability to distinguish closely related isoforms. Although these sequencing approaches typically involve many sequential enzymatic steps including reverse transcription, PCR amplification, ligation, and poly(A) extension, a number of recent studies have pinpointed adapter ligation as the main contributor to expression profile bias Due to the rising popularity of 2nd generation sequencing for small RNA detection and discovery, a number of studies have sought to benchmark microRNA expression profiles across various detection platforms and systematically look for sources of bias et al showed that consistent differential expression profiles can be seen across samples when the same adapter sequence is used but that large variations are seen when different adapter sequences are used even within the same sample et al demonstrated that microRNA species can appear over or under-expressed by multiple orders of magnitude due to biases in ligation efficiency Ligation bias is critical because it underlies such a large number of microRNA analysis methods. Ligation can introduce two distinct levels of bias to microRNA expression profiles. First, bias can be introduced across samples when different adapters are used on different individual samples. Alon et al found that two terminal bases on the 3′ adapter can have dramatic effect on ligation efficiency et al and Hafner et al demonstrated that secondary structure interactions can contribute significantly to variations in ligation efficiency et al have investigated the optimization of reaction conditions for bias suppression Recent studies have sought to identify the cause of ligation bias and remediate it In this study, we have designed a microRNA capture method based on 3′ adapter ligation that achieves very high efficiency (86% AVG) and low bias (10% SD) across all microRNA species tested. High efficiency capture is demonstrated even with microRNA that previous studies have had difficulty capturing and even based on the standard 3′ modban adapter that previous studies have shown to exhibit high ligation bias. Using a panel of 20 microRNA, we studied key assay parameters such as PEG%, enzyme selection, adapter saturation, and design and show that they can be used to suppress bias and nearly eliminate ligation preference given suitable optimization methodology. We demonstrate that optimization must be done in the presence of total RNA using a microRNA panel to minimize global bias, as erroneous conditions can be found if optimization is done using only a single microRNA or in synthetic conditions.et alMicroRNA targets were synthesized by Integrated DNA Technologies . They consist of HPLC purified, DNA oligonucleotides with 5′-Ph and either 3′-ddC blocking group or 3′-Cy5 label. The adapters are based on modified 3′ modban adapters www.mirbase.org) and are listed in MicroRNA were synthesized by Integrated DNA Technologies . The sequences were taken from miRBase , 5 µL of 50% PEG8000, 1 µL of synthetic target, 0.5 µL of total RNA, 1 µL of T4 RNA Ligase 2 truncated K227Q  and water into a 20 µL reaction volume. The reaction was then incubated at 25°C for 4 hours and heat denatured at 65°C for 20 minutes in a thermal cycler. In the experiments where different ligases were investigated, T4 RNA Ligase 2 truncated, T4 RNA Ligase 2 truncated R55K K227Q, and Thermostable 5′ App DNA/RNA Ligase were all obtained from New England Biolabs. In spiking experiments, 500 ng of human brain total RNA  was added to each sample.The samples were analyzed on precast 15% TBE-urea polyacrylamide gels . 5 µL of sample was mixed with 5 µL of loading buffer and heated for 5 minutes at 95°C. The sample was then loaded into the gel and run for either 30 min or 50 min at 300V. The separated gels were scanned using a Typhoon 9410 variable mode imager . The gel images were analyzed using ImageQuant  to obtain lane profiles. These profiles were then curve-fit with Gaussian curves using Origin  to precisely determine band position and intensity.MicroRNA consist of short RNA sequences that are typically 17–23 nt in length. Due to their short length, 5′ and/or 3′ adapter ligation is often used to label, capture, or lengthen the microRNA before downstream detection. A number of studies have suggested a pooled adapter approach to average out the intrinsic effects of ligation bias. However, by improving ligation reaction design and optimization methodology, we have developed a ligation based method that achieves high efficiency and low bias microRNA capture without the need for adapter pools.As shown in et al reported difficulties capturing miR-4803, miR-5183, and miR-567 while Hafner et al reported low capture efficiencies for miR-31, miR-712, and miR-338 et al reported that miR-106b could not be captured consistently under any of their experimental conditions. The final three microRNA were randomly selected . Each target microRNA was labeled with a Cy3 dye at the 5′ end to enable quantification of ligation efficiency under spiking conditions in total RNA. The reaction products were analyzed using denaturing PAGE, and the gels were scanned using a multimode imager. Image analysis was then used to obtain band positions and DNA quantity. Using this PAGE analysis method, we obtain excellent quantification and reproducibility. Quantification is linear from <5 amols to >10 pmols with an experiment to experiment CV of 10%  The first parameter we investigated was ligase type, as the ligases themselves likely have different intrinsic bias. T4 RNA ligase 2 truncated (T4 Rnl2 T) is a mutant enzyme that lacks the domain necessary for ATP incorporation, which should significantly reduce side product formation when used with pre-adenylated adapters in the absence of ATP T4 RNA ligase 2 truncated K227Q (T4 Rnl 2 TK) contains a point mutation that is designed to further reduce side product formation Methanobacterium thermoautotrophicum. This thermostable ligase is unable to incorporate ATP and works optimally at 65°C. Ligation at an elevated incubation temperature could serve to reduce bias from secondary structure interactions. Though the same adapters and microRNAs were used, a different pattern of ligation bias was seen. This likely arose from ligation preferences intrinsic to the ligase itself. In addition, despite the higher reaction temperature, neither ligation efficiency (30% AVG) nor bias (28% SD) was significantly improved over the T4 Rnl2 variants.Finally, we tried Thermostable 5′ App DNA/RNA Ligase (MthRnl) from New England Biolabs, which is a point mutant of RNA ligase isolated from Although T4 Rnl2 T had high ligation efficiency and low bias, the large amounts of background products complicate downstream processes and were deemed unacceptable. We chose to use T4 Rnl2 TK moving forward due to the belief that it would be easier to increase reaction efficiency and reduce bias than to suppress side product formation.Additives such as polyethylene glycol (PEG) Based on the initial results of When the microRNAs were spiked into idealized buffer conditions rather than total RNA, a different behavior was seen. Maximum capture efficiency was reached at lower PEG levels with miR-31 and miR-4803 behaving similarly again, reaching plateau at 20% PEG, and miR-155 reaching plateau at 15% PEG. The overall ligation efficiencies also increased significantly, particularly for miR-155. Total RNA likely contains inhibitors that prevent ligation from reaching completion such as: 1) RNA species that bind and sequester the microRNA, 2) RNA species that ligate competitively to the microRNA, 3) RNA species that ligate competitively to the adapter, 4) RNA species that ligate competitively to each other, and 5) inhibitors of the ligase. Though our and reaction design should minimize effects 2), 3), and 4), the large amount of background RNA can still occupy the ligase binding site even if the actual ligation cannot occur . In addition, the decrease in ligation efficiency seen at high PEG levels under spiking conditions is also absent or greatly reduced under idealized buffer conditions. It is unclear what the exact mechanism of PEG is and why different effects would be seen with and without total RNA spiking. Furthermore, the discrepancy in ligation behavior across the three microRNA also appears to decrease, with all three microRNA being well captured at 20% PEG.This data illustrates that the optimal assay parameters determined using 1) a single microRNA vs. a panel and 2) in idealized buffer vs. spiking conditions are quite different, highlighting the critical importance of optimization methodology and design. Most previous publications, as well as manufacturer protocols, have recommended 12–15% PEG. At this PEG level, only miR-155 is optimally captured under spiking conditions. In spiking conditions, ligation at 20–25% PEG is optimal whereas ligation at the recommended 15% PEG leads to very low efficiency. Of all the parameters investigated, PEG had the most dramatic effect on ligation bias and efficiency.Next we tested the effects of adapter concentration and ligase amount in conjunction with one another. Adapter concentration must be in excess to the ligated species to drive ligation forward. In addition to microRNA, samples often contain other RNA species such as mRNA, rRNA, and siRNA that can also be ligated. Having too few adapters will limit the ligation efficiency and increase bias. Having too large an excess of adapters will promote side product formation. In addition, large excesses of free adapters may also complicate downstream assay processes. The ligase amount also needs to be sufficient to obtain a high ligation efficiency in a reasonable amount of time. However, high ligase amounts can promote side product formation as well. Practically speaking, ligase is the most expensive reaction component and should be minimized to reduce costs.Adapter concentration and ligase amount were optimized by quantifying their effects on the capture of miR-31, both in presence and absence of total RNA. For the previous reactions, a 4 hour incubation was performed. We investigated whether this time was sufficient and whether it could be reduced. Using miR-31 as a model and the optimized protocol developed thus far, we tested incubation times from 30 minutes to 18 hours both in the presence and absence of total RNA. Secondary structure has been proposed as a main contributor to ligation bias We performed a second analysis using T4 Rnl2 TK while incubating at 4°C for 18 hours, 25°C for 4 hours, or 37°C for 4 hours. T4 Rnl2 TK is not thermostable and is denatured at 65°C so temperatures beyond 37°C were not tried. Incubation at 4°C allows for the reaction to proceed for an extended amount of time to compensate for decreased enzyme activity. Most enzymes recommend incubation at 25°C. We also performed ligation at 37°C to see if a modest increase in incubation temperature would have any effect on bias. The highest efficiency and lowest bias were seen at the 25°C condition where ligation efficiency across the 20 microRNA panel was 53.1% AVG ±13.7% SD. When the incubation temperature was decreased to 4°C, the ligation efficiency across the panel decreased to 23.6% AVG ±7.2% SD. Despite the increased ligation time (18 hours vs. 4 hours), the decreased enzyme activity at 4°C led to significantly reduced ligation efficiency. Interestingly, the capture efficiency CV for the 4°C condition (31% CV) was only slightly worse than at 25°C (26% CV), indicating that temperature does not have a large impact on bias across this range. The pattern in capture efficiency for each individual microRNA in the panel was fairly similar for the two conditions except that the capture efficiency at 25°C was 2× higher in most cases. When the incubation temperature was increased to 37°C, the ligation efficiency across the panel dropped to 34.7% AVG ±17.7% SD. The higher incubation temperature reduced the ligase activity but unexpectedly increased the bias over both the 4°C and 25°C conditions to nearly 51% CV. Incubation temperature likely results in a combination of effects on ligase activity, ligase degradation, and secondary structure formation that impact ligation efficiency in a complex manor. With a few exceptions, the pattern in ligation efficiency across the panel was generally similar to the 4°C and 25°C conditions.Thus far, we've investigated the use of reaction conditions to suppress the intrinsic bias of T4 Rnl2 TK. Adapter design can also play a large part in ligation bias due to primary sequence and/or secondary structure effects. However, the differences in microRNA capture efficiency seen across published studies illustrate the unpredictable nature of these interactions Encouraged by the previous results, we synthesized the SR1 and SR1-S adapters used by Zhuang Based on the optimized conditions determined in previous experiments, we performed ligation on the 20 microRNA panel using 300 units of T4 Rnl 2 TK, 25% PEG, 200 nM adapter incubated for 4 h at 25°C. Ligation was performed both in idealized buffer and in total RNA spiking conditions as shown in et al reported that miR-16 (92%), miR-21(66%), miR-155 (90%), and miR-567 (73%) were well captured while miR-31 (48%) and miR-338 (3%) were less well captured et alet al under any conditions, is now captured at 90% When compared to previous studies performed in idealized buffer background, a significant improvement in average capture efficiency and bias was seen. For example Hafner et el reported significantly reduced capture efficiencies under spiking conditions with efficiencies of 19%, 17%, 9%, 2% and 4% for let-7a, miR-31, miR-567, miR-4803, and miR-5183, respectively et alThese differences are further pronounced when comparing microRNA capture in realistic spiking conditions. The presence of total RNA likely reduces capture efficiency due to the aforementioned competitive reactions. Under realistic spiking conditions in presence of total RNA, our capture efficiency is slightly decreased and bias is slightly increased with a capture efficiency of 64% AVG ±17% SD across the panel. 16 of 20 microRNA have capture efficiencies greater than 50%. Only miR-155 had a low capture efficiency (27%). We experienced repeatability problems with miR-155, likely due to synthesis and degradation, as previously described. In comparison, Zhuang The experiments shown in Based on results across the 20 microRNA panel, we believe that ligation bias can be largely suppressed through suitable ligation optimization methodology, as opposed to solely focusing on adapter design. It is critical to design the reaction methodology to optimize for bias rather than just ligation efficiency. It is also critical to optimize using a panel of microRNA targets, rather than a single species, and to perform experiments both in idealized buffer and in realistic spiking conditions. Total RNA spiking has a large impact on the optimal reaction conditions, likely due to a number of competitive and inhibitive effects. Using this methodology, we were able to capture a panel of 20 microRNA at 86% AVG ±10% SD (12% CV) in buffer and 64%±16.5% SD (26% CV) in spiking conditions. This variability is below the typical variability in PCR amplification efficiency and means that expression profiles will likely vary very little due to this step. In contrast to previous methods that incorporated randomized adapter pools, the current method achieves low bias using only a single adapter sequence to capture all 20 microRNA. The elimination of adapter pools can greatly simplify downstream assay design. Key to this process was the use of very high PEG levels (25%) to suppress bias, particularly under total RNA spiking conditions. This PEG level is about double that which is typically recommended (10-15%). Molecular crowding by PEG has been demonstrated to affect both RNA enzyme activity Through this process we have also discovered that variability in the adapter and microRNA synthesis can lead to significant artifacts. The oligonucleotides that we ordered were always HPLC purified and had specified yields of >80%, yet we saw large variations in capture efficiency across different batches of adapters and microRNA targets. 3 microRNA targets that initially appeared poorly captured became consistently well captured after the targets were re-synthesized. However, miR-155 and, to a lesser degree miR-712, behaved increasingly erratically as the targets aged and required multiple re-synthesis rounds to obtain optimal results. Interestingly, all the other microRNA targets remained very stable over time. It is possible that some of the variability and inconsistencies seen in previous papers could be also attributed to synthesis issues. While the 20 microRNA panel used herein is not exhaustive of all microRNA species, we feel it is a good representative snapshot of what can be expected.File S1Table S1, Figures S1-S8.(DOCX)Click here for additional data file.