Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m6A or m5C modifications from MLV transcripts inhibits MLV replication in cis, and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors in trans. Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.The data presented in the present study demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m N6-methyladenosine (m6A), 5-methylcytosine (m5C), and 2′O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of  N6 position of adenosine (m6A), several other mRNA modifications, including cytidine methylation (m5C) and 2′O-methylation of the ribose moiety that forms part of all four ribonucleotides , have also been reported.Eukaryotic mRNAs are subject to a range of covalent modifications at the single nucleotide level, and it is now evident that these epitranscriptomic modifications can profoundly affect mRNA function –3. While–6A is the most intensively studied epitranscriptomic modification, and the protein complex responsible for m6A addition, or m6A “writer,” has been identified as a nuclear heterotrimer, consisting of the proteins METTL3, METTL14, and WTAP, that adds m6A to mRNA sites bearing the consensus sequence 5′-RA*C-3′, where R is a purine   gene expression and replication. Moreover, we further show that MLV gRNAs are also extensively epitrancriptomically modified, with m6A, m5C, and Nm residues all detected at levels that range from ∼9- to >20-fold higher than observed on cellular poly(A)+ RNA. Together, these observations are consistent with the hypothesis that sites of epitranscriptomic modification on viral mRNAs are under positive selection and suggest that many viruses may have independently evolved to use epitranscriptomic gene regulation as a mechanism to promote their replication and, hence, pathogenesis.Previously, we reported that m0 (SV40) –11, and lication , 13 and lication , 15. Morpression , and othpression . Consistlar mRNA . Here, wThe initial goal of this project was to quantify the epitranscriptomic RNA modifications present on MLV gRNA using ultrahigh-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) , 17. Thi6A (∼51 m6A residues per gRNA) and high levels of 2′O-methylated adenosine (Am), guanosine (Gm), and cytosine (Cm) , as well as a high level of m5C (∼13 residues per gRNA). These levels are considerably higher than previously reported for cellular poly(A)+ RNA + RNA is ∼0.24% of “A” residues, while ∼2.4% of all “A” residues in the MLV genome are m6A + RNA. We therefore conclude that m6A, m5C, Am, Gm, and Cm are all highly overrepresented on MLV gRNAs compared to the average cellular mRNA.To assess the purity of the MLV gRNA preparations, we performed transcriptome sequencing (RNA-seq) and then aligned the reads obtained first to the mouse genome and then to the MLV genome. As shown in (A)+ RNA  and quan are m6A . It was MLV gRNA . Similar1G) and 7-methylguanosine (m7G), the observed levels in the MLV genome are ∼10-fold higher than the very low levels detected on 3T3 poly(A)+ RNA. Similarly, 1-methyladenosine (m1A) was detected at an ∼20-fold higher level on MLV gRNAs than on cellular poly(A)+ RNA (0.20% versus 0.01%). While these three modified residues are all present at low levels on MLV gRNAs  or pseudouridine, we note that the UPLC-MS/MS method is less sensitive for detecting modified uridines at low concentrations of input RNA, as in this case, due to the comparatively inefficient ionization of uridine compared to other ribonucleosides. Nevertheless, our data do suggest that levels of Um in the MLV gRNA are <1nM, which equates to <20 Um residues per MLV gRNA.While our data identify the five epitranscriptomic modifications listed above as unusually prevalent on MLV gRNA, with >10 modified residues of each per gRNA, we also detected several other modified nucleotides at levels ranging from ∼2 to ∼4 residues per MLV genome  and D. ILV gRNAs , we notear mRNAs , thus no6A and m5C residues, we wished to map these residues not only on the gRNA but also on MLV RNAs expressed in infected 3T3 cells. For this purpose, we infected 3T3 cells with MLV virions rescued from the pNCS proviral clone  RNA fragments recovered, converted to cDNA, and subjected to deep sequencing. As may be observed in 6A peaks on the MLV gRNA, almost all of which were also observed on the MLV transcripts expressed in infected 3T3 cells. Consistent with the fact that all MLV virion RNAs are genome length, while ∼50% of the MLV transcripts expressed in infected cells are env mRNAs that have been spliced to remove the Gag and Pol open reading frames, we detected a 2- to 3-fold-lower level of m6A binding sites in the MLV gag and pol genes compared to the env gene and long terminal repeat (LTR), in the intracellular RNA sample  to measure the level of MLV gRNA isolated from the supernatant media of the transfected 293T cells and then normalized these data by qRT-PCR analysis of the level of cellular 7SL RNA, which is known to also be selectively packaged into MLV virions , as shown in 6A facilitates viral gene expression and replication for a range of distinct viruses, including HIV-1, influenza A virus, SV40, enterovirus 71, and KSHV + RNA isolated from human cells, with m5C (∼20× higher) and Nm (11× to 28× higher) being particularly enriched + RNA + RNA . Second,utations  and 3. WT3 cells . Finallyor HIV-1 , 9, we oor HIV-1 , and a sor HIV-1 . These Mor HIV-1 , 9, indinslation –10.ythdf2 gene (NP_663368) was PCR amplified from a cDNA library and cloned into the pLEX vector (puro) resistance gene, all driven by the cytomegalovirus immediate-early promoter. A previously described (NP_060225) expression plasmid was generated by PCR amplification of an NSUN2 cDNA that was then cloned into the pcDNA3.1 expression plasmid to generate pcDNA-FLAG-NSUN2. A mutant form of NSUN2 was generated by introducing the C271A mutation into pcDNA-FLAG-NSUN2. The NSUN2-C271A mutant spontaneously forms stable covalent bonds with target cytosines on RNA  and the second mutated to remove four m5C sites (Δm5C). Only silent mutations were introduced at these sites. Two DNA gBlocks were synthesized by IDT containing these silent mutations and cloned into pNCS to generate pNCS-Δm6A and pNCS-Δm5C, respectively. Mutations introduced into pNCS-Δm6A were as follows, counting from the start codon of Env, with introduced mutations indicated by lowercase letters: nt 354, 5′-GAAGAgCCTctcACCTCC-3′. Mutations introduced into pNCS-Δm5C are as follows, counting from the start codon of Gag, with introduced mutations again indicated in lowercase letters: nt 2898, 5′-GGtTTtTGTaGatTaTGGATt-3′; nt 3645, 5′-agtGCTCAGaGaGCTGAAtTGATAGCAtTgACt-3′; nt 4242, 5′-aGAACAtTaAAAAATATtACTGAGACtTGt-3′; and nt 4500, 5′-ATtTTtCCtAGGTTt-3′. Clone integrity was verified by Sanger sequencing.The pNCS MLV proviral expression vector has been described and was a gift from Stephen Goff . Two MLVTo generate infectious MLV, pNCS-based proviral expression vectors  were tra+ RNA purification, using cellulose oligo(dT) beads, and one round of rRNA depletion using a RiboZero Gold kit (Illumina). The bulk of the purified MLV RNA was then used for UPLC-MS/MS analysis of RNA modifications, while a small aliquot was retained for RNA-seq analysis, which was used to determine the purity of the MLV gRNA sample. RNA-seq was performed using the SMARTer stranded total RNA-Seq kit v2 (NEB) according to the manufacturer’s instructions.MLV virions were purified by a two-step method, as previously described . Briefly2 for 2 h at 37°C, followed by incubation with Antarctic phosphatase (NEB) for an additional 2 h at 37°C (+ RNA sample isolated from the producer 3T3 cell line.Nucleosides were generated from purified MLV RNA by nuclease P1 digestion (Sigma) in buffer containing 25 mM NaCl and 2.5 mM ZnCl at 37°C . Nucleos at 37°C , except 6A-seq and PA-m5C-seq were performed as previously described (–+ RNA was purified using oligo(dT) magnetic beads  and 10 μg of poly(A)+ RNA or virion gRNA was then used according to the previously reported PA-m6A-seq protocol  or m5C-specific  polyclonal antibody.PA-mescribed –10. Brieprotocol  using eiPlasmids expressing FLAG-tagged versions of the wild-type or C271A mutant form of murine NSUN2, or human A3G, were cotransfected with pNCS into 293T cells using PEI. At 72 h posttransfection, the supernatant media were passed through 0.45-μm-pore size filters, and virions were harvested by ultracentrifugation through a 20% sucrose cushion. Protein was extracted from the virion pellet in Laemmli buffer  before aProteins were extracted using Laemmli buffer, sonicated and denatured at 95°C for 10 min and then separated on Tris-glycine-SDS polyacrylamide gels (Invitrogen). After electrophoresis, proteins were transferred to a nitrocellulose membrane, and then blocked in 5% milk in PBS plus 0.1% Tween (PBS–0.1% Tween). Membranes were incubated in primary and secondary antibodies diluted in 5% milk in PBS–0.1% Tween for 1 h each and then washed in PBS–0.1% Tween. Each antibody was used at a 1:5,000 dilution. The antibody targeting MLV Gag has been described  and was To investigate the effect of reduced NSUN2 protein levels on MLV protein expression, RNAi was utilized to knockdown NSUN2 expression in 293T cells. An siRNA specific to NSUN2 (siNSUN2), or a control small interfering RNA , was transfected into 293T cells at a concentration of 25 pmol/ml using Lipofectamine RNAiMAX (Invitrogen). At 48 h posttransfection, cells underwent a second siRNA transfection and were then incubated for a further 24 h. Cells were then transfected with pNCS. At 72 h posttransfection, the cells were harvested for Western blot analysis.The lentiviral expression vectors pLEX-GFP and pLEX-YTHDF2 were transfected into 293T cells along with the packaging plasmids pMD2G  and pΔ8.74 . Media were changed at 24 h posttransfection. At 72 h posttransfection the supernatant media containing the lentiviral particles was harvested and passed through a 0.45-μm-pore size filter and overlaid on 3T3 cells . At 48 hpi, transduced 3T3 cells were selected by the addition of puromycin to the culture media. After a further 72 h, cells were single cell cloned, expanded, and assessed by Western blotting for FLAG-GFP or FLAG-YTHDF2 expression. A single clone was then selected to determine the effect of overexpression of YTHDF2 on MLV infection. The GFP- or YTHDF2-overexpressing cell lines were infected with MLV and, at 72 hpi, protein was harvested for Western blot analysis.TC method.Relative MLV gRNA expression levels were determined by qRT-PCR. The level of GAPDH  mRNA was used to normalize all cellular qRT-PCR experiments, while 7SL RNA, which is packaged by MLV virions , was useThe primers sequences for GAPDH mRNA detection were as follows: GAPDH forward: 5′-TGGGTGTGAACCATGAGAAG-3′; and GAPDH reverse, 5′-GATGGCATGGACTGTGGTC-3′. The primers used for MLV genomic RNA detection were as follows: MLV gag forward, 5′-AGGAATAACACAAGGGCCCA-3′; and MLV gag reverse, 5′-GGGTCCTCAGGGTCATAAGG-3′. The primers used for human 7SL RNA detection were as follows: 7SL forward, 5’GTGCGGACACCCGATCGGCA-3′; and 7SL reverse, 5′-TGAGGCTGGAGGATCGCTTGAG-3′.Read alignments were performed using Bowtie . Reads wThe raw sequencing data obtained by RNA-seq have been submitted to the NCBI expression omnibus and are available under GenBank accession number GSE129302.


Functionalized copolymers were synthesized and are proposed as hosts of RNA. The copolymers are based on carboxymethyl cellulose and poly-(ethylene glycol)-OH. These copolymers were functionalized with two amino acids, either lysine or histidine, through amide bond formation. The functionalized copolymer was then used to adsorb ribosomal RNA. The RNA loading was based on the nature of the amino acid functionalization of the copolymer. The array of RNA-copolymers was observed to be soft sphere-like, where the density of spheres was a function of the molecular weight of the carboxymethyl cellulose and the nature of the amino acid. Such RNA-copolymer systems are very sensitive to changes in pH. Nanoparticles are already being developed as effective carriers of drugs and gene delivery to target regions of the body that were previously hard to access using traditional drug formulation methods. Through manipulation of their elemental composition, charge, size, and chemical functionalization, it may be possible to target particles to specific organs .Gene therapy uses nucleic acids as a powerful tool to cure genetic deficiencies or diseases that currently have no cure. This includes a number of brain diseases (Alzheimer’s and Parkinson’s disease), viral infections and cancer , 3. TherComplex DNA nanostructures can be functionalized with different biomolecules or nanomaterials, such as different nanowires, nanoparticles, organic molecules, peptides or proteins, to combine the properties of both DNA and nanomaterials for achieving the aimed functionality . NucleicGiven their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery , 15. PolFurthermore, a polymer coating with polyethylene glycol (PEG) provides protection from uptake by human monocytes. Surface modification of nanoparticles with PEG prolongs the circulation time of the nanoparticles, temporarily avoiding the mononuclear phagocyte system . This poThe nucleic acid nanocarriers are usually captured by the cells and internalized via an endocytic uptake mechanism ; they shN-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), phosphate-buffered saline (PBS), sodium hydroxide, l-lysine, and l-histidine monohydrochloride monohydrate were purchased from Sigma-Aldrich. Quant-iT RiboGreen RNA Reagent was purchased from Fisher Scientific, Inc.Carboxymethyl cellulose  was obtained from CPkelco. Hydrochloric acid, acetonitrile, diethyl ether, methanol, poly(ethylene glycol) methyl ether , 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (EDC HCl), Copolymers of carboxymethyl cellulose-polyethylene glycol were synthesized as follows: CMC (1.2 mmol acid) was dissolved in water (30 ml) and pH adjusted to 5 (HCl solution), to which EDC HCl (1.4 mmol), NHS (1.4 mmol), and DMAP (0.2 mmol) were added. The solution was stirred for 1 h at room temperature under dark conditions. After an hour, PEG-OH (0.36 mmol) reagent was added to the CMC-PEG solution. After overnight reaction, the product was precipitated with methanol and washed with a mixture of acetonitrile and diethyl ether (90:10). It was subsequently dissolved in water and precipitated in methanol, filtered and dried at room temperature. EDC HCl, DMAP, and NHS were dissolved in water (2 ml), and mPEG-OH was dissolved in water (4 ml). Two copolymers were synthesized and labeled 7CMC-PEG or 3CMC-PEG according to the precursor of CMC utilized in synthesis , NHS (1.4 mmol), and DMAP (0.2 mmol) were added. Lastly, after 1 h, 0.36 mmol of amino acid, histidine or lysine was added  and Tween 80 solution, was prepared by dissolving 3 mg of copolymer in 2.5 ml of sterile solution. The solution at 6% was prepared by diluting Tween 80 in sterile deionized water. The samples were sonicated for 2 h and centrifuged at 12,000 rpm for 30 min. The nanoparticles were suspended in sterile deionized water, and the size of the nanoparticles was measured by dynamic light scattering.13C nuclear magnetic resonance (CP MAS NMR) spectroscopies. FTIR spectra (ATR mode) of samples were acquired using a Bruker Alpha FTIR spectrometer. The spectra were recorded at a resolution of 4 cm−1 in the spectral window 4000–400 cm−1. 13C CP MAS NMR spectra were obtained at a Larmor frequency of 75.4 MHz using a Bruker Avance 300 spectrometer equipped with a 4 mm cross-polarization (CP) MAS probe. The samples were spun at a rate of 5 kHz. Spectra were recorded using a contact time of 5 ms and π/2 pulses of 5 µs. The chemical shifts were referenced to TMS.The structures of the materials were characterized by infrared (FTIR) and The thermal properties of the materials were characterized by thermogravimetric analysis (TGA). TGA profiles were obtained using a TGA 500 system. Samples were heated at a heating rate of 10 °C/min from 30 to 900 °C under a nitrogen atmosphere with a flow rate of 1.0 ml/min.The average hydrodynamic diameter, zeta potential and polydispersity index (PDI) of the nanoparticles were measured by dynamic light scattering (DLS) analysis using a Zeta sizer Nano ZS90 . All measurements were carried out at 25 °C, at a detection angle of 90° (for size and PDI) and 120° for zeta potential.The nanoparticles were suspended in ribosomal RNA solution (2 ml with a 100 ng/ml concentration). The solution was incubated at 8 °C for 10 min. Then, the mixture was centrifuged at 12,000 rpm for 30 min to pellet the nanoparticles. The supernatant was removed and analyzed by the Quant-iT RiboGreen assay (Invitrogen). The amount of ribosomal RNA in the supernatant (w) was then subtracted from the total amount of ribosomal RNA added . The experiments were conducted in triplicate. The percentage efficiency (E) of ribosomal RNA entrapment to the nanoparticles was calculated using the following formula:Measurement data were expressed as the mean ± standard deviation; the comparison between two groups was analyzed using a two-sample t-test. Statistical analysis was performed using GraphPad Prism ver. 5.0 . P values ≤ 0.05 were considered statistically significant.The nanoparticles loaded with ribosomal RNA were suspended in sterile phosphate-buffered saline (PBS) at a pH of 5, 6 and 7.4. The pH of the PBS was modified using 0.1 M NaOH or 0.1 M HCl. The mixture was then sonicated for 1 h. The nanoparticles were stored at 8 °C for periods as long as 1 week. The particle size and zeta potential of polymer complexes were measured at various time points .2I(q) against q. Lastly, the fractal dimension of the scattering objects was estimated from the slope of the curve log I(q) vs log(q) , of the RNA-copolymers. The modified Kratky plots have a main peak as well as a number of secondary peaks, and these profiles indicate that scattered objects have a globular shape. No significant differences concerning the shape of scattered particles were observed among the copolymers under study. The copolymer particle sizes were estimated from the SAXS data curves by fitting to a poly-disperse system of spheres. Table Figure PEGylated link polymers of carboxymethyl cellulose were effectively synthesized by EDC-NHS reaction and coupling amino acids onto the side groups of CMC-PEG copolymers. Copolymers were formed as stable soft spheres. Functionalization with lysine and histidine is crucial to obtaining copolymers prone to adsorbing ribosomal RNA; the type of amino acid determines the electric charge on the external surface of the spherical copolymer where RNA is incorporated. RNA-copolymers are partially stable as the amino acid-RNA interactions vary with pH, which could facilitate the release of RNA; this is a promising result because this RNA-copolymer system could then be used for RNA delivery.


Picornavirales specifically package their genomic RNAs is poorly understood. Research with monopartite members of the order, such as poliovirus, indicated that packaging is linked to replication, although the presence of “packaging signals” along the length of the viral RNA has also been suggested. Thanks to the bipartite nature of the CPMV genome, which allows the manipulation of RNA-1 without modifying RNA-2, we show here that this specificity is due to a functional link between the two processes of viral replication and encapsidation. This has important implications for our understanding of the fundamental molecular biology of Picornavirales and opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.The mechanism whereby members of the order  Picornavirales, we have taken advantage of the bipartite nature of a plant-infecting member of this order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (empty virus-like particles [eVLPs]) can be generated by transiently coexpressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24,000-molecular-weight (24K) protease, in the absence of the replication machinery . We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effects of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication.To elucidate the linkage between replication and encapsidation in IMPORTANCE The mechanism whereby members of the order Picornavirales specifically package their genomic RNAs is poorly understood. Research with monopartite members of the order, such as poliovirus, indicated that packaging is linked to replication, although the presence of “packaging signals” along the length of the viral RNA has also been suggested. Thanks to the bipartite nature of the CPMV genome, which allows the manipulation of RNA-1 without modifying RNA-2, we show here that this specificity is due to a functional link between the two processes of viral replication and encapsidation. This has important implications for our understanding of the fundamental molecular biology of Picornavirales and opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies. Picornavirales are a large order of single-stranded positive-sense viruses that form icosahedral particles. They have genomes consisting of either one  or two (cowpea mosaic virus [CPMV]) strands of positive-sense, single-stranded RNA that are encapsidated within protein capsids of 28 to 30 nm in diameter. The viral capsids exhibit pseudo-T=3 (P=3) icosahedral symmetry, comprising 60 asymmetric units, each with 3 distinct β-barrel domains. These domains can either remain as a single polypeptide or be processed to give two or three distinct coat protein subtypes; in some cases, additional processing occurs to generate small proteins which are found within capsids. Because of their importance as pathogens and their relatively simple, nonenveloped capsids, structures of the Picornavirales have been extensively studied by both X-ray crystallography and cryo-electron microscopy . As a result, infections with CPMV produce three types of particles with identical protein compositions: top (T), middle (M), and bottom (B). These particles contain, respectively, no RNA, exclusively RNA-2, and exclusively RNA-1  via the transient expression of the CPMV coat protein precursor VP60 and RNA-1-derived proteins containing the CPMV 24K protease domain needed for its correct processing  RNA-1 to be encapsidated in coexpressed CPMV VLPs is due to its ability to self-replicate, plasmids encoding two different versions of CPMV RNA-1 were utilized: pBinPS1NT, containing an infectious full-length version of RNA-1 , and pBiNicotiana benthamiana leaves with either pEAQ-HT-VP60, a construct that encodes both the precursor of the L and S proteins (VP60) and the P19 suppressor of silencing , with negligible levels of contaminating host proteins being detected. In each case, the S coat protein is resolved into 2 bands as a result of the presence or absence of the labile C terminus , in the presence or absence of pBinPS1NT. Northern blot analysis of RNA extracted from the leaves at 1, 4, and 5 days postinfiltration (dpi) showed the clear presence of full-length RNA-2 at all times, even in the absence of RNA-1 . It seemThe above-described results strongly suggest that a replication-competent version of RNA-1 is required for encapsidation of both RNA-1 and RNA-2 but do not provide evidence for the direct involvement of the RNA-1-encoded replication-associated proteins in the process. To investigate this, point mutants within the region of RNA-1 encoding the 87K replicase were generated to create a replication-deficient version of RNA-1 with minimal sequence changes. The 87K replicase protein of RNA-1 contains a GDD motif, a motif that is highly conserved across numerous viral RNA polymerases and is thought to be critical for replicase function through its interaction with divalent cations . Thus, pEscherichia coli and suffer mutations and sequence rearrangements (Petunia × hybrida (GenBank accession number AJ224165.1), and the four introns that were chosen would introduce frameshift mutations into the coding sequence of RNA-1 if they were not correctly spliced; the insertion sites were chosen as described previously , while in the second, it was mutated to AAA (pEAQ-RNA-1-Int-AAA).Initial attempts to create mutants in the GDD motif using pBinPS1NT were unsuccessful, as plasmids containing full-length RNA-1 are unstable in ngements . To allengements , 35. Theeviously , with thN. benthamiana plants in the presence of pEAQ-RNA-2. After 30 days, the only plants to show symptoms characteristic of a systemic CPMV infection (curling and mottled yellowing of the upper leaves) were those coinfiltrated with pEAQ-RNA-1-Int and pEAQ-RNA-2  mutations (HT (with the AUG codons eliminated) was coexpressed with pBinPS1NT for provision of RNA-1 and pEAQ-HT-VP60-24K to guarantee consistent quantities of processed capsid protein in both samples. RNA was extracted from particles purified from the infiltrated leaves, and equal amounts of nucleic acid were separated on a denaturing agarose gel  to excise a part of the RNA-2 coding sequence and religate the ends to yield XhoI-RNA-2 (∼3.3 kbp) and MfeI-RNA-2 (1.5 kbp). In addition to this, a construct (green fluorescent protein [GFP]) was generated by replacing the entire RNA-2 coding sequence (from the AUG at position 512) with the coding sequence for GFP , leaving37–HT-VP60-24K in the presence or absence of RNA-1  and HT-24K (∼1.3 kbp) from the HT-VP60-24K construct, suggesting that these RNAs are also encapsidated to a minor extent. This low-level encapsidation is unsurprising, since HT mutations do not abolish encapsidation and that the coding region is irrelevant. Northern blotting experiments confirmed the identities of these RNAs (The bands corresponding to RNA-2 truncation products at ∼3.3 kbp (XhoI) and ∼1.5 kbp (MfeI) and GFP (∼1.5 kbp) were of an intensity similar to those of the wild-type RNA-2 band in the positive control . Thus, tese RNAs .HT mutations (HT-GFP) or an entirely synthetic 5′ UTR (S-GFP), a 64-nucleotide sequence designed to be an efficient translational enhancer but without any sequence similarity to CPMV RNA-2 or any other viral RNA . The expression of S-GFP was verified by Agrobacterium-mediated transient expression in N. benthamiana and visualization of GFP fluorescence in the infiltrated leaves under UV light , with the HT construct being encapsidated to a much lesser extent than the construct with a wild-type 5′ UTR. No signal was seen at the position of S-GFP (∼1 kbp), regardless of whether the particles were produced with or without RNA-1. The gel prior to transfer shows that RNA at a size corresponding to RNA-1 was present in all three samples in which pBinPS1NT was coinfiltrated, confirming the occurrence of viral replication and the production of encapsidation-competent particles in these samples. Because leaves expressing S-GFP show GFP fluorescence , no RNA was detected within particles. In contrast, a mutation that reduced but did not abolish the replication of RNA-1 (RNA-1-Int-GAD) concomitantly reduced the amount of RNA encapsidated. Similarly, the HT mutations in the 5′ UTR of RNA-2 were found to knock down, but not abolish, replication of RNA-2, with a parallel knockdown effect on encapsidation. Finally, it was found that truncations within the coding region of RNA-2, and, indeed, even the complete substitution of the RNA-2 open reading frame, did not abolish packaging. Taken together, these data demonstrate that packaging of both RNA-1 and RNA-2 requires replication-competent RNA-1 and that the 5′ UTRs of both RNA-1 and RNA-2 play a crucial role in replication and packaging.The work presented here establishes the linkage between the processes of viral replication and RNA packaging in CPMV and, by extension, other members of the order Picornavirales, suggested that the aspartate residues of the GDD motif are usually essential for replicase function, and only mutations in the glycine residue are typically tolerated . In this case, it seems likely that low-level viral replication will lead to a low level of reversion to the wild-type sequence. This would result in the expression of a fully functional revertant 87K replicase, but the revertant RNA-1-Int RNA itself would have no selective advantage over RNA still carrying the GAD mutation in terms of its ability to be replicated. It would therefore remain at a constant low level within the RNA population of the cell and would not reach levels detectable by the methods used in this study. Nevertheless, the fact that only replicating RNA can be packaged provides a means for ensuring that only viral RNAs are found in particles, which explains why CPMV capsids produced in the absence of replication appear to be devoid of any nucleic acid , where, in the absence of the authentic genomic RNA, significant quantities of host RNA are encapsidated . It is possible that the colocalization of newly replicated RNA molecules and newly translated coat protein molecules in the cytopathological structures associated with virus infection , 45 simpPicornavirales. Moreover, the observation that only replication-competent versions of CPMV RNA-2 in which the open reading frame has been replaced with GFP can be packaged suggests that the linkage could be used to specifically incorporate heterologous RNAs expressed in plant cells within particles. The ability to efficiently encapsidate specific plant-expressed RNAs has profound implications for the future development of CPMV for bionanotechnological applications.Whatever its mechanism, the coupling of RNA replication to encapsidation provides a route to the selective packaging of viral RNAs in the Petunia × hybrida PSK7 gene (GenBank accession number AJ224165.1), according to a previously described protocol (X00206.1)  terminator sequences used in the pEAQ-HT expression vector (GenBank accession number GQ497234.1)  . The int97234.1) , flankedHT, directing the expression of RNA-2-HT, was created by amplifying the coding sequence of RNA-2 starting from the AUG at position 512; this sequence was inserted into pEAQ-HT using the restriction sites AgeI and StuI in its polylinker, which places the coding region between the CPMV RNA-2 UTRs with the HT mutations (mutations of the AUG codons in the 5′ UTR at positions 115 and 161) in the 5′ UTR , but its sequence is TTTAAGAGACGCAACCACAACGCTCTAACGCAATCAATCTACATTATATTAAACGTCTCTAAAA.Full-length wild-type CPMV RNA-2 was expressed from either pBinPS2NT  or pEAQ-e 5′ UTR . GFP wase 5′ UTR  into pEAQ-HT-GFP . GFP witHT-VP60  for VP60 and/or 24K. In all cases, when a DNA construct is being referred to, its full name, including the name of the plasmid, is given. When transcribed RNA is discussed, the designation of the plasmid is omitted.To provide a source of the CPMV capsid proteins in the absence of or in addition to RNA-2, pEAQ-HT-VP60  or pEAQeHT-VP60  was usedE. coli one shot TOP10 cells (Life Technologies) grown at 37°C in LB medium containing 50 μg/ml kanamycin for plasmid selection. For intron-containing RNA-1 constructs, E. coli cells were grown at 28°C in LB medium containing kanamycin and glucose . Colonies were verified by colony PCR and sequence analysis (Eurofins Scientific). Plasmids having the correct sequence were transformed into electrocompetent Agrobacterium tumefaciens strain LBA4404 and used for transient expression in Nicotiana benthamiana plants via agroinfiltration using a needleless syringe as described previously (All gene constructs were cloned and maintained in eviously . Plants Particles were purified from infiltrated leaves by polyethylene glycol (PEG) precipitation and pelleting as described previously . The prog for 15 to 30 min at 8°C to recover the RNA pellets. The pellets were washed with 70% (vol/vol) ice-cold ethanol and centrifuged again for 10 min, the ethanol wash was removed, and the pellets were allowed to air dry before being resuspended in a small volume of sterile nuclease-free water.RNA was extracted from equal amounts of purified particles (as determined by protein contents of purified particle preparations). The purified particles were first treated with micrococcal nuclease (NEB) (4 μl of enzyme in 50-μl reaction mixtures) at 37°C for 15 min. The reaction was terminated by adding NETS buffer  supplemented with EGTA . The nuclease-treated particles were then heated to 65°C for 10 min, and the nucleic acid was extracted by phenol-chloroform extraction as previously described . RNA wasTaq high-fidelity DNA polymerase (Life Technologies). The reverse-transcribed and amplified DNA thus generated was sequenced (Eurofins Scientific), and the resulting sequences were analyzed using Vector NTI Advance 11.5.3.RNA extracted from equal amounts of particles (as measured by protein content) was analyzed by electrophoresis under denaturing conditions in formaldehyde-containing agarose gels, in the presence of ethidium bromide . For NorNicotiana benthamiana (Taq qPCR master mix (Promega) and loaded in Thermofast white 96-well qPCR plates (Thermo Fisher Scientific). qRT-PCRs were carried out in a Bio-Rad CFX96 real-time C1000 touch thermal cycler, and Bio-Rad CFX Manager analysis software was used to automate analysis of the data using the 2−ΔΔCT method, using the four reference genes described above. For both RNA-1 and RNA-2 quantification, quantification was normalized to the value obtained with the RNA-1-32E-containing sample. The efficiency of all primer pairs was analyzed using pooled cDNA from infiltrated leaf tissue to confirm that the amplification factor value for each pair was between 1.8 and 2.2. The primers used and their sequences are available upon request.For qRT-PCR, total leaf RNA from agroinfiltrated leaves from three experimental repeats was purified using the Qiagen RNeasy plant minikit (including the DNase I digestion step) and then further treated with Turbo DNase (Thermo Fisher Scientific) using the thorough treatment protocol recommended by the manufacturer. The selected reference genes were PP2A, FBox, and L23, which have been previously identified as robust reference genes for qRT-PCR in virus-infected thamiana , along wThe authors agree that any materials and data that are reasonably requested by others are available from a publicly accessible collection or will be made available in a timely fashion, at reasonable cost, and in limited quantities to members of the scientific community for noncommercial purposes. The authors guarantee that they have the authority to comply with this ASM policy either directly or by means of material transfer agreements.


Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA ‘seed’ region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2′-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2′-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2′-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.CRISPR  endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar–phosphate backbone of  Streptococcus pyogenes (S. pyogenes crRNA is complementary to target DNA at its 5′ guide and base-pairs with a trans-activating crRNA (tracrRNA) via its 3′ repeat sequence. The tracrRNA uses 3′ stem-loops to bind Cas9 while the crRNA makes several direct Cas9 contacts, including six predicted 2′-hydroxyl contacts  where the crRNA and tracrRNA are artificially fused together  is an adaptive immune defence system that evolved to combat foreign invading nucleic acids in bacteria and archaea . Their cpyogenes ,9. The Ss Figure   repeat region is compatible with a broader array of modifications, including DNA, 2′F-ANA and 2′,5′-RNA. Despite strong A-form conformational preference, relatively bulky modifications such as C2′- or C4′- of Cas9 ,33,34. C2Cl2 for 110 s. Capping of failure sequences was achieved with acetic anhydride in tetrahydrofuran (THF) and 16% N-methylimidazole in THF. Oxidation was done using 0.1M I2 in 1:2:10 pyridine:water:THF. Coupling times were 10–15 min for RNA, 2′F-ANA, 2′F-RNA, 2′F,4′OMe-RNA, 2′,4′-diOMe-RNA and LNA phosphoramidites. Mixed sugar modifications were prepared using premixed 1:1 equivalents of RNA 2′-amidite with RNA 3′-amidite, or 1:1 equivalents of RNA 2′-amidite or DNA 3′-amidite, or 1:1 equivalents of RNA 2′-amidite with 2′F-RNA 3′-amidite. This 1:1 ratio results in ∼0.77:1 incorporation of 2′-amidite to 3′-amidite. Deprotection and cleavage from the solid support was accomplished with either 3:1 NH4OH:EtOH for 48 h at room temperature (RT), or at 55°C for 16 h. Oligonucleotides containing RNA were synthesized with standard 2′-TBDMS phosphoramidites, and desilylation was achieved with either neat triethylamine trihydrofluoride for 48 h at RT, or with triethylamine trihydrofluoride/N-methyl pyrrolidone/triethylamine (1.5:0.75:1 by volume) for 2.5 h at 65°C. Crude oligonucleotides were purified by anion exchange HPLC on an Agilent 1200 Series Instrument using a Protein-Pak DEAE 5PW column (7.5 × 75 mm) at a flow rate of 1 ml/min. The gradient was 0–24% of 1 M LiClO4 over 30 min at 60°C. Samples were desalted on NAP-25 desalting columns according to manufacturer protocol. Modified crRNAs were prepared for RNP assembly by heating to 95°C then placing on ice to prevent formation of stable secondary structures.Standard phosphoramidite solid-phase synthesis conditions were used for the synthesis of all modified and unmodified oligonucleotides. Syntheses were performed on an Applied Biosystems 3400 or Expedite DNA Synthesizer at a 1 micromole scale using Unylink CPG support (ChemGenes). All phosphoramidites were prepared as 0.15 M solutions in acetonitrile (ACN), except DNA, which was prepared as 0.1 M solutions. 5-Ethylthiotetrazole (0.25 M in ACN) was used to activate phosphoramidites for coupling. Detritylations were accomplished with 3% trichloroacetic acid in CHin vitro transcription with DNA templates synthesized by IDT. Single-stranded DNA templates were annealed to T7 promoter oligo to generate double-stranded promoter regions, which support in vitro transcription by T7 RNA polymerase. In vitro transcriptions were performed by standard protocols for 2 h. Briefly, reactions contained purified T7 RNA polymerase (4 μM), 30 mM Tris (at pH 7.9), 12.5 mM NaCl, 40 mM MgCl2, 2% PEG 8000, 0.05% Triton X-100, 2 mM spermidine and 2.5 μM T7-DNA template. Afterward, the DNA template was degraded by DNase I treatment. Reactions were phenol–chloroform extracted and gel-purified from denaturing polyacrylamide gels. Purified RNA was refolded by heating to 95°C for 5 min in a heating block followed by slow cooling the block to RT (∼40 min). RNA was quantified by measuring absorbance at 260 nm and calculated extinction coefficients using nearest neighbor approximations and Beer’s Law.DNA oligonucleotides and DNA–RNA chimeric oligonucleotides were synthesized by Integrated DNA Technologies (IDT). Chimeric crRNAs were prepared for RNP assembly by heating to 95°C then placing on ice to prevent formation of stable secondary structures. Cas9 tracrRNAs were prepared by T7 RNA polymerase SpCas9 (simply referred to as Cas9) with a C-terminal fusion of a nuclear localization signal (NLS) and a 6x-Histidine tag (pET-Cas9-NLS-6xHis) was obtained from Addgene (62933). A dead Cas9 (dCas9) version was prepared by performing site-directed mutagenesis on this plasmid to generate H840A and D10A mutations (pET-dCas9-NLS-6xHis). Cas9 proteins were prepared similar to that previously described (−1 cm−1) and Beer’s law.Plasmid encoding an escribed . BrieflyIn vitro cleavage assays were performed as previously described (2) supplemented with 0.1 mg/ml of purified yeast tRNA. The concentration of tracrRNA was purposely set as the limiting component of the RNP complex and used to predict final RNP concentration. Molar excess of Cas9 and crRNA will ensure complete assembly of tracrRNA into RNP complexes and does not result in aggregation at concentrations used in our assays . Error bars for all quantified data represent experimental replicates, not technical replicates. Sample size was selected based on the expectation that three or more replicates will be representative of typical in vitro assay conditions.escribed . Linearir assays . StandarA total of 100 pmols of antisense DNA target strand was radiolabeled with [γ- 32P]-ATP using T4 polynucleotide kinase following the manufacturer’s recommended enzyme protocol (Thermo Fisher). Reactions were phenol-chloroform extracted and radiolabeled DNA was gel-purified on 15% denaturing polyacrylamide gels by the crush-and-soak method. Gel-purified radiolabeled RNA and DNA was quantified by scintillation counting.For target binding by Cas9 RNP complexes, radiolabeled duplex target DNA (1000 cpm/reaction) was combined with increasing concentrations of a pre-assembled dCas9–tracrRNA–crRNA complex in a final reaction of 20 μl 1× cleavage buffer and 0.1 mg/ml tRNA. After incubation at 37°C for 15 min, reactions were vacuum filtered over nitrocellulose membrane  using a 96-well dot blot apparatus. Wells were washed twice with 200 μl of 1× cleavage buffer. Membrane was then removed and washed with 1× PBS solution three times and air dried at RT. Binding of radiolabeled DNA was then visualized by GE Typhoon phosphorimager. Spots were quantified with ImageQuant software, values plotted in Prism (GraphPad) and data fit to a one-site binding hyperbola equation.2). Samples were melted in a Cary 400 UV/Vis spectrophotometer at a ramp rate of 1°C/min while UV absorbance at 280 nm was collected every 1 min. Experiments were repeated in duplicate. Melting temperatures were determined using Van’t Hoff calculations and error determined by standard error of the mean using two experimental replicates for each sample. Melt data was plotted using Prism (GraphPad) software.Thermal denaturation was as previously described . Cas9 alSpCas9 were a kind gift from Wen Xue  . At least 20 000 events were collected and analyzed by Accuri CFlow Plus software. The cells were first gated based on forward and side scattering (FSC-A/SSC-A) to remove cell debris, then gated to select single cells (FSC-H/FSC-A). At last, cells were gated to select EGFP positive cells. The quadrant gate was established using the signal from non-EGFP expressing control cells. Untreated HEK 293T cells expressing EGFP and Cas9 contained ∼6% non-fluorescent cells. The average from four replicates was used for background subtraction to determine the extent of cell-based editing after treatment.S. pyogenes CRISPR-Cas9 to probe the role of the guide RNA in enzyme activity  y Figure . As oppoy Figure , dual RNy Figure ,37. Givey Figure . To faciy Figure . For thiy Figure . To isol plasmid  32)..S. pyogeTo establish a baseline for modification effects, we completely modified a native crRNA (crTR1) to 2′F-RNA (crTR2), a modification that strongly mimics RNA sugar pucker but cannot donate hydrogen bonds  Figure . A DNA–D35 (or 3.44 × 1010) isomers. A similar isomeric effect is observed with phosphorothioate linkages, which has not been a major hurdle for their use in development of FDA-approved drugs like Nusinersen , the 2′ epimer of 2′F-RNA, is known to acquire a C2′/rm helix ,46, whicrm helix ,11. We trm helix , it is lWithin the crRNA guide region is a ‘seed’ sequence where crRNA pairing to a target DNA is nucleated . PreviouBased on the above results, we tested a variety of ‘altimers’ where two or more modifications are alternated in a pattern . Altimerin vitro enzyme activity by Cas9  and 2′F-4′-Cα-OMe RNA (crTR46) (endo) sugar pucker previously reported for 2′,4′-di-Cα-OMe and 2′F-4′-Cα-OMe  with 2′F-RNA (crTR41), 2′,5′-RNA (crTR42), 2′F-ANA (crTR43), DNA (crTR44) (to distinguish thymine versus uracil effects), 2′,4′-di-Cα-(crTR46) . Other t′-Cα-OMe ,50,51. MOMe (crTR50), or 2′F-4′-Cα-OMe (crTR51) also resulted in no activity. Thus, RNA was unable to offset the unfavorable effects of these modifications. In contrast, combining RNA with 2′F-RNA (crTR52 and crTR53) and LNA (crTR54), a 2′-4′ bridging modification that locks C3′-endo sugar pucker 2′F-ANA was well tolerated in the tracr-pairing region, as long as a few of the first 5′ nucleotides were DNA (crTR13 and crTR59). However, placement of 2′F-ANA at the second position  in the tracr-pairing region (crTR60) abrogated activity. Combined with the activity of 2′F-ANA-containing crRNAs crTR12, crTR13, crTR25, crTR27 and crTR28, this result suggests that one or more positions closest to the seed must assume A-form structure, either through conformational predisposition or flexibility. DNA may temper the formation of A/B junctions or rigid versus flexible A-form transitions between the seed and tracr-pairing region. Placement of 2′F-ANA outside of the seed but still in the guide region (crTR61 and crTR62) was also detrimental to activity. Conversely, placing 2′F-RNA in the same guide and tracr-pairing positions resulted in robust activity (crTR63 and crTR64). These results reinforce a strong preference for A-form helical structure in the guide region, especially the seed sequence and a greater tolerance for modification in most of the tracr-pairing region. This difference may derive from hybridization of the crRNA 5′ guide region to a DNA target versus pairing of the 3′ region to a tracrRNA partner.OMe (crTR67), 2′,4′-di-Cα-OMe (crTR68) and even 2′F-ANA (crTR69) were well-tolerated. The incorporation of 2′-O-methyl, a known RNA conformational mimic  of 44°C. Upon assembly with a tracrRNA, the Tm increased to 46.6°C. Higher order assembly with native crRNA (crTR1) resulted in an additional increase in the Tm, up to 49.5°C. We selected a handful of representative crRNAs to determine their impact on RNP stability. Two crRNAs that provided high enzyme activity, crTR2 and crTR35, fell into a high Tm regime like native crRNA. One exception was crTR3, composed entirely of DNA. This modified oligonucleotide seems to assemble stable RNP complexes but does not support catalysis . However, crTR39, with 2′F-ANA in the seed, also fell into the intermediate Tm range. This result and the Tm of crTR3 illustrate the modular nature of crRNAs where tracrRNA-pairing might support assembly, yet the guide cannot support catalysis. Only crTR4, composed entirely of 2′-5′ linkages, did not increase RNP Tm. This crRNA may be unable to efficiently assemble a stable RNP complex with Cas9-tracrRNA, explaining its lack of activity.To rationalize the effect of modifications, we assembled Cas9 RNP complexes and determined their thermal stability Figure  and theiatalysis . crRNAs The ability of these chemically modified crRNAs to stably engage target DNA mirrored the observations from RNP stability Figure . Native in vitro cleavage assays as before but using a linearized plasmid harboring the EGFP gene as substrate  or 2′F-RNA and DNA (crEG6) into HEK 293T cells stably expressing EGFP and Cas9. These modified guides target EGFP and should result in loss of EGFP expression if gene editing was successful. Editing was measured by flow cytometry five days after transfection. Despite high activity y Figure . A receny Figure .in vitro in Figure To focus on guides that might reveal the impact of chemical modifications at these positions, we generated a new set of modified crRNAs (crEG7-crEG12) against the same EGFP sequence. In this case, however, only two predicted 2′-hydroxyl contact positions were modified. These modified crRNAs are similar to a subset tested In vitro cleavage revealed that these crRNAs either supported high activity , moderate activity (crEG8), or no activity (crEG11)  Figure . All exco Figure . These rO-methyl, 2′F-RNA, and phosphorothioate (PS) or thiophosphonoacetate linkages at the termini  revealed a clear reduction in editing efficiency for all modification types tested. Thus, our results further emphasize a disconnect between the intrinsic biochemical activity of Cas9 and its ability to induce gene editing in cells that centers on a handful of 2′-hydroxyl contacts with the guide RNA. While several mechanistic causes are possible, the difference in substrate complexity between in vitro (plasmid DNA) and cell-based editing (chromatinized DNA) experiments is evident. Characterizing the source of discrepancy between in vitro and cell-based activity should provide a path forward for the generation of CRISPR-Cas9 enzymes that can utilize completely modified guides with high efficiency for gene editing applications in the future.Previous reports have suggested a difficulty in replacing predicted Cas9–2′-hydroxyl contacts with chemically modified nucleotides when trying to preserve gene editing activity in cells ,28,31. Oin vitro, pose restrictions to highly efficient gene editing by fully modified guides (This study establishes A-form helical structure and steric constraint as the primary determinants of the crRNA structure–function relationship with Cas9. Based on this observation, we identify rational guidelines for chemical modification of the crRNA sugar–phosphate backbone that are compatible with Cas9 enzyme activity. In summary, the guide region prefers an A-form-like architecture, primarily due to nucleation of target DNA pairing at the seed. The tracr-pairing region is compatible with a broader array of modifications due to hybridization with tracrRNA and possible roles in conformational regulation of Cas9 activity. Potentially bulky modifications should be avoided in the seed sequence while a broad variety of modifications are likely to be well-tolerated at crRNA 5′ and 3′ termini . The comd guides ,31,32. TSupplementary DataClick here for additional data file.


Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA-seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by rationally designing RNA-seq adapters that minimize adapter dimer formation. Additionally, we developed biochemical and computational methods for remediating 5′- and 3′-end biases, the latter based on a random forest regression model that provides insight into the contribution of different factors to these biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5′- and 3′-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation. A weakness of most current RNA-seq methods is their use of a retroviral reverse transcriptase (RT) to copy target RNAs into cDNAs for sequencing on various high-throughput DNA sequencing platforms4. Retroviral RTs have inherently low fidelity and processivity, and the extent to which these properties can be improved by protein engineering or in vitro evolution is limited by the retroviral RT structural framework5.High-throughput RNA sequencing (RNA-seq) has revolutionized biology and will become ever more powerful as new methods that address weaknesses and expand capabilities of current methods are developed9. Unlike retroviral RTs, which evolved to help retroviruses evade host defenses by introducing and propagating mutational variations5, group II intron RTs evolved to function in retrohoming, a retrotranposition mechanism that requires faithful synthesis of a full-length cDNA of a long, highly structured group II intron RNA10. Their beneficial properties for RNA-seq include high fidelity, processivity, and strand displacement activity, along with a proficient template-switching activity that is minimally dependent upon base pairing and enables the seamless attachment of RNA-seq adapters to target RNAs without RNA tailing or ligation11. Thermostable group II intron RTs (TGIRTs) from bacterial thermophiles combine these beneficial properties with the ability to function at high temperatures (60–65° C), which help melt out stable RNA secondary structures that can impede reverse transcription6. A recent crystal structure of a full-length TGIRT enzyme  in active conformation with bound substrates revealed that group II intron RTs are closely related to RNA-dependent RNA polymerases, as expected for an ancestral RT, and identified a series of novel structural features that may contribute to their high fidelity and processivity12. These features include more constrained binding pockets than retroviral RTs for the templating RNA base, 3′ end of the DNA primer, and the incoming dNTP, as well as a larger fingers region that enables more extensive contact with the template-primer substrate than is possible for retroviral RTs12.To address this weakness, we have been developing RNA-seq methods using the RTs encoded by mobile group II introns, bacterial retrotransposons that are evolutionary ancestors of introns and retroelements in eukaryotes14; quantitative tRNA-seq based on the ability of the TGIRT enzyme to give full-length end-to-end reads of tRNAs with or without demethylase treatment16; determination of tRNA aminoacylation levels17; high-throughput mapping of post-transcriptional modifications by distinctive patterns of misincorporation20; identification of protein-bound RNAs by RIP-Seq or CLIP21; and RNA-structure mapping by DMS-MaPseq23 or SHAPE24. A study comparing TGIRT-seq to benchmark TruSeq v3 datasets of rRNA depleted (ribo-depleted) fragmented Universal Human Reference RNA (UHRR) with External RNA Control Consortium (ERCC) spike-ins showed that TGIRT-seq: (i) better recapitulates the relative abundance of mRNAs and ERCC spike-ins; (ii) is more strand-specific; (iii) gives more uniform 5′- to 3′-gene coverage and detects more splice junctions, particularly near the 5′ ends of genes, even from fragmented RNAs; and (iv) eliminates sequence biases due to random hexamer priming, which are inherent in TruSeq7. Other recent studies have shown that TGIRT-seq more accurately depicts the quantitative relationship between mRNAs and structured small ncRNAs than other tested methods14 and eliminates artifacts due to RT mispriming in RNA-seq reactions25.GsI-IIC RT (TGIRT-III) has been used for a variety of applications, including the comprehensive profiling of whole-cell, exosomal and plasma RNAs6. For Illumina RNA-seq, the initial RNA template/DNA primer consists of a 34-nt RNA oligonucleotide containing an Illumina Read 2 sequence (R2 RNA) with a 3′ blocking group  annealed to a 35-nt DNA primer (R2R DNA) that leaves a single nucleotide 3′-DNA overhang. The latter can base pair to the 3′ end of the target RNA, serving as a springboard for TGIRT template-switching and the initiation of cDNA synthesis6. To capture heterogeneous 3′ ends in a pool of RNAs, this single nucleotide 3′ overhang is an equimolar mixture of A, C, G, and T (denoted N) and is added in excess to the target RNA. After reverse transcription, a second RNA-seq adapter  is ligated to the opposite end of the cDNA by a single-stranded DNA ligation with thermostable 5′ App RNA/DNA ligase (New England Biolabs), and this is followed by minimal PCR amplification with primers that add Illumina capture sites and sequencing indices. By avoiding gel-purification steps, TGIRT-seq libraries can be generated rapidly from small amounts of starting material (1–2 ng input RNA).The TGIRT-seq method currently used for comprehensive transcriptome profiling  is outlined in Fig. 14. This problem is particularly acute for low abundance RNA samples where multiple rounds of AMPure beads clean-up may be required to sufficiently decrease the ratio of adapter dimers to a small amount of library products8. Due in part to this limitation, miRNAs and other very small RNAs have been analyzed by an alternative TGIRT-seq method (the TGIRT CircLigase method), which was patterned after the method used for ribosome profiling with retroviral RTs27. In the TGIRT-based version of this method, template-switching rather than RNA ligation is used to add an adapter containing both R1R and R2R sequences, and the resulting cDNAs with the linked R1R/R2R adapter are gel-purified and circularized with CircLigase for RNA-seq library construction18.Unlike retroviral RTs, which have been studied extensively and optimized for biotechnological applications for decades, the recently introduced TGIRT enzymes and TGIRT-seq methods are potentially subject to substantial improvement. In this regard, a weakness of the TGIRT Total RNA-seq method is the thermostable 5′ App RNA/DNA ligase used to attach the R1R adapter to the 3′ end of the cDNA, which introduces sampling biases for cDNA ends and produces unwanted adapter dimers by ligating the R1R adapter to residual R2R adapter carried over from previous steps. To avoid wasting reads, these adapter dimers are removed by AMPure beads clean-up of the library prior to sequencing Fig. , a step 29 to design an R2/R2R adapter with just a single nucleotide change that dramatically decreases the formation of adapter dimers, thereby improving the recovery of miRNA sequences and enabling the construction of TGIRT-seq libraries from even smaller amounts of starting material. Additionally, using a miRNA reference set containing an equimolar mixture of 962 human miRNAs, we systematically analyzed 5′- and 3′-end biases in TGIRT-seq, and developed biochemical and computational methods for ameliorating these biases. We found that the 5′-sequence biases introduced mainly by the thermostable 5′ App RNA/DNA ligase could be computationally corrected and that 3′-biases introduced by TGIRT template-switching could be corrected either computationally or by employing an altered ratio of 3′-overhang nucleotides in the R2 RNA/R2R DNA primer mix.Here, we used the previously determined ligation biases of the thermostable 5′ App RNA/DNA ligase29. We noticed that the R2R adapter used previously for TGIRT-seq (denoted NTC based on its 3′ end sequence) has a C-residue at position −3 from its 3′ end, which favors the formation of R1R-R2R adapter dimers during the ligation step  in which a single T residue was inserted at position −3, thereby replacing the favored C at this position with a disfavored T, but leaving the remainder of the R2R sequence unchanged Fig. . This in7. The TGIRT-seq libraries were constructed in triplicate with one round of 1.4X AMPure beads clean-up to remove adapter dimers and sequenced on an Illumina NextSeq 500 to obtain 61–105 million 75-nt paired-end reads (Supplementary Table 7 were downloaded (NCBI SRA accession number SRP066009) and processed using the same bioinformatic pipeline.To assess the performance of the NTT R2R adapter, we used it for TGIRT-seq of ribo-depleted fragmented UHRR with ERCC spike-ins, as done previously for TGIRT-seq with the NTC adapter30 , with similar proportions of the mapped reads mapping concordantly in the correct orientation to annotated genomic features  and compared its performance to that of the NTC adapter tested in parallel. Libraries prepared using each adapter were constructed in triplicate, with the libraries constructed using the NTT adapter requiring 1 round of 1.4X AMPure beads clean-up prior to sequencing compared to 4 rounds for those constructed using the NTC adapter , while the 3′ bias due primarily to template-switching favors reference set miRNAs with a 3′ G residue and strongly disfavors miRNAs with a 3′ U residue  values were ≥1 standard deviation higher and lower, respectively, than the mean log10 CPM for all of the miRNAs in the reference set  of the measurement errors .As the random forest regression model predicts the full spectrum of miRNA measurement errors, we could use it to quantitatively assess the contributions of each of the first three 5′- and 3′-end positions to the measurement errors Fig. . The resHaving investigated the sources of the 5′- and 3′-end bias in the TGIRT-seq protocol, we next explored biochemical and computational approaches for mitigating these biases. For the 3′ bias, we first thought that the preference for a G residue and against a U residue at position −1 might reflect the strength of the base-pairing interaction between that nucleotide and the 3′-overhang nucleotide of the DNA primer that is used to direct TGIRT template-switching, with a strong rG/dC base pair favored over a weak rU/dA pair. However, changing the 3′-A overhang in the NTT primer mix to a diaminopurine (denoted MTT) to enable a stronger base pair with 3 instead of 2 H-bonds to a 3′ U only slightly ameliorated this bias  did not decrease the ligation bias , with such adapters giving lower bias and better coverage at low sequencing depths than those with invariant sequences at their ends32, did not ameliorate end biases nearly as well as the random forest regression model , and Fig. 10CPM values for the reference set miRNAs obtained by the different methods. The plots confirmed the previous finding36 that the 4N protocols perform better than other small RNA-seq methods both in sampling miRNAs  with smaller variance across the measured miRNA CPM values , the TGIRT CircLigase method, employing TGRT template-switching by TeI4c RT instead of GsI-IIC RT and a cDNA gel-purification step prior to circularization, performed about as well as the 4N protocols both in miRNA recovery as a function of sequencing depth and in overall sample bias and variance  or lower (n = 27) than the mean log10CPM, and then compared several potentially bias-inducing characteristics of these miRNAs to the remaining 927 more uniformly represented miRNAs  5′ and 3′ nucleotides in the most stable predicted secondary structure.To identify other factors that might have contributed to the biased representation of these outlier miRNAs in TGIRT-seq, we defined over- and under-represented miRNAs as those whose logR2 = 0.073; Fig. Violin plots of the distribution of miRNAs in each of the above group as a function of the compared characteristic showed that miRNA length is the only tested factor that contributes significantly to the under- or over-representation of these outlier miRNAs in TGIRT-seq  and/or confirmation by orthogonal approaches, such as RT-qPCR, microarrays, or bead-based hybridization assays 42. Longer term, the bias in 5′-adapter addition might be addressed by using a modified or different ligase, but a less biased ligase might also lead to increased production of adapter dimers. Thus, a preferable approach may be to use an alternative method for 5′-adapter addition, such as leveraging the ability of TGIRT-III to add non-templated A residues to the 3′ of cDNAs to enable template-switching to an acceptor oligonucleotide with 3′ U residues, analogous to the Clontech/Takara SMART-seq protocols44. The 3′ bias in TGIRT template-switching seems less problematic as it can be addressed either computationally or by using an adjusted ratio of 3′ overhangs in R2 RNA/R2R DNA starter duplex. Longer term, it might also be addressed by using a different or modified TGIRT enzyme with less sequence bias. The recently determined crystal structure of full-length GsI-IIC RT in an active conformation with bound substrates12 provides a platform for detailed analysis of the structural basis and possible alleviation of this 3′-end bias.As noted previously by Giraldez 46; comparison of expression levels of small ncRNAs and mRNAs encoding components of the same RNP complexes14; and the analysis of tRNAs and tRNA fragments or mature, pre-, and pri-miRNA in the same RNA-seq47. Previous work showed that the total RNA-seq protocol with TGIRT-III works well for quantitation of small RNAs down to ~60 nt14, and the use of the new NTT adapter substantially improves the recovery of miRNAs sequences, both for the miRNA reference sets used here and in cellular, exosomal, and human plasma RNA samples analyzed in our laboratory. We note, however, that even with the NTT adapters, the recovery of miRNA sequences in the TGIRT Total RNA-seq method with GsI-IIC RT (TGIRT-III) is less efficient than that of larger RNAs  to confirm quantitative inferences about miRNA abundance. Additionally, based on comparison of published datasets, we find that alternative TGIRT-CircLigase method, which includes a gel-purification step, performed similarly to 4N protocols in both the recovery of miRNA sequences as a function of sequencing depth and overall variance from the expected CPM values  at 1 μg/μl was mixed with 1 μl of ERCC ExFold Mix 1  prepared according to the provided protocol. 2 μl of the resulting UHRR sample with ERCC spike-ins was ribo-depleted by using a Human/Mouse/Rat Ribo-zero rRNA removal kit (Illumina), fragmented to 70–100 nt by using an NEBNext Magnesium RNA Fragmentation Module , and treated with T4 polynucleotide kinase (Epicentre) to remove 3′ phosphates that impede TGIRT template-switching7. After each of the above steps, the RNA was cleaned-up by using a Zymo RNA Clean & Concentrator kit, with 8 volumes of ethanol added to the input RNA to maximize the recovery of small RNAs7. The fragment size range and RNA concentration were verified by using a 2100 Bioanalyzer (Agilent) with an Agilent 6000 RNA pico chip and aliquoted into 6 ng/3 μl portions for storage in −80 °C.The miRXplore miRNA reference set was purchased from Miltenyi Biotech. The RNA was dissolved in nuclease-free water (Invitrogen), adjusted to 1 μM, and aliquoted for storage at −80 °C. Fragmented human reference RNA samples were prepared as described8 using 6 ng of fragmented Universal Human Reference RNA (UHRR) with ERCC spike-ins or 50 nM Miltenyi miRXplore RNA prepared as described above. The template-switching and reverse transcription reactions were done as described8 with 1 μM TGIRT-III (InGex) and 100 nM pre-annealed R2 RNA/R2R DNA in 20 μl of reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, 5 mM DTT, pH 7.5. Reactions were set up with all components except dNTPs, pre-incubated for 30 min at room temperature, a step that increases the efficiency of template-switching and reverse transcription, and then initiated by adding dNTPs . The template-switching reactions were incubated for 15 min at 60 °C and then terminated by adding 1 μl 5 M NaOH to degrade RNA and heating at 95 °C for 5 min followed by neutralization with 1 μl 5 M HCl and two rounds of MinElute column clean-up (Qiagen) to decrease the amount of unused R2R DNA adapter. The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C. The ligated products were purified by using a MinElute Reaction Cleanup Kit and amplified by PCR with Phusion High-Fidelity DNA polymerase . The PCR products were cleaned up by using Agencourt AMPure XP beads  and sequenced on an Illumina NextSeq 500 instrument to obtain 2 × 75-nt paired-end reads.TGIRT-seq libraries were prepared as described7). After removing the extra T residue introduced by the NTT adapter at the 5′ end of Read 2, reads from datasets obtained by using the NTT and NTC adapters were trimmed with cutadapt48 1.16 to remove Illumina TruSeq adapters and PCR primer sequences , and reads <15-nt after trimming were discarded. Reads were then mapped by using HISAT249 v2.0.2 with default settings to a human genome reference sequence (Ensembl GRCh38 Release 76) combined with additional contigs for 5S and 45S rRNA genes and the E. coli genome sequence (Genebank: NC_000913) (denoted Pass 1). The additional contigs for the 5S and 45S rRNA genes included the 2.2-kb 5S rRNA repeats from the 5S rRNA cluster on chromosome 1  and the 43-kb 45S rRNA repeats that contained 5.8S, 18S and 28S rRNAs from clusters on chromosomes 13, 14, 15, 21, and 22 (GeneBank: U13369). Unmapped reads from Pass 1 were re-mapped to Ensembl GRCh38 Release 76 by Bowtie 250 v2.2.6 with local alignment to improve the mapping rate for reads containing post-transcriptionally added 5′ or 3′ nucleotides ), short untrimmed adapter sequences, or non-templated nucleotides added to the 3′ end of the cDNAs by the TGIRT enzyme (denoted Pass 2). The uniquely mapped reads from Passes 1 and 2 were combined using Samtools51 v1.8. To process multiply mapped reads, we examined different alignments with the highest mapping score and selected the alignment with the shortest distance between the two paired ends . In the case of ties between reads mapping to rRNA and non-rRNA sequences, the read was assigned to the rRNA sequence, and in other cases, the read was assigned randomly to one of the tied choices. Uniquely mapped reads and the filtered multiply mapped reads were combined and intersected with gene annotations (Ensembl GRCh38 Release 76) supplemented with the RNY5 gene and its 10 pseudogene sequences, which were not annotated in this release, to generate the counts for individual features. Coverage of each feature was calculated by Bedtools52. To avoid miscounting of embedded sncRNAs, the reads were first intersected with sncRNA annotations, and the remaining reads were then intersected with the annotations for protein-coding genes, lincRNAs, antisense, and other lncRNAs. To further improve the mapping rate for tRNAs and rRNAs, we combined reads that were uniquely or multiply mapped to tRNAs or rRNAs in the initial alignments and re-mapped them to tRNA (Genomic tRNA Database and UCSC genome browser website) or rRNA (GeneBank: X12811 and U13369) reference sequences using Bowtie 2 local alignment.Datasets obtained for ribo-depleted, fragmented UHRR plus ERCC spike-ins using the NTT adapter were compared with published datasets for identically prepared samples using the NTC adapter downloaded from NCBI  to calculate the percentage of bases in CDS, UTR, intron, and intergenic regions.For correlation analysis, RNA-seq datasets were normalized for the total number of mapped reads by using DESeq22 transformed, and used to generate scatter plots, empirical cumulative distribution function (ECDF) plots, and nucleotide frequency plots in R. RMSE was calculated using log2 transformed median normalized counts.For datasets obtained for the Miltenyi miRXplore miRNA reference set, reads obtained using the NTT and NTC adapters were processed as described above for UHRR datasets and then mapped with Bowtie2 using local alignment with default settings to the Miltenyi miRXplore reference sequences. Uniquely mapped reads with lengths between 15 and 40 nt . The predicted deviations between the expected log10 miRNA abundance for each miRNA  and measured log10 abundance for that miRNA were calculated by using a random forest regression model implemented in R54 according to the following equation:miRNA sequence biases were analyzed with customized scripts using pysam10CPM and expected log10CPM for miRNA m; f indicates the random forest regression function; and xm,i indicates the nucleotide of miRNA m at position i. Only the first 3 bases (i = 1 to 3) and the last 3 bases (i = −3 to −1) of each miRNA were considered in this model.where 10CPM for each miRNA. The corrected read count was then converted into a corrected pseudo-count (non-integer) by taking a power of 10, via:m and 10CPM for miRNA m. Codes have been deposited in GitHub: https://github.com/wckdouglas/tgirt_smRNA.Correction of miRNA abundances was done by subtracting 36), and counts from the 962 Miltenyi miRXplore RNAs were extracted for the comparisons. Raw reads obtained by the TGIRT-CircLigase method6 were downloaded from NCBI (SRA accession number SRR8337756) and aligned to the Miltenyi miRXplore reference sequences using Bowtie2  to generate a miRNA count table. miRNA counts from TGIRT-seq datasets and the downloaded datasets were normalized to CPM for the comparisons. The predicted RNA folding and co-folding patterns and minimum free energies were computed by the ViennaRNA package55.miRNA count tables for 4N ligation, NEXTflex, TruSeq, NEBNext and CleanTag were downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA accession number SRP126845The datasets generated and analyzed in the current study are available in the National Center for Biotechnology Information Sequence Read Archive under SRA accession number SRP168562.Supplementary Information


To investigate the risk factors of cervical lymph node (LN) metastasis in papillary thyroid microcarcinoma (PTMC) patients.We retrospectively analyzed the clinicopathologic data of all patients who received standard lobectomy for PTMC at our institution between October 2017 and January 2019. Central LNs were dissected in all patients. Lateral LNs were dissected if metastasis to the lateral LNs was suggested based on pre-op fine-needle aspiration biopsy. The relationship between variables available prior to surgery and cervical LN metastasis was examined using multivariate regression.P = 0.021) and had significantly larger tumor diameter on pathology . Multivariate regression analysis identified the following independent risks for cervical LN metastasis: male sex , age  and ultrasound tumor diameter at > 5 mm .Post-op pathologic examination revealed cervical LN metastasis in 79 (29.5%) patients. Seventy subjects had metastasis only to central LNs, and 4 (1.5%) patients had metastasis only to lateral LNs. Five patients had metastasis to both central and lateral LNs. In comparison to patients without cervical LN metastasis, those with LN metastasis were significantly younger (40.63 ± 13.07 vs. 44.52 ± 12.23 years; Cervical LN metastasis occurs in a non-insignificant proportion of PTMC patients. Independent risks included male sex, younger age and larger tumor diameter on ultrasound. According to the World Health Organization (WHO) report, the number of new cases of thyroid cancer in China accounted for 15.6% of that worldwide, and the number of deaths accounted for 13.8% . SeveralThe most common site of PTMC metastasis is cervical lymph nodes, especially the central lymph nodes (known as level 6) . SurgeryIn the current retrospective analysis, we examined the potential correlation between pre-operative variables with cervical LN metastasis in 268 PTMC patients.The current study (including access to raw data) was approved by the Ethics Committee of Shanghai Ruijin Rehabilitation Hospital (Committee Chairman: Ms. Yuezhen Dai) and performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Patient consent was not required because of the retrospective nature of the study. Anonymized patient data (in the Chinese language) are available upon request.This retrospective study analyzed the clinical data of pathologically proven PTMC patients who were initially treated at Shanghai Ruijin Rehabilitation Hospital, Shanghai, China, between October 2017 and January 2019. Patients were included if 1) they received initial diagnosis and treatment for thyroid nodules; 2) they were diagnosed with PTMC by preoperative fine-needle biopsy and underwent conventional lobectomy of thyroid carcinoma; 3) they had pathologically proven solitary PTMC; 4) the maximum tumor diameter on pathology was ≤10 mm.The following data were retrieved from the hospital electronic record systems: name, sex and age and clinicopathologic and surgical data including 1) time to surgery from initial presentation; 2) surgical procedure received; 3) pathological type, maximum diameter and location  of the tumor; 4) histological results of involved central and/or lateral lymph nodes; 5) concurrent Hashimoto’s thyroiditis; 6) routine laboratory results such as blood chemistries, thyroid function, parathyroid hormone and blood calcium before and following operation.All patients underwent preoperative physical examination, high-quality thyroid ultrasonography (US), and US-guided fine-needle aspiration biopsy of suspected thyroid nodule or lymph nodes. Solitary PTMC was considered if the tumor showed no pathological evidence of multifocality within the thyroid gland. The maximum diameter and location of the primary tumor within the thyroid were determined by pathological examination.All patients underwent standard lobectomy. Central LN dissection was conducted in all subjects. In patients suspected of metastasis to lateral LNs based ultrasound examination prior to surgery, lateral LNs were also dissected. The excised tissue was then sent for pathological examination. Patients received routine rehydration as well as symptomatic treatment postoperatively and were closely observed for incision hemorrhage, hoarseness, or numbness.t-test and χ2 test were used to examine potential differences between subjects with vs. without cervical LN metastasis. Multivariate logistic regression was used to evaluate the correlation between pre-operative variables and cervical LN metastasis. P < 0.05 was considered statistically significant.The data was analyzed with SPSS 20.0 software . Student’s The study flowchart is shown in Fig. P = 0.021) and had significantly larger tumor diameter on pathology  (Table Seventy-nine 29.5%) patients had cervical LN metastasis: 70 subjects had metastasis only to central LNs, and 4 (1.5%) patients had metastasis only to lateral LNs. Five patients had metastasis to both central and lateral LNs. Compared to patients without LN metastasis, those with cervical LN metastasis were significantly younger , younger age (P = 0.021) and larger tumor diameter on ultrasound (P = 0.010). The multivariate analysis confirmed the following risks: male sex , younger age  and ultrasound tumor diameter at > 5 mm  . The current study also revealed an association between cervical LN metastasis with younger age  and the male sex . We failed to show an association between cervical LN metastasis with tumor location, capsule invasion or Hashimoto’s thyroiditis, possibly due to insufficient sample size.Based on previous literature, independent risk factors of cervical LN metastasis in PTMC patients include age over 45 years , male seV600E mutation was associated with extra-thyroid infiltration, lymph node and distant metastasis, and advanced TNM staging. Therefore, determination of BRAF mutation status preoperatively may allow preoperative evaluation of capsule invasion and cervical LN metastasis. However, more studies are required to validate of molecular markers such as BRAF.The availability of gene sequencing data may allow molecular stratification of cancer patients. Lai et al. showed in a meta-analysis that BRAFWith the development of medical technology, PTMC patients now have more options for treatment. In addition to the more aesthetically pleasing surgical methods such as thyroidectomy with laparoscopy or robot-assisted surgery, radiofrequency ablation therapy has also garnered the interest of clinicians. Based on the findings from the current study, we believe that ablation may be suitable for selected patients  with low risk of cervical LN metastasis.Cervical LN metastasis is not a rare occurrence in PTMC patients. Independent risks for metastasis include the male sex, younger age and tumor diameter on ultrasound at > 5 mm.