The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5′-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes. The analytical performance of biosensors has been greatly enhanced through the development of nanomaterials by nanotechnology engineering . Due to The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) genome engineering technology has facilitated editing the genome of any species . RecogniIn addition to CRISPR-dependent HDR, there are some other CRISPR-Cas-based tools that are precise in gene editing and do not depend on double-strand break repair mechanisms. For example, CRISPR base editors can introduce single base substitutions to target DNA sites in a programmable manner without double-strand break formation ,21. In aInosine-substituted probes have been used for the detection of genomic mutations ,26. The Here for the first time, electrochemical nanogenosensing based on inosine-substituted probes and disposable pencil graphite electrodes was used to verify the presence of CRISPR-Cas9-introduced mutations in murine cells. This method allows for efficient detection of the desired outcome created by HDR in CRISPR-Cas9 studies with high selectivity and specificity.The differential pulse voltammetry (DPV) technique was used for analysis with an Autolab PGSTAT-30  electrochemical analysis system and GPES 4.9 software package . A three-electrode system involving disposable graphite electrodes (PGEs) with diameters of 0.5 mm and lengths of 3 cm long as working electrodes , a platinum wire as the counter electrode and a reference electrode (Ag/AgCl).N-hydroxysulfosuccinimide sodium salt (NHS), [N-(3-dimethylamino)propyl)]–N′-ethylcarbodiimide (EDC) and Trizma hydrochloride were purchased from Sigma-Aldrich Chemical Company , Sodium dodecyl sulfate (SDS) and tri-sodium citrate were purchased from Merck . Other chemicals were of analytical reagent grade and supplied by Merck and Sigma. All experiments were performed at 25 °C. The synthetic oligonucleotides were provided from Invitrogen  by Thermo Fisher Scientific as a lyophilized powder. The base sequences of the oligonucleotides are as follows (I = inosine):2-C6-CICtAAITICTCTIIAIaIIT-3′S-331 genome wild-type (WT) probe: 5′-NHTCTCCAGAGCACTTAGCG-3′Synthetic WT target: 5′-ACC2-C6-CICCAAITICTCTIIAICIIT-3′S-331 genome mutant-type (MT) probe: 5′-NHGCTCCAGAGCACTTGGCG-3′Synthetic MT target: 5′-ACCSynthetic non-complementary sequence: 5′-GGCAGCGGTGACTATGGCACC-3′ACCGCTCCAGAGCACTTGGCGCCCCAGCAGGATGTCCTGGAG-3′CRISPR-Cas9-edited S-331 gene PCR amplicon (underlined bases refer to the introduced point mutation region via CRISPR/Cas9 system): 5′-TTAGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTGGAACTGGGTCAAAGGCCTCTGGGAAGATAGAGCTTTGGTCTTCTTGGATTTGCTGGTTTGTTTTCATTTTTGAGACAATCTTGGCTGACCTAGAACTCACTATGTAGACCAGGCTGGCCTCAACTCTTCAGAAGAGATCCGCCTGTCTCTTCCTCCCTAGGGTCAGGATCAAAGGCATAGACCACCACAACTGGCTTTTTGCTTATCTTTGGATCTTTGCTAGCTCAGAGGAGTCCACCGAGAAAGGCCCTACAGGGCAGCCACAAGCAAGGGTCCAGCCTCAGACCCAGATGACAGCACCAAAGCAGACACAGACCCCGGATCGGCTGCCTGAGCCACCAGAAGTCCAAATGCTGCCGCGTATCCAGCCACAGGCACTGCAGATCCAGACCCAGCCAAAGCTGCTTTGGCTGGGTCTGAGGCAGGCACAGACACAGE. coli): 5′-AAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCGATGAATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGAGCCGACGTTAAACACCGCCATTCCGGGCGATCCGCGTGATACCACTTCACCTCGGGCAATGGCGCAAACTCTGCGGAATCTGACGCTGGGTAAAGCATTGGGCGACAGCCAACGGGCGCAGCTGGTGACATGGATGAAAGGCAATACCACCGGTGCAGCGAGCATTCAGGCTGGACTGCCTGCTTCCTGGGTTGTGGGGGATAAAACCGGCAGCGGTGACTATGGCACCACCAACGATATCGCGGTGATCTGGCCAAAAGATCGTGCGCCGCTGATTCTGGTCAC-3′Non complementary PCR amplicon (All oligonucleotide and primer stock solutions (1000 µg/mL) were prepared with ultrapure water  and stored at −20 °C until use. Immobilization of probes onto the surfaces of electrodes was achieved via carbodiimide chemistry (5 mM EDC and 8 mM NHS). Dilutions of the capture probe (CP) were prepared with an aqueous acetic acid (0.5 M) with NaCl (20 mM)  buffer. Synthetic target sequences and the PCR products and non-complementary amplicons were diluted with hybridization buffer (HB) containing 5 × saline sodium citrate (SSC) + 0.05% solution , and 1× SSC + 0.1% SDS solution  was used as a washing buffer unless otherwise indicated.CIZ1 gene and proximal to a protospacer adjacent motif (PAM). The guide sequence was introduced into the linearized GeneArt® CRISPR nuclease vector-containing Cas9 and the CD4 reporter using the manufacturer’s instructions (Invitrogen). Amplification of the plasmid was performed by transformation of One Shot® TOP10 chemically competent E. coli, antibiotic selection and overnight cultures. DNA sequencing of the plasmid was performed to verify the construct (Eurofins). Single strand oligonucleotide-directed mutagenesis was used to introduce mutations into the CIZ1 gene using HDR with a 98 nt single-stranded oligodeoxynucleotide (ssODN): 5′-GCAGATCCAGACCCAGCCAAGCTGCTGAGGCAGGCACAGACACAGACCgCTCCAGAGCACTTgGCGCCCCAGCAGGATCAGGTAGAGCCACAGGTAC-3′.The following oligonucleotides were used for the construction of the guide sequence  were complementary to the region surrounding the PAM site to aid in efficient HDR. ® CRISPR nuclease (CD4 reporter) Vector using Dynabeads® CD4 magnetic beads (Invitrogen). Single cells were plated into a 96-well plate, and mutated cells were identified using a restriction endonuclease specific for desired mutations.Transfection of 3T3 embryonic mouse fibroblast cells with 1 µg of CRISPR-Cas9 expression vector and 2.5 µL of 50 µM ssODN, using 100 µL of transfection reagent Kit-R , was carried out by electroporation using the Nucleofector™ program U-030 (Lonza). Cell enrichment was carried out to isolate the transfected cells with the GeneArtSingle cells were cultured in a flat bottomed 96-well plate for 2 weeks, replacing media every 2–3 days and passaging cells on to 24 well plates. Genomic DNA (gDNA) was extracted using 50 μL of QuickExtract™ DNA extraction solution (Epicentre-Lucigen). The sequences of interest were screened after digestion with BanI (S331) restriction enzymes. gDNA was PCR amplified using One Taq Quick-Load 2× master mix , 94 °C-30 s, 30 cycles of 94 °C-30 s, 68 °C-30 s, 68 °C-60 s, the final extension was carried out at of 68 °C-5 min. PCR products were DNA sequenced to verify the presence of mutations.® TOPO® PCR cloning kit—Thermo Fisher Scientific, Waltham, MA, USA) and DNA sequenced  for multiple clones to confirm the presence of desired mutations.Identification of colonies of interest was identified by restriction endonuclease digestion with BanI according to manufacturer’s instructions. Clones that showed the correct restriction digest pattern were inserted into the TOPO10 Blunt cloning vector  H2. Then, the temperature was set to 1000 °C with the same ramp rate. After this, CNT synthesis was commenced by passing CH4 with a flow rate of 50 sccm and H2 with 200 sccm over the catalyst. CNT synthesis lasted 40 min under atmospheric pressure. To end the synthesis, the CH4 flow was turned off, and the CNTs obtained were left to cool under an atmosphere of H2.In this study, the carbon nanotubes (CNTs) were synthesized by thermal chemical vapor deposition (CVD)  utilizinPrior to probe immobilization, PGE electrodes were activated at 1.40 V for 30 s in acetate buffer solution . Probe immobilization was carried out on the surface of the PGEs. The probe and CNTs were allowed to interact in solution as previously described . BrieflyDenaturation of the PCR amplicons was carried out at 95 °C/8 min and 0 °C/2 min to separate the strands. Hybridization buffers containing the 5 µg/mL of complementary and non-complementary target sequences and amplicons were used for the hybridization between the probe and target sequences. Hybridizations were carried out on electrode surfaces at room temperature by placing the electrodes inside the vials containing 30 µL of the target or non-complementary solutions in a hybridization buffer for 30 min. The electrodes were then immersed in a washing buffer for 1 min to remove the unhybridized DNA.DPV was used to measure the oxidation signal of guanine after placing the electrode in ACB (pH 4.8) by scanning from +0.75 V to +1.40 V at ambient conditions (25 °C). The raw data were obtained as the analytical signal after moving average baseline fitting using a peak width of 0.01 V. All results presented in this paper are the means of at least five measurements, and the error bars show the standard deviations. The procedure for electrochemical biosensing is presented in CIZ1 protein sequence [CIZ1 is a protein that cooperates with cyclin A-CDK2 to promote the initiation of DNA replication [CIZ1 sequence by using HDR, including a point mutation that provides the S331A substitution in the CIZ1 protein and a silent point mutation that allows the formation of a BanI restriction site  to determine the selectivity of the designed genoassay. CNT-free ssDNAs were immobilized on the surface of unmodified PGEs, and guanine oxidation signals were found to be 220 nA. However, after the modification of ssDNA with CNTs, the guanine signal increased to 750 nA, resulting in a (3 times) increase over the unmodified PGEs  synthetic target sequence was obtained. To assess the efficiency of mutation detection, differences in guanine signals obtained from hybridization with a complementary synthetic target and from hybridization with a synthetic target containing two base mutations in the sequence region of interest (FM/MM = mismatch) was obtained. By varying only the probe concentrations ranging from 0.1 to 10 µg/mL, we found that the highest FM/NC and FM/MM values were obtained at 5 µg/mL probe concentration . Then weFinally, the optimized nanogenosensor has been used on real PCR amplicons . The guaCIZ1 gene with sufficient sensitivity and specificity.Electrochemical biosensing methods at the DNA level are used in a broad range of areas from disease-causing organisms to mutated genes and even detection of food contaminants, DNA/drug interactions and monitoring of the environment. The principle of the proposed detection strategy relies on a well-established method based on electrochemical genosensors. On the other hand, the emergence of the CRISPR/Cas9 system paved the way to alter the genome and control gene expression. The outcomes of any CRISPR-Cas9 assay should be investigated to perform safe and effective genome editing. This study demonstrates the possible use of inosine-substituted probes along with CNT/PGEs for label-free electrochemical genosensing of CRISPR-Cas9-mediated genomic mutations. The techniques used in this study are based on established genosensing methods capable of detecting nucleic acids with high sensitivity and sequence-specificity ,26,33,37Higher levels of guanine oxidation signals were obtained with PCR amplicons than with 21-base synthetic targets . This isThe genosensor developed here has potential applications in the evaluation of CRISPR-mediated genome editing results. For example, in any HDR-based gene editing assay, the edited clones can be precisely detected by sequencing all the clones; however, this is time-consuming and expensive. Using this genosensor, correctly edited clones can be quickly selected from among tens or hundreds of clones prior to precise verification by sequencing. Moreover, the use of nanogenosensor can be exploited for the detection of single point mutations that cause disease and for detection of CRISPR-Cas9-mediated reversion of the mutation. This method can also be further developed for the detection of other types of CRISPR-based manipulations, including small insertions and deletions and multiple base substitutions generated by HDR, base editing, or prime editing. This approach is also suitable for expansion by employing an array system of sequence-specific probes to detect multiple gene editing outcomes. Further studies could be designed to detect off-target mutations caused by HDR, NHEJ, or other CRISPR-based tools to screen genome editing outcomes more comprehensively.CIZ1 gene was edited using CRISPR-Cas9, and the introduced point mutations were detected using the genosensor as a proof-of-concept. Therefore, future work is needed to fully establish this nanogenosensor as a platform to detect CRISPR-based genome editing outcomes. To address these limitations, several other genes will be targeted, and the nanogenosensor will be used to detect other types of mutations such as multiple base substitutions, insertions, and deletions introduced via CRISPR/Cas9-HDR, base editing, and prime editing. Another limitation of this genosensor is that it may not work efficiently if the GC content of the edited DNA site is quite low. To prevent this, localization of the probe should be optimized such that it matches at least several guanines. Further investigation of this system will provide a basis for exploring other potential applications of the method.Although promising, this study includes some limitations. The results are preliminary, and only the CIZ1 gene, and the presence of the mutations was confirmed based on electrochemical nanogenosensor by monitoring guanine oxidation signals using disposable pencil graphite electrodes. Some parameters, such as probe concentration, target concentration, hybridization buffer, and washing time, were also optimized in order to improve the performance of the nanogenosensor. The presence of DNA hybridization on the pencil graphite electrodes was confirmed by the observation of an increase in guanine oxidation signal. The irreversible oxidation signal of the guanine base at +1.0 V vs. Ag/AgCl reference electrode was monitored using the DPV technique. The detection method relies on the measurement of increased oxidation currents of the guanine bases only in the presence of the hybrid due to the electrochemical inactivity of the inosine base in the probe sequence. Thus, a simple, sensitive, selective, non-time-consuming and cost-effective electrochemical-based “yes/no” detection system was developed. This hybridization signal was able to differentiate between non-complementary, complementary, and mismatched sequences. The hybridization signal of non-complementary DNA was relatively small because due to minimal interactions. This study has, for the first time, demonstrated direct measurement of CRISPR-Cas9-mediated mutations within a target gene using a nanogenosensor.Here, CRISPR-Cas9 was used to edit the


Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function.cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function.We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci. Noncoding RNAs have been shown to play central roles in biology. Key cellular machines such as telomerase and the ribosome are comprised of proteins and noncoding RNAs and serve as classic examples of RNA-based functionalities , 2. WhilTug1 for further analysis because of its high conservation [We previously reported the generation of 20 lncRNA loci knockout mouse strains, five of which displayed either viability, growth, or brain phenotypes . From thervation  as well ervation .Tug1 was first identified in a microarray screen for genes upregulated in response to taurine in a heterogenous culture of retinal cells [Tug1 is transcriptionally regulated by p53 [Tug1, including but not limited to the development of photoreceptors [Tug1 in cancer by acting as a tumor suppressor in human gliomas [Tug1 underscores the importance of this locus.al cells . In addid by p53 . A numbeeceptors  and in receptors –27. Ther gliomas , 29 and  gliomas . Thus, tTug1 locus using multiple genetic approaches and describe a physiological function in spermatogenesis and male fertility. We show that deletion of the Tug1 locus in mice leads to male sterility and also report an underappreciated molecular complexity at the Tug1 locus. Using several complementary genetic approaches , we provide evidence of a DNA-based repressive element within the Tug1 locus that regulates several genes in cis. We show that a gene expression program dysregulated in Tug1-knockout testes can be partially rescued by ectopic expression of Tug1 RNA in vivo. Finally, we show that the Tug1 locus contains an evolutionarily conserved ORF, which can be translated into a stable protein that impacts mitochondrial membrane potential upon overexpression. Collectively, this study implicates the Tug1 locus as essential in male fertility and provides evidence that the Tug1 locus contains at least two noncoding regulatory activities and a putative coding function.Here, we characterize the Tug1 lncRNA locus is located on chromosome 11 and has three annotated transcripts  is located approximately 680 bp upstream of the first Tug1 TSS. The Tug1 locus is enriched with hallmarks of active transcription, such as RNA polymerase II (Pol II) and histone H3 lysine 4-trimethylation (H3K4me3) at its promoter, H3K36me3 across its gene body, and abundant transcription as shown by RNA-seq  and higher than other well-characterized lncRNAs including Hottip (71%), Neat1 (69%), Xist (30%), and Firre (4%)    mouse model where the gene body of the Tug1 locus was removed and replaced with a lacZ reporter cassette downstream of the endogenous promoter, thereby preserving the act of transcription . Therefore, we sought to investigate the fertility of Tug1−/− mutants in more detail. We separately mated Tug1−/−, Tug1+/−, and wild-type males or females to C57BL/6J mice. We did not observe a difference in the mounting behavior between wild-type and Tug1−/− mice, as assessed by the presence of a vaginal plug. Strikingly, matings between Tug1−/− males (n = 8) and C57BL/6J females did not produce any offspring, whereas matings involving either Tug1+/− males (n = 8) or wild-type males (n = 8) with C57BL/6J females resulted in similar numbers of offspring  in Tug1−/−  compared to wild type  , which produced on average only 40% as many sperm as wild-type mice  .To further understand the underlying fertility defect in Tug1−/− males. We examined the morphological features of sperm and quantified the frequency of 15 different abnormalities  compared to wild-type males   and abnormal sperm morphology (teratozoospermia).Based on these results, we investigated whether perturbations in sperm morphology could also contribute to the complete sterility observed in  9) Fig. d. We obsTug1 locus leads to abnormal sperm morphology, we examined the timing of Tug1 expression at different stages of spermatogenesis. To this end, we took advantage of the knock-in lacZ reporter driven by the endogenous Tug1 promoter and assessed the expression by lacZ staining of histological sections of Tug1+/− testis and epididymis. From stages IX to XI of spermatogenesis in the testis, lacZ staining was restricted to the excess cytoplasm, known as residual bodies, which are phagocytosed toward the basement membrane by Sertoli cells  [Tug1−/− model enables us to test for potential cis-regulatory activity within the Tug1 locus because the gene-ablation design removes potential cis-acting elements yet keeps the act of transcription intact   were significantly upregulated in Tug1−/− testes compared to wild type  in embryonic stem cells [We next sought to investigate what, if any, molecular activities  are present at the -acting) –12. The em cells , 40.Figcis-effect upon deletion of the Tug1 locus is more widespread, we performed RNA-seq on 6 additional tissues ) as well as re-analyzed an existing brain dataset from wild-type and Tug1−/− mice , 9 genes were dysregulated in one or more tissues (7 upregulated and 2 downregulated) , and 8430429K09Rik (6 of 8 samples)  Fig. a. Of theTug1 locus, we reasoned that the repressive activity could be mediated either directly by the Tug1 transcript or by regulatory DNA elements within the locus. To determine if the repressive effect of Tug1 on neighboring genes occurs on the same allele (cis-acting), we performed allele-specific RNA-seq using a hybrid mouse strain. To generate this strain, we crossed Tug1+/− C57BL/6J females with Mus castaneus (Cast/EiJ) males  and identified significant changes in the gene expression relative to wild type. Deletion of the Tug1 locus was accompanied by 2139 significantly dysregulated genes across all samples examined. We observed global changes in the gene expression clustered by tissue type, indicating tissue-specific gene dysregulation , we identified an isoform of Tug1 that lacks the 5′ region, thus ensuring we would address the role of Tug1 RNA alone. To this end, we generated a doxycycline (dox)-inducible Tug1 transgenic mouse by cloning a Tug1 isoform downstream of a tet-responsive element )  in the Tug1−/− background that also contained an allele that constitutively expresses the reverse tetracycline transcriptional activator gene (CAG-rtTA3)  . Notably, 53 (including Tug1) of the 1051 genes that were significantly dysregulated in Tug1−/− testes were found significantly and reciprocally regulated in the testes from dox-fed Tug1rescue mice  and C57BL6/J female mice (n = 12) did not produce any progeny , similar to the levels observed in Tug1−/− males (mean = 4.69 × 105 ± 1.6 × 104 cells/mL) compared to wild type (mean = 9.32 × 105 ± 3.9 × 103 cells/mL)  .To further characterize the potential translated regions in ing data , a techning data , 50–52. nt snRNA , 54, is n = 50)    Fig. a. NotablF1) Fig. b. In conRNA Fig. : Fig. S9Since CMXR is commonly used to measure mitochondrial membrane potential, we reasoned that either impaired mitochondrial integrity or impaired redox potential at the mitochondrial membrane could account for the accumulation defect of CMXR in mitochondria upon TUG1-BOAT overexpression. To address these possibilities, we immunostained for TOM20, a redox independent translocase located on the outer mitochondrial membrane . We obseTug1 lncRNA locus in vivo and also report an underappreciated molecular complexity at the Tug1 locus  cis-acting DNA repressive element, (ii) a lncRNA that acts on gene expression in trans, and (iii) a conserved putative ORF which when overexpressed affects mitochondrial membrane potential , each with the potential to exert function. In this study, we report an essential role in male fertility for the cus Fig. . We findial Fig. . TogetheTug1 locus has an essential role in male fertility in mice. Based on our results, we speculate that the sterility of Tug1−/− males arises from a combination of oligozoospermia (low sperm count) and teratozoospermia . Notably, there is some evidence that Tug1/TUG1 may have a conserved role in male fertility in humans. Microarray profiling of sperm from sterile men  has significantly less TUG1 expression compared with sperm from fertile males  have been found to be important in spermatogenesis, but in contrast to the Tug1-knockout phenotype reported in this study, none lead to male sterility when disrupted in mouse models [Tug1, may be less common.Indeed, studies have performed systematic gene expression profiling at defined stages during spermatogenesis and have identified many developmentally regulated lncRNAs, suggesting that lncRNAs may have important roles during spermatogenesis . Four ote models –61. On te models . Thus, oTug1 locus harbors at least two distinct noncoding activities, and potentially a third coding one. Several lines of evidence indicate that the Tug1 locus has a cis-acting repressive DNA function. We observed that upon deletion of the Tug1 locus, several genes located downstream of Tug1 were consistently upregulated across multiple tissues, and in an allele-specific manner in the testes. This dysregulation is consistent with a previous study from our group in which genes located near the Tug1 locus were dysregulated in the brain of Tug1−/− mice [cis-effect is mediated by DNA regulatory elements within the Tug1 locus. Consistent with a repressive modality of the Tug1 locus, a recent study which systematically tested for enhancer activity across lncRNA loci, reported a lack of enhancer activity across the Tug1 gene body but found enhancer activity at all other lncRNA loci examined [Tug1 locus.Second, we found that the −/− mice . Collectexamined . Contrarexamined –66. FurtTug1 in the testes by a trans mechanism. In support of this conclusion, we found that a subset of genes dysregulated in Tug1−/− testes could be rescued by ectopic expression of Tug1 RNA, even at low levels. While the Tug1 transgene was expressed at lower levels than wild-type Tug1 RNA, other lncRNAs such as Hottip and Xist have been shown to exert a biological activity at relatively low copy numbers [Tug1 RNA appears to have functional activity even at low levels. Consistent with our finding of a trans-acting role for Tug1 RNA on gene expression, a previous study found that Tug1 RNA can regulate the levels of Ppargc1a mRNA in cultured podocytes [Ppargc1a was not significantly dysregulated, but it is important to note that our dataset does not include the kidney. We also note that low levels of Tug1 transgene RNA were not sufficient to rescue the Tug1 male fertility defect. More studies will be needed to determine the direct or indirect mechanisms of Tug1 RNA on gene expression in a male germ cell context in vivo.In addition to a DNA regulatory modality, this study finds evidence for an RNA-based role for  numbers , 68; thuodocytes . In our Tug1 locus. We demonstrate that the 5′ region of the Tug1 locus contains an evolutionarily conserved ORF that when overexpressed, impacts mitochondrial membrane potential. The protein product which we named TUG1-BOAT is predicted to have a high positive charge (net charge ~ + 16.5). Thus, we speculate that the accumulation of such a positively charged protein at the mitochondria in an overexpression context could lead to depolarization of the mitochondrial membrane. Consistent with this finding, a recent study also identified a conserved ORF in the 5′ region of the human TUG1 locus and showed that the ORF produces a stable protein that localizes to the mitochondria [Tug1 does not appear to be limited to the putative protein. One study found that overexpression of a Tug1 RNA isoform lacking ORF1 affects mitochondrial bioenergetics in cultured podocytes derived from a diabetic nephropathy mouse model [Tug1 RNA (lacking the ORF1 sequence) overexpression on mitochondrial membrane potential by CMXR staining in cell-based assays. Identifying endogenous and disease contexts in which Tug1 RNA and TUG1-BOAT are detected and function endogenously will be important to further explore the potential link between Tug1 and the mitochondria and determine whether the locus does contain a third coding function.A number of recent studies have identified ORFs at lncRNA loci and have also demonstrated that some ORFs can produce proteins , 50, 51.chondria . In addise model . In our Tug1 DNA, RNA, or putative protein could individually or in combination mediate the observed male fertility defect in Tug1-knockout mice. There are few biological contexts in the literature describing noncoding loci implicated in human disease with potential DNA, RNA, and protein functionalities. However, one intriguing example is the human D4Z4 locus, which is a repeat region that is reduced in copy number in humans with facioscapulohumeral dystrophy (FSHD) [D4Z4 locus, including a lncRNA, a protein that is detected in the disease context, and a DNA repressor element [Tug1-knockout mice, there is evidence to support a potential role in male fertility for each modality, thus warranting further investigation.Finally, this study opens the possibility that y (FSHD) , 70. Inty (FSHD) , 72. Mec element –76. Whilcis genes dysregulated upon deletion of the Tug1 locus have a role in male fertility. Loss-of-function mutations in 2 of the 6 cis genes upregulated in Tug1−/− testes, Smtn and Pla2g3, are characterized to have male fertility and sperm maturation defects [cis genes upon deletion of Tug1  have not yet been reported. In addition, there is some evidence for a potential role of Tug1 RNA in male fertility. Mice with a loss-of-function mutation for Selenop, a gene that was found significantly upregulated in Tug1−/− testes and significantly and reciprocally regulated in Tug1rescue testes, are reported to have reduced male fertility [There is some evidence that the  defects , 78. Howertility . As for Tug1 locus in male fertility. This study also highlights the molecular complexity embedded at lncRNA loci as our findings provide evidence that the Tug1 locus harbors two distinct noncoding activities: (i) a cis DNA repressor and (ii) a trans-acting lncRNA, and we find a potential third protein-coding activity. Therefore, going forward, it will be important to investigate the individual and/or combined roles of Tug1 DNA, RNA, and/or putative protein in male fertility as well as in disease contexts in which Tug1 is altered.In summary, this study provides genetic evidence for an essential role of the Tug1tm1.1Vlcg-knockout mice have been described previously [loxP-flanked neomycin resistance gene included in the targeting construct, we crossed Tugtm1.1Vlcg mice to C57BL6/J mice and then to a cre-recombinase strain (B6.C-Tg(CMV-cre)1Cgn/J, The Jackson Laboratory, 006054). Mice free of both the neomycin-resistance and cre-recombinase genes were selected for colony expansion and subsequently backcrossed to C57BL/6J mice. The Tug1-knockout allele was maintained by heterozygous breeding, and mutant mice were identified by genotyping for loss of the Tug1 allele and gain of the lacZ cassette .eviously , 41. To Tug1BL6-KO/Cast-WT mice by crossing inbred Mus castaneus (Cast/EiJ) males  with inbred heterozygote Tug1 females. The F1 hybrid male progeny  were used for allele-specific expression studies.For allele-specific gene expression analyses, we generated Tug1-overexpression mouse, tg(Tug1), we cloned Tug1 cDNA (see the “Tug1 (Ensembl ID: ENSMUST00000153313.2) was amplified from Riken cDNA clone E330021M17 (Source Bioscience) using specific primers containing MluI and EcoRV restriction sites (see the “Tug1 cDNA by sequencing (see the “Tug1) cassette were identified by genotyping for the pTRE allele and individually mated to female C57BL/6J mice  to expand the colonies. Next, we generated quadruple allele transgenic mice to test the functionality of the Tug1 RNA by the following strategy. We mated tg(Tug1) males to Tug1tm1.1Vlcg females and identified male progeny that were Tug1+/−; tg(Tug1). These mice were then mated to female rtTA mice (B6N.FVB(Cg)-Tg(CAG-rtTA3)4288Slowe/J mice ), and we identified male progeny that were Tug1+/−; tg(Tug1), rtTA. Finally, we mated male Tug1+/−; tg(Tug1), rtTA mice to Tug1+/− females, and at the plug date, females were put on 625 mg/kg doxycycline-containing food . We genotyped progeny from the above matings  and identified male progeny that were Tug1−/−; tg(Tug1), rtTA, and maintained these mice on the doxycycline diet until the experimental end point.To generate an inducible see the “” sectionsee the “” sectionsee the “” sectionTug1−/− mouse embryonic fibroblasts (MEFs) from E14.5 littermates from timed Tug1+/− intercrosses as described [l-glutamine, and non-essential amino acids. We genotyped MEFs derived from each embryo and used only male Tug1−/− and wild-type littermate MEFs at passage 2 for all experiments. 3T3 , HeLa , and BJ  cell lines were purchased from ATCC and cultured as recommended.We derived primary wild-type and escribed . We mainTug1 (see the “Tug1 cDNA (Ensembl id: ENSMUST00000153313.2) and performed in situ hybridization on a minimum of three C57BL6/J embryos per embryonic stage. For whole-mount staining, embryos were fixed in 4% paraformaldehyde for 18 h at 4 °C, followed by three washes in PBS for 10 min. We then dehydrated embryos through a graded series of 25%, 50%, and 75% methanol/0.85% NaCl incubations and then finally stored embryos in 100% methanol at − 20 °C. Embryos were then rehydrated through a graded series of 75%, 50%, 25%, and methanol/0.85% NaCl incubations and washed twice with PBS with 0.1% Tween-20 (PBST). Embryos were treated with 10 mg/mL proteinase K in PBST for 10 min  or 30 min . Samples were fixed in 4% paraformaldehyde/0.2% glutaraldehyde in PBST for 20 min at room temperature and washed twice with PBST. We then incubated samples in pre-hybridization solution for 1 h at 68 °C and then incubated samples in 500 ng/mL of Tug1 antisense or sense riboprobe at 68 °C for 16 h. Post-hybridization, samples were washed in stringency washes and incubated in 100 μg/mL RNaseA at 37 °C for 1 h. Samples were washed in 1× maleic acid buffer with 0.1% Tween-20 (MBST) and then incubated in Roche Blocking Reagent  with 10% heat-inactivated sheep serum  for 4 h at room temperature. An anti-digoxigenin antibody  was used at 1:5000 and incubated for 18 h at 4 °C. Samples were washed 8 times with MBST for 15 min, 5 times in MBST for 1 h, and then once in MBST for 16 h at 4 °C. Prior to developing, the samples were washed three times with NTMT , 50 mM MgCl2, 0.1% Tween-20, 2 mM levamisole). The in situ hybridization signal was developed by adding BM Purple  for 4, 6, 8, and 12 h. After the colorimetric development, samples were fixed in 4% paraformaldehyde and cleared through a graded series of glycerol/1× PBS and stored in 80% glycerol. Imaging was performed on a Leica M216FA stereomicroscope (Leica Microsystems) equipped with a DFC300 FX digital imaging camera.We generated an antisense riboprobe against see the “” sectionTug1 single-molecule RNA FISH as described previously [Tug1 transcripts were designed with LGC Biosearch Technologies’ Stellaris probe designer (Stellaris Probe Designer version 4.2) and manufactured by LGC Biosearch Technologies.We performed eviously . Brieflyl-lysine (10 μg/mL) diluted in PBS. Prior to hybridization, coverslips were washed twice with PBS, fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, and washed twice more with PBS. Coverslips were immersed in ice-cold 70% EtOH and incubated at 4 °C for a minimum of 1 h. We then washed the coverslips with 2 mL of Wash Buffer A (LGC Biosearch Technologies) at room temperature for 5 min. Next, we hybridized cells with 80 μL hybridization buffer (LGC Biosearch Technologies) containing Tug1 probes (1:100) overnight at 37 °C in a humid chamber. The following day, we washed the cells with 1 mL of Wash Buffer A for 30 min at 37 °C, followed by another wash with Wash Buffer A containing Hoechst DNA stain  for 30 min at 37 °C. Coverslips were washed with 1 mL of Wash Buffer B (LGC Biosearch Technologies) for 5 min at room temperature, mounted with ProlongGold (Life Technologies) on glass slides, and left to curate overnight at 4 °C before proceeding to image acquisition (see below).Human foreskin fibroblasts (ATCC® CRL-2522™) and mouse 3T3 fibroblasts  were seeded on glass coverslips previously coated with poly-Tug1−/− (n = 8) and wild-type (n = 9) males between 8 and 41 weeks of age were sacrificed and weighed. We then dissected the entire male reproductive tract in phosphate-buffered saline (PBS). One testis was removed, weighed, and fixed in 4% paraformaldehyde (PFA) for histology (see below). Sperm were collected from one cauda epididymis by bisecting and suspending the tissue in a solution of Biggers-Whitten-Whittingham (BWW) sperm media at 37 °C. After a 15-min incubation, we used the collected sperm solutions to analyze sperm morphology and counts.We characterized sperm morphology by fixing sperm in 2% PFA in PBS, mounting 20 μL of suspended sperm in Fluoromount-G media (Southern Biotech) on Superfrost glass slides (Thermo Fisher Scientific) and scanning each slide in a linear transect, recording the morphology as normal or abnormal for each sperm cell encountered (between 30 and 120 sperm). When abnormal, we also recorded the type of morphological defects: headless, head angle aberrant, head bent back to midpiece, debris on the head, debris on the hook, head misshapen, midpiece curled, midpiece kinked, midpiece stripped, debris on the midpiece, tailless, tail curled, tail kinked, broken tail, or multiple cells annealed together.Tug1−/− (n = 7) and wild-type (n = 9) mice were determined using a Countess Automated Cell Counter according to the manufacturer’s protocol . For the Tug1rescue experiment, sperm counts for control (WT and Tug1+/−) (n = 2), Tug1−/− (n = 2), and Tug1−/−; tg(Tug1); rtTA mice (n = 3) were determined by manual counts using a hemocytometer. For all analyses, statistical comparisons between Tug1−/− and wild type were performed using the two-tailed Wilcoxon rank-sum tests with an a = 0.05. The results for testis, sperm count, and morphological parameters are presented in Additional file Tug1−/− versus wild type for relative testis size, sperm morphology, and sperm counts were performed using R ).Sperm counts for each lacZ reporter and histological staining for morphological analysis of male reproductive tissues was conducted on the testes and epididymis from Tug1−/− (n = 2) and wild-type (n = 2) mice. We fixed the testis and epididymis in 4% paraformaldehyde in PBS overnight at 4 °C and washed the tissues three times in PBS. For lacZ staining, we rinsed Tug1+/− and wild-type tissues three times at room temperature in PBS with 2 mM MgCl2, 0.01% deoxycholic acid, and 0.02% NP-40. We performed X-gal staining by incubating the tissues for up to 16 h at 37 °C in the same buffer supplemented with 5 mM potassium ferrocyanide and 1 mg/mL X-gal. The staining reaction was stopped by washing three times in PBS at room temperature, followed by 2 h post-fixation in 4% paraformaldehyde at 4 °C.Expression of the knock-in We then embedded the organs in paraffin, sectioned the organs at 6 μm thickness, and then mounted the sectioned samples onto glass microscope slides. The testis sections were additionally stained with Mayer’s hematoxylin, periodic acid, and Schiff’s reagent , and the epididymis sections were stained with eosin . Images were collected using a Zeiss AxioImager.A1 upright microscope or on an Axio Scan Z.1 (Zeiss).Tug1 mutant and wild-type samples, on the Illumina HiSeq platform using the rapid-full flow cell with the 101-bp paired-end reads sequencing protocol .We isolated total RNA from mouse tissues, mouse embryonic fibroblasts (MEFs), and blood cells using TRIzol  by chloroform extraction followed by spin-column purification  according to the manufacturer’s instructions. RNA concentration and purity were determined using a Nanodrop. We assessed RNA integrity on a Bioanalyzer (Agilent) using the RNA 6000 chip. High-quality RNA samples (RNA integrity number ≥ 8) were used for library preparation. We then constructed mRNA-seq libraries using the TruSeq RNA Sample Preparation Kit (Illumina) as previously described . The libWe mapped sequencing reads to the reference mouse genome (GRCm38) by STAR  with theTug1-/- and  Tug1-/- vs. Tug1rescue) using the generalized linear model. Statistical significance was calculated with the assumption of the negative binomial distribution of the read counts and with the empirical estimation of variance by using the R packages DESeq2 [p values by the false discovery rate (FDR) method were smaller than 0.05.We identified differentially expressed genes by comparing the mean read counts of biological replicates between the groups  to evaluate the enrichment of the gene sets available from MSigDB  after ma, CAMERA .C57BL/6J, Cast/EiJ diploid genome by incorporating single nucleotide polymorphisms and indels (obtained from the Mouse Genome Project: ftp://ftp-mouse.sanger.ac.uk/REL-1303-SNPs_Indels-GRCm38) from both strains into the M. musculus GRCm38 reference genome sequence. We created a transcriptome annotation set as follows. The gencode.vM2.annotation GTF file was downloaded, and Mt_rRNA, Mt_tRNA, miRNA, rRNA, snRNA, snoRNA, Mt_tRNA_pseudogene, tRNA_pseudogene, snoRNA_pseudogene, snRNA_pseudogene, scRNA_pseudogene, rRNA_pseudogene, and miRNA_pseudogene were removed (not enriched in our RNA-seq libraries). To create an extensive set of transcripts, we added to the gencode.vM2.annotation all transcripts from the UCSC knownGene mm10 annotation file, which are not represented in the gencode.vM2.annotation set. We also added all functional RNAs from the Functional RNA database (fRNAdb) [C57BL/6J, Cast/EiJ diploid transcriptome set.We performed allele-specific expression analysis as previously described . For mou(fRNAdb) , which dhttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore) with stringency level 3. We then mapped each of the C57BL/6J::Cast/EiJ hybrid RNA-seq libraries to the C57BL/6J and Cast/EiJ diploid genome and transcriptome splice junctions using STAR RNA-seq aligner [C57BL/6J and Cast/EiJ diploid transcriptome using MMSEQ [Each RNA-seq library was first subjected to quality and adapter trimming using the Trim Galore utility  from Riken cDNA clone E330021M17 (Source Bioscience) using specific primers containing MluI and EcoRV restriction sites  (see below).We amplified the full-length see the “” sectionTug1−/− mice (112 days old) and were homogenized in TRIzol  with a gentle MACS Dissociator . RNA was isolated by column purification  using a QiaCube (Qiagen). RNA was assessed and quantified on a Bioanalyzer (Agilent). cDNA was synthesized using 500 ng of total RNA as input with SuperScript IV VILO Master Mix with and without RT . PCR was performed on the synthesized cDNA (RT and no RT samples) using MyTaq Red Mix  with the cycling parameters: 95 °C for 1 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 10 s. PCR products were run a 1% agarose gel and verified by Sanger sequencing (GeneWiz). The following are the primers used for RT PCR: 7SK F1: GACATCTGTCACCCCATTGA and R1: TCCTCTATTCGGGGAAGGTC; Tug1 F1: CGGAGGAGCCATCTTGTCTTGTC and R1: GCTTCCAATTCCATACACACACTG; Tug1 F2: CTCTGGAGGTGGACGTTTTGT and R2: GTGAGTCGTGTCTCTCTTTTCTC.The testes were collected from wild-type and Tug1 cDNA sequences with CLC Genomics Workbench (Qiagen) for open reading frames (ORFs), allowing both canonical and non-canonical start codons . After, sequences with annotated ORFs were aligned using MUSCLE alignment. All further sequence and amino acid alignments were performed with CLC Genomics Workbench.We analyzed human and mouse Tug1 ORF1 that contained an in-frame 3xFLAG epitope tag prior to the stop codon, with and without the 5′ leader sequence (GeneWiz). We also synthesized a construct containing mouse ORF1 with an HA tag after the 3xFLAG before the stop codon, with and without the 5′ leader sequence (GeneWiz).We generated a synthesized construct for human Tug1 cDNA sequence with primers (see the “Tug1 vector plasmid using Q5 polymerase (Roche) and under the following conditions: 96 °C for 2 min, 35 cycles of , 72 °C for 4 min, and gel purified the amplicon. We digested the inserts and pcDNA3.1(+) plasmid with proper restriction enzymes according to the manufacturer’s instructions. After digestion, the plasmid was dephosphorylated using alkaline phosphatase. We then ligated the plasmid and inserts using T4 ligase (NEB) in a 1:3 ratio, respectively, followed by bacterial transformation, culture growth, and plasmid isolation (Qiagen Mini-Prep Kit).We amplified the see the “” sectionl-lysine-coated 18-mm glass coverslips. Next, we transfected the cells with 14 μg of plasmid (pcDNA3.1(+) containing each of the inserts) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) per manufacturer’s recommendations. Forty-eight hours post-transfection, cell pellets were harvested for protein extraction (see below) and coverslips were processed for RNA FISH and/or immunofluorescence (see below).We seeded 3T3 and HeLa cells in 10-cm plates containing poly-We resuspended 3T3 and HeLa cell pellets in RIPA Lysis and Extraction Buffer 48 h post-transfection (Thermo Fisher Scientific). Total protein was quantified with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). We then separated a total of 20–25 μg of denatured protein on a 12.5% SDS polyacrylamide gel for 100 min at 120 V. We transferred proteins to an Immobilon-PSQ PVDF membrane  at 400 mA for 75 min. After blocking in 5% dried milk in TBST, the membrane was incubated with properly diluted primary antibody  in 5% dried milk/TBST overnight at 4 °C. The next day, we washed the membrane three times for 5 min each in TBST (0.5% Tween-20). We then incubated the membrane with horse radish peroxidase-conjugated secondary antibody , diluted in 5% dried milk/TBST for 1 h at room temperature. Following three 5-min washes in TBST, SuperSignal™ West Pico PLUS chemiluminescent substrate  was added and chemiluminescence was detected using ImageQuant™ LAS 4000 imager. Original, uncropped images of western blots are provided in Additional file l-lysine-coated coverslips. Forty-eight hours post-transfection, we rinsed the coverslips twice with PBS and fixed the cells with 3.7% formaldehyde in PBS for 10 min at room temperature. After 2 washes with PBS, we permeabilized the cells with PBT  for 15 min at room temperature. Next, we blocked the coverslips with 5% BSA in PBT for 1 h at room temperature and then incubated the coverslips with properly diluted primary antibody  in 5% BSA in PBT for 3 h at 37 °C in a humid chamber. Coverslips were washed three times for 5 min each with PBT and incubated with diluted secondary antibody  in 5% BSA in PBT for 1 h at room temperature. Cells were then washed twice for 5 min with PBS, once for 20 min with PBS containing Hoechst DNA stain , rinsed in PBS, and then mounted on glass slides with ProLong Gold (Thermo Fisher Scientific).We plated HeLa and 3T3 cells on poly-l-lysisne-coated coverslips and transfected as described in the previous sections. Forty-eight hours post-transfection, cells were incubated with 200 nM MitoTracker red chloromethyl-X-rosamine  in 1 mL FBS-free growth media for 40 min. We then washed the cells twice with PBS, fixed with 3.7% formaldehyde for 10 min at room temperature, and processed for immunofluorescence and/or RNA FISH (as described previously).We plated cells on poly-We acquired z-stacks (200 nm z-step) capturing the entire cell volume for single-molecule RNA FISH, single-molecule RNA FISH/CMXR staining, 3xFLAG tag immunofluorescence/CMXR staining, and/or Tom20 immunofluorescence with a GE wide-field DeltaVision Elite microscope with an Olympus UPlanSApo 100x/1.40-NA Oil Objective lens and a PCO Edge sCMOS camera using corresponding filters. 3D stacks were deconvolved using the built-in DeltaVision SoftWoRx Imaging software. Maximum intensity projections of each image were subjected for quantification using Fiji.Age- and sex-matched adult mice were used in all flow cytometry experiments. We obtained peripheral blood by cardiac puncture and collected blood into a 1.5-mL Eppendorf tube containing 4% citrate solution. Next, we added the blood-citrate mixture to 3 mL of 2% dextran/1× PBS solution and incubated for 30 min at 37 °C. The upper layer was transferred to a new 5-mL polystyrene FACS tube  and centrifuged at 1200 rpm for 5 min at 4 °C. We then lysed red blood cells for 15 min at room temperature using BD Pharm Lyse . Cells were washed twice with staining media  containing 2% FBS and 2 mM EDTA). The following antibodies were added (1:100) to each sample and incubated for 30 min at room temperature: Alexa Fluor 700 anti-mouse CD8a , PE/Dazzle-594 anti-mouse CD4 , APC anti-mouse CD19 , Alexa Fluor 488 anti-mouse NK-1.1 , and PE anti-mouse CD3 , and Zombie Aqua Fixable Viability Kit  was used as a live-dead stain. We washed samples twice with staining media and sorted directly into TRIzol LS using a BD Aria FACS.We isolated and quantified RNA from sorted blood populations as described in the RNA Isolation and RNA-Seq Library Preparation. One hundred nanograms of total RNA was used as input to generate cDNA using SuperScript IV VILO Master Mix , according to the manufacturer’s protocol. cDNA was diluted 1:3 with DNase- and RNase-free water, and 1 μL was used per each reaction. We performed qRT-PCR using FastStart Universal SYBR Green Master Mix with ROX  on a ViiA 7 Real-Time PCR System (Thermo Fisher). Analysis was performed using the ∆∆Ct method . PrimersAll primary RNA-seq data are available at the Gene Expression Omnibus (GSE124745 and GSE88819).GAGACACGACUCACCAAGCACUGCCACCAGCACUGUCACUGGGAACUUGAAGAUCCAAGUUUCUGUCCAGAACCUCAGUGCAAACUGACAACACUCCAUCCAAAGUGAACUACGUCCCGUGCCUCCUGAUUGCUGAAUGUUCACCUGGACCUGCCAAUGACCUUCCUUCUGCUACUCCAUCAGCCUACAGACCUGGUACUUGGAUUUUUGUCCAUGGUGAUUCCUUCCACCUUACUACUGAAGAAGACACCAUUCCAGUGGACCACUGUGACCCAAGAAGCAUUCAGCCAUCAUGAUGUGGCCUUUACCUCCACUCCUGUCCUACUCUGCCCAGAUUCAGCACAGCCCUUUAUAGUGCAGUCAAGAGUCUUCAAGCCAAAUAACUGAAGCUAUUUUAUCACAACAAAGGCCAGGUUUAUUCCAUAAAUGUACAGUUCAUUUCUGCAGUUUAUUCUUCAGAGACACAUAGUAAAUUUGGACCAGGGGAUUUUGTug1_5190-5166TD_Forward:TGACTGGCCCAGAAGTTGTAAGTug1_5190-5166TD_Reverse:GCAAGCAGGTCTGTGAGACTATTClacZ_5_Forward:TTGAAAATGGTCTGCTGCTGlacZ_5_Reverse:TATTGGCTTCATCCACCACAYchr_Forward:TCTTAAACTCTGAAGAAGAGACYchr_Reverse:GTCTTGCCTGTATGTGATGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGGGGTTTTTTGTTTTGTTTTTTAAATTGAAGGCTAAAGTTTTTGAAAAAACTTTGTTGGACTCTGGCTGGGACACAAAATCAGATATTTGGAATCATTTTGAAGCTTAACTTTTTCCTAACCAGCCTTGTATTCTAATTGCTTGCAAATGTGAGACTGAATGGCCAAAATGCCGTTTGTTTGTTTGTTTATTGTCAGCTGCTTTTATCAAATTCCAGGCCATTATCCAGCAAACACTATTAAAATGTTTGAACAGTTGGGTTTCAAACATTTTTGTTTTGTGGAGTGGTGCTTATTAAGTGGTACAGCTCTCTAAGCAAGTGAACACAAACATATTTAAGTGTATTTTGTATGATTAGATGTTACCAATTCTGATATTTTATTCAAATGTCTAAAAAAATAAGTTGACTTATTCCCTTTACCAAAGGGCCAGAGACAAATGGTTTCCTTTCAAGAGAAATGACTGTTTTGAAGAAAAACTCTGTTGGTCTTAGCTCTTTTGTAATTAAATCTGGATGTACCTCAAAAGACTCTTTAAGACTGTGGTGTTAAAAGGCTTTCCTCTGGAGAAGGAGAAAAAATAAAATCAACTGGAACTTAAAAGCTTGAAATTCCATGACAAAACACAGATGTCCAGGATTGGAGGTTCATAAAGTACATGCAGTAGTTGGAGTGGATTCCATTTTCAGTGTAGCTGCCACCATGGACTCCAGGCTCCCAGATTTTCAAGAACTGGACCTGTGACCCAGAAGAGCTTGTCAAGATATGACAGGAACTCTGGAGGTGGACGTTTTGTATTCAATTTTGGAACTGTTGATCTTGCCGTGAGAAAAGAGAGACACGACTCACCAAGCACTGCCACCAGCACTGTCACTGGGAACTTGAAGATCCAAGTTTCTGTCCAGAACCTCAGTGCAAACTGACAACACTCCATCCAAAGTGAACTACGTCCCGTGCCTCCTGATTGCTGAATGTTCACCTGGACCTGCCAATGACCTTCCTTCTGCTACTCCATCAGCCTACAGACCTGGTACTTGGATTTTTGTCCATGGTGATTCCTTCCACCTTACTACTGAAGAAGACACCATTCCAGTGGACCACTGTGACCCAAGAAGCATTCAGCCATCATGATGTGGCCTTTACCTCCACTCCTGTCCTACTCTGCCCAGATTCAGCACAGCCCTTTATAGTGCAGTCAAGAGTCTTCAAGCCAAATAACTGAAGCTATTTTATCACAACAAAGGCCAGGTTTATTCCATAAATGTACAGTTCATTTCTGCAGTTTATTCTTCAGAGACACATAGTAAATTTGGACCAGGGGATTTTGTTTTGTTTATATTGTCAACACTGTCTGAAGAAAGGCATCTCTGAGAACAGCATTGGACCCTACTCCACAATCTCAAATGATTGAAGTTTCATAAACTGCCTAGGATCCTGTCAAGGCCACTGGACTCTTGTTCTTTTCCTACTTCAAAATCTGTAGCTGTCTACTAAATGACAAAGCAGATATTCTGACCCATTGGGATCAAAACCAAGGCATTTTGAATTCCTCATAGTATCATCTTCGGGTTACTCAGGAACCAAAACTTTTCACACCAATTTAAGAAATTCTACTGAGGAATCCCTTTACCTAACCATCTCACAAGGCTTCAACCAGATTCCTGAAAAGGCCTCTTGATATATCAAGATAGAACCTACATGCATTTTGTGAACAACTTATCACTGATTTTCCAAAGGCTTTGTGCTCTTGAAGTTCTTTGAAGGAAAGCTGTGTGGAAGTCCAGAGTAAAGTGAAGCTGCTCTGGATGAAGTAGTGAAGTGGGAGTTGAGGTCTACAACCTGCCACAACCATCTTCCTTTACCACCATGGTGATGCCAAAAGGGACTTCCTTAAAGCTCTTCAGAAAATCCTGCTTGAAACCACTACCCTAGACAACATGTTTGACCTGGATGGCATTCTCTTCAAAACAATTCATATTCAGTTGATGCTCAACATGTTTGGAGATGCTTTATTCAGAGAATGATGATAATTACAGCATTGTCTAATGAAGTTTTATTAATAGCATTCCATCCAAGGTGGACTTCCTGGAGTTGGATATAACCAGAGAGCAATTCATATGTATCCTACACTGAAGAACACCATTAACTTTCAGCAACCTATAGCTAGTGGTACTAGAAGTACGTGTCTTGGAAGTCTATGAGAGCTGGTATTGAAGCTGATGCCTCCTTAAGGCCATCTTAGACCAAGTTGTTTGTTTGACCTCTCCTCATTAACTATGGAGCAGAATTGAAATACAAATTTTTCCTAAAGGGACTTGCAACCTGGTTATCATTCATTATCTCAAGTTTCAAGTCATGTTGATGCAACCAGTAGTTATTAAACTGCTCCATGGTTTTTTGTTATTTAATACTTTTTCCAGGGCTTAAAAAAACAAAATTAAATTTCTCCAACACGTCTATACTTGTCTGTTCAAAAGTAACTACTCACCACTATATGGAACAGATGATTCTGAAGACACTCTGAGCATCCTTTATGATATTTGTGACTTAAAATGTGGCTGGAAATTTTCCTTCTACCCAGTGAAATATTTAATGATTAGTCTTCATGCCTGATACCATCAACTGTATATGCGTGATAGGCAAAGTTTGACATAGGCATTTGACTCTAGGCTATGATAGCTTGCTAGTAACTTCAAGTAGCATATTGTCAACCTGTTTGCTGGAAAAGTAGAGTAACTTGGAAAAAAAACTAAATGGCAGCTAAGGATTTTTTTCAGTATTCCTGAGTTTCTGTCCTTGGGATATTTCAATGAAATTTTCACCTGTCTCTTCACTTAACAGAGTGACTGACTCCTTACTATGAAGTATTCTTAAGACATTAAGATTACTTTTGTAGAAAGGATAAAATTCCTGACCATCCAAATCATCATAGTGAACAAGACTTCAATTTGTGACCTGAGAAAATCTCATTTCTCTACTTCGTAGTCAATGTAAGGGCCAATGCTATCAGCTACTCTGAGTGCACTGGGTAAACGTTGGAACTGCCTTCTTTATATCATTACTTTTTATCCTCTAAATTAATCATGGTTATGTAATTCTCGCCACAAATCAGCAAATCAGACTCAGATCTGGTTATTCTAGACTGCTCACAGTTAACAAATCAAACTCTGGATGACTTCTGCTTGTATATGCAACTACTATTTGTAAAGAAATTGCAAATTCACTTTTCTATTACCTCTACATTGCTAGCTCTTTCTTTTGTGTTTGTATTAAAAACAAAAATAAGCTACACTGCCAGCTATTCCCTCCTGCCATACTCAGTTAAAATGAAGAATCGGGAATCTAACCAGTGAATGGATAAGTAGAAAAAACTAAAACTTAAGGCAAAAGCCTTAATCTAGGGCCTTTTCTACTATCTTCATGTCTTGGATTTCATCTAAAATCAACAGTGCCACCCAACCAGTCTGAGGTCTTGACTTGCTTTTAAGATGATTCTTAGAGATGGGCTGTATTACAGAAGGTGAAGACTTGATTACCAAAGAAAGTAGAGCCAACTTTGACAAACCTGGCTCTACAATCCTATTGCTTCCAGATGTAGCATAGACTCATAACTAGAACCTCAAGTCTGCATTGAGGATATAGCCTTCTAAGCTGACAGTTCTTGCAACAGGTGAGCAAGAAAATGAAAGCTGTTATACCCAACTGGCCCTTTAAGATCCAAAAATAATGTCTGGACTAAACCCTATGGAGTACCCAGGACAAAAACTAATTTACAGAGCTTCATTATTAATCTGCCTGTTCTTCTAGCTTAATTATTGGTATGGCTGGCCCTACTGAAGTAGTTTGTCTGTTTACCTGTCTTCAGCTCTTAACCTGGCTATTTTGACATGCTACTGCAATTAGACTAACTGGCTTTGAGAAGACTACAATCAGTTTCAGCCTCTCCTTTGCCCAATTTCACCAAGGAATTTTGATAAGAGGAACCCATACCTCACCCCACCAGAACAGAAAGGACCATGCTGCATATTCCTTGACCAGCAACTTTAAGTAGAGAACAACCCTGCTTGTTTTCAACATCTGAAACACCATTTGATCTAATAGGAGTATAGAAGGTTGACAGCAGAGTACACTACTTACTTCTTTCATAACTCAGAAATGAATATGACTGGCCCAGAAGTTGTAAGTTCACCTTGACAAGAAACAGCAACACCAGAAGTTTACTGCTGAACTTAACTTGCCACTTACTCGAATAGTCTCACAGACCTGCTTGCCAAGTAGGAGGCTAGTTTTCCTGCTTCATATCACCATTGGAGTGGGGCTCAATGGGGTCAATGTTAATACTGACTTGAATGGGGACCTTATGGTGAATCCTAGACTATGAGGCTAATGGAAATTATTGTCTATTCAAGTGGATTATAGATTTCCTGAGGACAGAACAGACATCACTCCTGGTGATTTTTAGAACTTGATTACCAAGGAAGAAATACCAGCTGCTAACAGTCAACTTCATGGGCAAAGATTAAGCTCTCTATATCTGGTCGTATCCTGGATGCTAGTTTTTTATTGCCCAGTGACCATTTCCATCTCACGCTTAACTTCCTGATGTTTTTTGGAACCATCTCTTCCAATTTTCAGTCCTGGTGATTTAGACAGTCTTTTCATGCTGGACATTTTGTTGCAACCTCATCAATCACAGCAAAGTCCATCTTGACTTTAGTGATTAGTTCAGGAATGGATGCATGATTCAAGTTTGTCCAATGATAATCAACCCTAGGTGTTTTCTCAGTTGTGGAGAAGTTCTCTTAGATGCTTTAGCTTTGTAGGAGAAAACTCAAACCAACAGGGCCTACCTACTATGTTGAATGATTGTAGGAGAAAACTCAAACCAACCAGGCCTACCTACTATGTTGAATGAGCCAGGCAGAAAATGAAGCCAGTACAGAGGGAAATGGAGCCAAAAGAGGAAGAGACTTGAGTTCTGATGATCACATTTATGCCCCTGTATCCAACTGTGCCTGAAGCTAATAGTACATCACCTGGACTTTTCAGTTATGTGAACCAATAAATTCCCCTTTTTGTTTAAGTTACTTTGAGTTTug1_MluI_cDNA_Fwd gagaacgcgtTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGGGGTTTTTTGTTTTGTTTTTTAAATTGAAGGCTAAAGTTTTTGAAAAAACTTTGTTGGACTCTGGCTGGTug1_EcoRV_cDNA_RevgagagatatcAACTCAAAGTAACTTAAACAAAAAGGGLNCXAGCTCGTTTAGTGAACCGTCAGATCTUG1_76TTTAAATTGAAGGCTAAAGTTTTTGAATUG1_266GGCCATTATCCAGCAAACACTUG1_755ACTCCAGGCTCCCAGATTTTTUG1_1254TCTTCAAGCCAAATAACTGAAGCTUG1_1741AGAACCTACATGCATTTTGTGAATUG1_2268ATGCCTCCTTAAGGCCATCTTUG1_2754TGTCAACCTGTTTGCTGGAATUG1_3267TTGCAAATTCACTTTTCTATTACCTCTUG1_3746CCCAACTGGCCCTTTAAGATTUG1_4241TGACAAGAAACAGCAACACCATUG1_4740TCACAGCAAAGTCCATCTTGATug1_Tg F/KpnIataggtaccGCCCCGAATTCACGCGTTTug1_Tg R/NotIatagcggccgcACCTGAGGAGTGAAGATug1_FwdCTCTGGAGGTGGACGTTTTGTTug1_RevGTGAGTCGTGTCTCTCTTTTCTCGapdh_Fwd GGTGAAGGTCGGTGTGAACGGapdh_Rev CTCGCTCCTGGAAGATGGTGHuman ORF1:Human ORF1+UTR:Mouse ORF1:Mouse ORF1+UTR:Additional file 1:Fig. S1. Mouse and human Tug1 locus and chromatin context in different cell types. (A)Tug1 mouse and (B) human genomic loci. Evolutionary nucleotide conservation (PhyloP) of the locus are presented along with the chromatin context  and protein binding ChIP-seq peaks  from ENCODE  datasets in the indicated cell types.Additional file 2: Fig. S2. In vivo expression pattern of Tug1 during murine embryogenesis. RNA in situ hybridization of Tug1 RNA using a digoxigenin-labeled antisense RNA probe in mouse embryos at different developmental stages. Embryonic day (E)8.5, E9.5, E10.5, E11.5, and E12.5 are shown.Additional file 3: Fig. S3. Overview of the Tug1 locus in mouse. UCSC genome browser showing the murineTug1 locus. The three predominate Tug1 isoforms are depicted (black) and the Tug1 transgene (tg(Tug1)) is shown (blue). For Tug1 knockout, the longest annotated Tug1 isoform was replaced by a lacZ reporter cassette, leaving the promotor and first exon intact. The deleted region is indicated by red dashed lines. The open reading frame (ORF) encoding the TUG1-BOAT protein and PhyloCSF scores for the (-2) frame across the locus are depicted (grey). Chromosomal coordinates (mm10) are shown.Additional file 4: Fig. S4. Morphology analysis of Tug1-/- mice and sperm (A) Body mass (g) measurements over 11 weeks of male and female Tug1-/- mice compared to wild type littermates. Males: Tug1-/- (n = 7); WT (n = 8). Females: Tug1-/- (n = 3), WT (n = 7). Significant p values at specific time points are indicated (*). (B) Representative images from adult male mice (12 weeks old) show normal physiological appearance of external genitalia and reproductive tracks in Tug1-/- compared to WT. Seminal vesicles (SV), vas deferens (VD), bladder (B), testicle (T), epididymis (E), anterior prostate (AP). (C) Box plots of body mass (g) (left panel), relative testis mass  and total sperm count for wild type (n = 9) and Tug1-/- males. (D) Box plots of the percentage of different sperm morphological abnormalities for wild type (n = 9) and Tug1-/- (n = 8) males. Significant (*) p value (Wilcoxon rank sum test) is indicated.Additional file 5: Table S1.Tug1-/- and wild type sperm morphological defects.Additional file 6: Table S2. Testes RNA-seq and Tug1rescue RNA-seq in testes.Additional file 7: Table S3. Prostate, spleen, eyes, brain, heart, liver, and MEF RNA-seq.Additional file 8: Table S4. Allele-specific RNA-seq in testes, expression data.Additional file 9: Table S5. Allele-specific RNA-seq in testes, differentially expressed genes.Additional file 10: Fig. S5. Gene expression of multiple tissues in Tug1 WT and KO mice. Gene expression of multiple samples (columns) were described with log scale of TPM: Log2(TPM+1). Genes (rows) are clustered by hierarchical clustering with Ward’s method based on Euclidean distance. Annotation of samples are provided in the top panel in terms of genotypes and tissues.Additional file 11: Fig. S6.Tug1 transgene expression and fertility assessment. (A) qRT-PCR for Tug1 RNA expression in testes and sorted peripheral blood populations: WT (n = 1), Tug1+/- (n = 1), Tug1-/- (n = 1), and Tug1rescue (n = 1) and sorted peripheral blood populations. Error bars indicate the relative quantification minimum and maximum confidence interval at 98%. Not detected (n.d.). (B) Representative flow cytometry gating strategy for NK, CD4, and CD8 cells in peripheral blood from WT, Tug1+/-, Tug1-/-, and Tug1rescue mice . (C) Scatter dot plot (mean with standard error of the mean shown) of the number of pups at birth per copulatory plug for matings using male wild type, Tug1+/-; tg(Tug1); rtTA, Tug1-/-, or Tug1rescue (on dox diet) with wild type C57BL/6J females. Each dot represents a litter from a different mouse. (D) Sperm count from control , Tug1-/- (n = 2), and Tug1resuce (n = 3) mice. Each dot represents a different mouse and the error bars indicate the standard error of the mean. (E) Hematoxylin and eosin staining in Tug1+/-, Tug1-/-, and Tug1rescue testes and epididymis. (F) Morphological analysis of sperm from Tug1-/- (n = 2), and Tug1rescue (n = 3) mice.Additional file 12: Fig. S7. Changes of gene expression in two comparisons of Tug1-/- (KO) vs. WT and Tug1rescue (Rescue) vs. Tug1-/- (KO). Each dot represents a gene whose x-axis value is the fold change of gene expression between KO and WT and y-axis value shows the fold change in Rescue vs. KO. Color describes statistical significance of fold change : no statistical significance in either comparison ; significance in KO vs. WT ; significance in Rescue vs. KO ; significance in both comparisons, KO vs. WT and Rescue vs. KO .Additional file 13: Fig. S8. The 5’ region of human TUG1 contains a conserved ORF. (A) GWIPS-viz tracks for human TUG1 genomic locus (hg38) is shown. Global aggregate of ribosome occupancy (ribosome profile), RNA-seq (mRNA coverage), and evolutionary protein-coding potential (PhyloCSF) across the TUG1 locus is shown. ORF1 and ORF2 are outlined with red and gray boxes, respectively. Tracks surrounding both ORFs are zoomed in for clarity (bottom). (B) Scheme showing human ORF1 construct design. hORF1 (labeled with a 3xFLAG epitope tag prior the stop codon) with the 5’UTR was inserted into pcDNA3.1(+) and transfected into HeLa cells. 48 hours post-transfection, TUG1-BOAT-3xFLAG localization was analyzed by immunofluorescence (IF) (shown in C). (C) Maximum intensity projection of HeLa cells expressing human 5’UTR-hORF1-3xFLAG. Localization of 3xFLAG tagged TUG1-BOAT was assessed by immunostaining against the 3xFLAG (green). Nucleus was monitored by DAPI (blue) and mitochondria was monitored by immunostaining against mitochondrial membrane translocase TOM20 (gray). Bar plot shows localization analysis of TUG1-BOAT. Scale bar is 5 μm.Additional file 14: Fig. S9. GFP over-expression does not compromise mitochondrial membrane potential. Maximum intensity projections of z-stacks acquired 48 h post-transfection of 3T3 cells with GFP cloned into pcDNA3.1(+) under CMV promoter and staining with Chloromethyl-X-rosamine (CMXR). GFP (green) was used as control. CMXR is shown in red, DAPI in blue. On the right, quantification of cells expressing GFP and mitochondria membrane potential by CMXR (n = 50). Scale bar is 5 μm.Additional file 15: Fig. S10. Loss of TUG1 expression in infertile human males. Heatmap of microarray data from three different probe sets showing decreased expression of TUG1 in sperm from infertile teratozoospermic men (T) compared to fertile (normospermic) individuals (ND). In all cases, p < 4.39 x10-4.Additional file 16: Fig. S11. Uncropped western blot images from Figure (A) Western blot of constructs overexpressed in 3T3 cells targeting the 3xFLAG tag (top). GAPDH is used as a loading control (bottom). (B) Western blot of constructs overexpressed in HeLa cells targeting the 3xFLAG tag (top). GAPDH is used as a loading control (bottom).Additional file 17. Review history.


In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm Single molecule observation has been limited to tethered molecules to ensure that the target remains in the field of view (FOV). Here, the authors develop a real-time tracking method that locks onto rapidly diffusing targets and tracks them in a 3D volume, enabling single molecules to remain in the FOV for minutes at a time. Valuable insights have been gained in a wide range of sub-disciplines, ranging from DNA transcription5 to enzyme catalysis8, among many others. To date, the vast majority of single-molecule investigations have been restricted either to short times (using a tightly focused laser spot to monitor single molecules which transiently diffuse through the observation volume) or rely on tethering the molecule to a surface . The former solution-phase measurements acquire limited information due to the rapid diffusion of molecules through the focal spot. The latter tethered methods require isolation of the molecule from its environment, precluding observation of the native functions of molecules such as enzymes, which may critically rely on the crowded and non-equilibrium surroundings of the cellular interior. Several methods have sought to overcome this need to tether molecules, including liposomes9, convex lens induced confinement (CLIC)11 and the anti-Brownian electrokinetic (ABEL) trap16. Although each of these methods remove the tether, they still restrict the molecule in an isolated environment and cannot continuously monitor unbound diffusing molecules, such as an enzyme freely diffusing through the cellular interior.Single-molecule spectroscopy has been a critical tool in investigating molecular dynamics in biological systems17. RT-3D-SPT acquires high-speed position measurement of a diffusing particle and implements a closed feedback loop to effectively lock the rapidly moving target in the observation volume. Feedback is applied using either piezoelectric nanopositioners or galvo mirrors18, meaning the target molecule or particle is followed at very high speed, rather than being physically confined. Pioneering work has shown that RT-3D-SPT can lock-on to a wide range of targets, from gold nanoparticles20 and single quantum dots23 all the way to viruses26, and is easily extended to live cell tracking28. This is because the “confinement” is not a physical impediment to the target, but rather a high-speed chase which follows the particle without perturbation.A group of methods with the promise to break this tether is real-time 3D single particle tracking (RT-3D-SPT)27. While truly a groundbreaking achievement for RT-3D-SMT, the trajectories were limited to several 100 ms. The same group followed-up 2 years later, showing the ability to disentangle the oligomerization states of azami green, though still in 90 wt% glycerol and still for only several 100 ms29. Further experiments with the same method have shown an expansion of the target scope (FRET), but with no increase in the trajectory duration despite still working in highly viscous solutions30. Similar results were observed by Liu and coworkers for single-labeled DNA31.While the pioneering work on RT-3D-SPT by several groups occurred more than a decade ago, the extension of this promising method to real-time 3D single-molecule tracking (RT-3D-SMT) has been limited  or the bleaching rate . Starting with the latter case, it is certainly possible that these methods suffered from bleaching, leading to the short trajectories. However, it is also possible that the reduced emission rate of single molecules, compared to multiply labeled fluorescent beads, could lead to an inability to lock-on to single molecules for any appreciable period of time.What has limited RT-3D-SPT methods from achieving long and stable RT-3D-SMT trajectories? A commonality among the methods introduced above is the need to slow down molecular diffusion by increasing the solution viscosity for successful tracking. This is not surprising and would presumably be necessary, given that a piezoelectric nanopositioner is typically bandwidth limited to about 1 kHz . Photon arrival times detected by an avalanche photodiode (APD) are converted to particle positions in real-time on a field-programmable gate array (FPGA) using a Kalman update filter with assumed Gaussian density of all likelihoods34. The results of 3D-SMART can be seen in Fig. 37. As a result, avoiding the excitation and allowing relaxation of the triplet state can reduce the rate of photobleaching. Pulse optimization to allow for dark state relaxation has led to increased photon yields in both confocal laser scanning39 and STED microscopies41. The fast, periodic illumination pattern of 3D-SMART likely reduces photobleaching in a similar way, where the relatively long time intervals between illumination of the tracked molecule permits triplet relaxation. The high concentration of glycerol may also play a role, as has been demonstrated in fluorescent proteins42.3D single-molecule active real-time tracking (3D-SMART) was achieved by applying these optimized parameters to the previously reported 3D-DyPLoT2/s, corresponding to a hydrodynamic diameter of 0.98 nm, well in line with what is expected for a single molecule . Applying the Stokes–Einstein relation reveals a measured diffusion coefficient of 2.44 μmule Fig. . A furthule Fig. –7. PhotoAn important consideration in any single-molecule study is the throughput. For a method which collects serial single-molecule traces to compile statistics, the microscope must be capable of capturing many molecules in a row, with minimal down time between trajectories. This is true of the 3D-SMART method. Figure The data presented above demonstrate that 3D-SMART only requires a single fluorophore for continuous observation of a molecule diffusion three dimensions. As such, the application possibilities are expansive. Here we demonstrate the applicability of this method to proteins, DNA, RNA, and even multicolor dynamic processes such as DNA transcription. Figure 44 as compared to a more sophisticated wormlike chain model which takes chain stiffness into account45. We found that the 3D diffusion of the various lengths of dsDNA, as measured by 3D-SMART, did not conform to the simple model of a flexible polymer put forth by Rouse and Zimm, but agreed almost exactly with the WLC model developed by Yamakawa and Fujii  in solution at high temporal resolution for long times enables examination of different models for the diffusion of semiflexible polymers. By tracking different lengths of dsDNA , longer dsDNA (547 bp) still shows good agreement with the WLC model, indicating that no change in the dsDNA conformation as function of glycerol concentration can be detected within the resolution of these measurements. This demonstrates that single fluorophore tracking is achievable in low viscosity environments at high diffusive speeds.Furthermore, dsDNA tracking is useful for extrapolating the speed and size limits of 3D-SMART. For single dye molecules, the required glycerol concentration for successful active feedback tracking is 90 wt% glycerol. For single proteins (BSA), it is 73.5 wt% glycerol. For dsDNA at a length of 1385 bp, with a single fluorophore labeled at the terminus, active feedback tracking is achieved in PBS . Upon binding, the chromophores are stabilized by the aptamer and go from nonfluorescent to fluorescent. By tracking individual aptamers, the number of bound chromophores can be extracted  pair to act as a fluorescence in situ hybridization (FISH) marker. Upon transcription of the DNA by the RNA polymerases, the FISH probe complements to the nascently transcribed RNA, separating the fluorophore from the quencher and resulting in a burst of fluorescence  to reduce fluorescence background, resulting in a low transcription detection rate in these experiments (6 of 628 trajectories) since hybridization rate is only 1.2 × 10−3 s−1 without the presence of glycerol. Moreover, the presence of glycerol will further decrease the hybridization kinetics55. Considering the low probe binding rate in the experiment, the start points of the transcription signal in Fig. 54 the binding of the fastFISH probe after the transcription has already started. On the other hand, the Alexa 488 bleaching time , so the disappearance of the signal should indicate dissociation of the transcribed RNA from the diffusing dsDNA. The results of this assay can be seen in Fig. 2/s to D ~2.5 µm2/s, Fig. E.coli RNAP resulted with no transcription bursts associated with probe binding observed.As a final demonstration of the power of 3D-SMART, we show the observation of a compete biochemical reaction at the single-molecule level in solution Fig. . To do skept low 0 pM to r2/s, all with photon-limited temporal resolution. 3D-SMART has the power to completely untether single-molecule experiments. This opens the path towards measuring the fast internal molecular motions of single proteins in the complex cytoplasmic milieu, though this represents only a tiny portion of what 3D-SMART can enable. One reason single-molecule observation has been so powerful in biological systems is that molecules are intrinsically “confined” to the cellular container, making observation over long times more feasible. 3D-SMART can extend single-molecule spectroscopy to completely unconfined systems, going beyond molecular biology to synthetic chemistry  and condensed matter physics . Untethering single-molecule spectroscopy in this way is critical towards increasing the application scope of these powerful methods.The above demonstration of 3D-SMART shows a dramatic improvement in tracking speed and information acquired from single molecules in solution, enabling continuous observation of a single, freely diffusing dye molecule for up to minutes at a time and diffusion coefficients up to 10 µmf = 150 mm  and f = 200 mm , and a pinhole with 75 µm diameters  are used to collimate and purify the 488 nm beam. The polarization state of 488 nm beam is cleaned by a Glan-Thompson Polarizer  and tuned by a half-wave plate . For 640 nm laser beam, two lenses with focal length f = 100 mm  and f = 150 mm  and a pinhole with 100 µm diameters  are used for collimation. The polarization state of the 640 nm beam was cleaned by a polarizer  and tuned by a half-wave plate . A dichroic mirror  is used to combine the 488-nm beam and 640-nm beam. The beams then travel through two electro-optic deflectors . The EODs are used to produce the designed scanning pattern. A lens pair  is placed after the EOD to act as beam expander. The TAG lens  is used to modulate the beams focal position dynamically. The TAG lens has a dynamically changing focus with low aberration performance and up to megahertz frequencies. Here we run the TAG lens at the ~70 kHz resonance. The beam from the TAG lens is relayed to the microscope objective lens  by two f = 200 mm focal length lenses . A dichroic mirror  reflects the beam towards the back aperture of the objective lens. A bandpass filter  separates the fluorescence emission from the excitation lasers. For two color experiments, a dichroic filter  separates the fluorescence onto different detection paths. Two APD are used to measure fluorescence signal with a lens with focal length 50 mm  installed before each APD. A micropositioning system  enables coarse sample positioning. A 3D piezoelectric stage system  is used to move the sample and objective lens for active feedback 3D tracking. In photon antibunching experiment, a 50/50 beamsplitter  was used to split Atto 647N fluorescence onto the two APDs and a time-correlated single-photon counting model  tags the arrival times of fluorescence photons in each APD. An IR blocking filter  is placed in front of one APD for preventing optical cross talk between two detectors.The 3D-SMART setup uses a 488 nm frequency-doubled solid-state laser  and 640 nm diode laser  for excitation. A telecentric system, consisting of two lenses with focal length A field-programmable gate array  based data-processing unit controls the EOD scanning, collects photon counts from the APD, interpolates the TAG lens signal, calculates the particle position estimates, applies position feedback with the piezoelectric stage, and records the particle position information. The system operates in custom software implemented with LabView 15 .For Alexa 488 tracking, the laser power was ~1.7 µW at the objective focus. For Atto 647 N tracking, the laser power was ~2 µW. For mRNA-DFHBI-1T tracking, the laser power was 1.9 µW. All the tracking experiments were performed at room temperature (~23 °C).Atto 647 N NHS ester  or Alexa 488  was first dissolved in Dimethyl Sulfoxide  at 5 mg/ml and aliquoted into 5 µl for each vial and stored in −80 °C. Then the dye was diluted into 10 pM with distilled water. Then 1 mL dye solution was mixed with 9 g glycerol  and added 400 µl solution to a home-made sample holder for 3D-SMART experiments  was dissolved in DPBS  at 2.6 mg/ml (40 μM). Then 40 μl BSA was mixed with 1 μL Atto 647 N NHS ester or Alexa 488 NHS ester (5 mg/ml). The mixture was incubated for 2 h (25 °C) on shaking stage. The labeled BSA solution was then purified by Zeba spin desalting columns  according to the manufacturer’s procedure. The purified BSA solution was diluted to 2–5 pM in 73.5 wt% glycerol-PBS solution. 400 µL of the solution was added to the home-made sample holder for 3D-SMART experiments.To tag the DNAs with fluorescent molecules, DNAs were modified with amino (synthesized by Integrated DNA Technologies). To label the amino modified DNA with Atto 647 N NHS or Alexa 488 NHS, the amino modified DNA was dissolved in PBS at a concentration of 100 µM, then incubated with dye at a molar ratio of ~1:5 for 2 h (25 °C) while shaking. The labeled dsDNA was then purified with NAP-5 G25 column  according to the product manual. All the amino modified DNA premers and single DNA strand were labeled with fluorescent molecules before the PCR amplification and hybridization.For 99 bp dsDNA tracking the following sequence was used (synthesized by Integrated DNA Technologies): 5′-TAT GTA ATT GGA GTG GTT AAG ATA AGG GAT AGG GTG AAA TTG TTA T /amino/ C CGC TCT CAC AAT TCC ACA CAT TAT ACG AGC CGA AGC ATA AAG TGT CAA GCC T-3′. Two complementary DNAs were mixed in PBS buffer with a concentration of 1 μM and then went through the following annealing procedure: (1) heat to 95 °C and maintain the temperature for 5 min; (2) Cool to 4 °C maintain the temperature for temporary storage.42), except for a small alteration in the initial transcribed region (ITR). Region −200 to +325 containing the T7A1 promoter region and the probe binding site was synthesized by Integrated DNA Technologies (IDT) as double-stranded DNA delivered on a pUCIDT plasmid. The sequence was then excised with XbaI and HindIII and inserted into pT7-7 plasmid (Addgene #36046) between the XbaI and HindIII restriction sites to generate a plasmid template construct (pT7-fastFISH (pT7-fF)).For 547 bp dsDNA tracking, the following sequence was used: 5′-CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAaaagacgccttgttgttagccataaagtgataacctttaatcattgtctttattaatacaactcactataaggagagacaacttaaagagacttaaaagattaatttaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccatgtagtaaggaggttctaatagccatcccaatcgacaCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAACCTTTTAGATACCTGATACAAAGTCCATTATGATTTTTAGATTTCGTATATTTACACTTGCACCATACGCATGTAAAATTAGAAGCAAAGTACGATTCTTAGACCGATGTATAATATAATTATGTAGATGTGATGAGTTTCTTTTATATGCTTCACCTGTCGGATCGGTCTGCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGAA-3′. Here, lower case letters indicate the T7A1 promoter region (−163 to +38 from the transcription start site represented by the bold typed a). Region −221 to +91 is the same template sequence as previously reported . The underlined portion indicates the “Spinach 2” aptamer sequence40. Region +95 to +374 containing (linker-Spinach 2-linker-Spinach 2-linker) was synthesized by GenScript as double-stranded DNA delivered on a pUC57 plasmid. This construct was inserted between the probe-binding site and HindIII restriction site of pT7-fF to create pT7-Spi2-2R. The final plasmid template construct pT7-Spi2-8R, containing eight “Spinach 2” aptamers, was created using the repeat expansion method57. The 1385 bp DNA transcription template was amplified by PCR from pT7-Spi2-8R using the Primer 1 and Primer 2.For 1385 bp dsDNA tracking and DFHBI-1T-mRNA tracking experiments, the following sequencing containing eight spinach2For ensemble and single-molecule diffusion fastFISH experiments, the following sequence containing the single probe-binding site was used:atgtagtaaggaggttctaatagccatcccaatcgacaCCCTATCCCTTATCTTAACCACTCCAATTACATACACCTTTCAAAACTTCAAA-3′. The 1218 bp DNA transcription template was amplified by PCR from pT7-fF using the following primers:5′-GCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGATCTGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCCCAGCGATTCGAACTTCTGATAGACTTCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATAaaagacgccttgttgttagccataaagtgataacctttaatcattgtctttattaatacaactcactataaggagagacaacttaaagagacttaaaagattaatttaaaatttatcaaaaagagtattgacttaaagtctaacctataggatacttacagccPrimer 3: 5′-GCTAATCCTGTTACCAGTGG-3′Primer 4: 5′-/amino/TTTGAAGTTTTGAAAGGTGTATG-3′a). Here, the underlined portion indicates the oligo probe-binding site. The self-quenched oligo probe is 5′-/Alexa488/GTTAAGATAAGGGATAGGG/RQ/-3′. RQ indicates Iowa Black RQ.As above, lower case letters indicate the T7A1 promoter region .For 1385 base pairs DNA tracking in PBS, the DNA was firstly dissolved in PBS and incubated for 30 min. For 99 and 546 bp DNA tracking, 70, 40 or 25 glycerol (by weight) was added after incubating the dsDNA in PBS for 30 min or by going through the annealing procedure as described above. The sample was then further diluted for tracking via 3D-SMART. The final concentration of DNA used in this work was 1–5 pM.2). The tracking solution was made by adding 30 µL of the mixture to 750 mg glycerol, 200 µL 5X binding buffer and 20 µL H2O.DFHBI-1T  was dissolved in DMSO at 31 mM concentration. Before mixing the DFHBI-1T with mRNA, the DFHBI-1T was diluted to 0.31 mM with DI water and titrated to neutral pH with KOH. Then 1.2 k bps mRNA (200 nM) was incubated with DFHBI-1T (10 µM) for 1 h in binding buffer (40 mM HEPES (PH 7.2–7.5), 125 mM KCl, 10 mM MgCln = (ImRNA – background) × IDFHBI−1, in which ImRNAis the mean intensity of first 100 ms trajectory and IDFHBI is the single DFHBI-1T intensity. Single DFHBI-1T fluorescence intensity is derived from the step-wise bleaching intensity. The mean of three stepwise bleaching steps from three trajectories was measured to be 1323 ± 93 Hz.The number is calculated by 2, 2.5 mM rNTPs , 20% (v/v) glycerol, 2.5 mM spermidine, 0.01% Triton X-100, 0.05 units YIPP (NEB #M2403S), 1 unit RNaseOUT , 1 mM DTT, and 200 ng of the 1218 bp DNA template. 2.5 units of T7 RNA Polymerase (New England Biolabs #M0251S) was added to initiate the reaction, which was then allowed to proceed at 37 °C for 2 h. To this reaction mixture the self-quenched oligo probe was added at a final concentration of 1.5 µM. The mixture was incubated at 37 °C for 30 min prior to acquiring fluorescent emission spectra.To validate transcription and oligo probe binding, bulk experiments were performed in buffer containing 40 mM Tris-HCl pH = 8.0, 25 mM MgClE.coli RNAP , 200 pM probe DNA, 80 U RNase Ribonuclease Inhibitor  and 12 pM template DNA. Before the transcription experiment, the double-strand template DNA was diluted into PBS with a concentration of 6 nM and incubated for 30 min in room temperature. Hundred and ten milligrams of glycerol was mixed with 314 µL DI water and 50 µL 10x transcription buffer. The sequentially added NTPs, RNase Ribonuclease Inhibitor, template DNA, RNAP and probe DNA. For in vitro transcription control experiment, all the experiment conditions were same as transcription experiments except without E.coli RNAP. All these experiments were performed in room temperature (23 °C).All the in vitro transcription experiments were performed in NEB’s transcription buffer  with 1 mM NTPs , 22 wt% glycerol, 100 U The transcription bursts are determined by inspection. As can be seen Fig. The 1388 bp dsDNA labeled with Alexa 488 was used to measure the photo-bleaching time of Alexa 488. The 1388 bp dsDNA was synthesized by PCR using Primer 2 and Primer 5, and pT7-Spi2-8R as the template.Primer 5: 5′-/Amino/ TTT CCA CAA CGG TTT CCC TCT AG.Primer 5 was labeled with Alexa Fluor 488 NHS using the same labeling procedure as described above, prior to PCR. 12 pM DNA was dissolved in PBS solution and then 500 μL of solution was applied to a quartz coverslip before the experiment. The DNA can bind to the coverslip non-specificity. In order to locate the dye in the center of laser volume, the feedback tracking was turned on at the beginning of measurement. Once the dye was captured, the feedback tracking was turned off and the laser will continually illuminate on the dye molecule. The used laser power is the same with that in the transcription tracking experiment.1388 bp dsDNA labeled with Atto 647 N was used to measure the tracking precision (Supplementary Fig. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Reporting Summary


The authors would like to draw the reader’s attention to the errors in the following article.J Diabetes Investig 2020; 11: 699–706.Sakurai K, Kato T, Tanabe H, Naoko Taguchi‐Atarashi N, Sato Y, Eguchi A, Watanabe M, Ohno H, Mori C. Association between gut microbiota composition and glycoalbumin level during pregnancy in Japanese women: Pilot study from Chiba Study of Mother and Child Health. On page 700, the primers used in the 16S rRNA sequencing V1‐2 region were incorrectly stated. The correct primers should have been as follows:27Fmod: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGTTTGATYMTGGCTCAG338R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCTGCCTCCCGTAGGAGTThe authors apologize for these errors and any confusion they may have caused.