Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium.We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids.The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures. There are several known methods of RNA extraction. Most require reagents such as phenol-chloroform, which according to the US Environmental Protection Agency, are toxic and have a negative effect on the environment -3. A rapLogemann et al.  describeThe quality of RNA extracted from plant tissues using the above protocol is generally high. However, this is not the case with plant species that have high phenol or cellulose contents, from which RNA is difficult to extract. RNA is even harder to extract from fungus-infected tissues, showing only variable success, with very low relative yields and quality ,7.In this work, we report on a simple, economical, fast, and relatively non-toxic high-yielding method for RNA extraction, termed LogSpin. This method combines the RNA extraction protocol described by Logemann et al.  with a sSpin columns contain a silica resin that selectively binds DNA/RNA, depending on the salt setting and other aspects influenced by the extraction method. Silica-based nucleic acid purification approaches make use of chaotrophic salts that denature proteins (including DNases and RNases) but also denature nucleic acids by disrupting their hydrogen bonding. This leads to selective binding of the nucleic acids to the silica resin in the column, and their effective separation from the rest of the sample. The nucleic acids are then washed with chaotropic salts to remove protein and pigment contaminants, and with ethanol to remove salts. After washing, nucleic acids are eluted from the column with water or low-salt solution, which induce its renaturation and thus eliminates their affinity for the silica resin.Since all nucleic acid spin columns are essentially based on the same silica matrix technology, we set out to verify whether plasmid DNA columns can be used to purify RNA. The use of a plasmid DNA extraction column as an RNA-binding device for RNA extraction was first tested by comparing plasmid DNA extraction spin columns  with RNA extraction spin columns from the commercial plant RNA extraction kit RNeasy . We compared the efficiencies of the two columns using reagents supplied with the RNeasy Plant Mini Kit, with some modifications. We extracted Arabidopsis plant tissue using 8 M guanidine hydrochloride buffer, a chaotropic salt that denatures proteins, thus inhibiting RNase, and denatures RNA so that under these conditions, the RNA will selectively bind to the silica resin in the column, separating it from the rest of the sample. The extraction buffer was also supplemented with 20 mM MES hydrate, to adjust the acidity for favored partitioning of RNA in the aqueous phase, and 20 mM EDTA, which also serves as an RNase inhibitor (β-mercaptoethanol was not added) [260/OD280 and OD260/OD230 to estimate the amount of RNA (ng/μl) and levels of protein and salt in the samples, respectively. Equivalent results were obtained for RNA from the two column types , we compared the efficiencies of five solution combinations for the washing step: (1) RW1 and RPE , 2) 3 M Na-acetate and 75% EtOH, (3) 96% EtOH, (4) 3 M Na-acetate and PB buffer , and (5) PB only Figure . We foun 3 M Na-aBotrytis cinerea. We extracted RNA from tomato seedlings and Arabidopsis leaves using the LogSpin, Tri-reagent and RNeasy protocols and obtained similar yields and purity , the RNeasy Plant Mini Kit for plant tissues and the Norgen kit for ty Table . RNA wasty Table , being cB. cinerea-infected and uninfected Arabidopsis leaves using the commercial RNeasy Plant Mini Kit yielded significantly lower RNA yield from infected tissue (9.3 ± 1.7 μg) than from its uninfected counterpart (Table ± 4.2 μg) from Arabidopsis leaves infected with B. cinerea, similar to the amount obtained from uninfected tissue (Table B. cinerea and Alternaria brassicicola) using the LogSpin protocol   and β-tubulin 2 (Tub2) genes . RNA stability after DNase I treatment was analyzed by gel electrophoresis: the RNA was found to be highly stable at 37°C for 30 min and at 72°C for 15 min. cDNA products obtained from the reverse transcriptase reaction were used in a PCR with primers for We compared RNA extraction using our protocol on different plasmid mini prep columns . From the results, we assume that the columns have the selective ability (low cutoff) to bind RNA nucleic acids preferentially over genomic DNA. Plasmid DNA extraction columns are designed to bind small DNA nucleic acid fragments . Finally, real-time PCR on cDNA synthesized from DNase I-treated Arabidopsis and rose RNA extracted by our LogSpin protocol showed consistently high-quality results  in less than 1 h (after plant tissue grinding). This allows several rounds of RNA production in the same day, providing numerous samples for treatment or use in further applications  at the same time.The third advantage of this protocol is the absence of phenol/chloroform/isoamylalcohol mixture and β-mercaptoethanol, compounds that are harmful to both humans and the environment  and vernalized at 4°C for 2 days before transferring to a controlled environment. Plants were kept in the growth room at 18°C under an 8/16 h light/dark photoperiod using fluorescent lamps . Tomato, petunia and rose plants were grown in the greenhouse at 25°C under natural daylight. Barley plants were collected from the field.B. cinerea strain B05.10 and A. brassicicola isolated in Israel were grown on potato dextrose agar  in a controlled-environment chamber at 22°C under illumination with fluorescent and incandescent light, at a photofluency rate of approximately 120 μmol/m2 s and 12 h day length. Conidia were harvested in sterile distilled water and filtered through four layers of sterile gauze to remove hyphae. For inoculation, conidial suspensions were adjusted to 3000 conidia/μl. B. cinerea conidial suspensions were prepared in half-strength filtered (0.45-μm) grape juice. Infected leaves were collected 72 h post-inoculation for RNA extraction.Guanidine hydrochloride for molecular biology (≥ 99% pure), MES hydrate, minimum 99.5% titration, and diethyl pyrocarbonate (DEPC) were from Sigma-Aldrich. EDTA was supplied from J.T. Baker , and Na-acetate was purchased from Merck . All chemicals were dissolved in DEPC-treated water.Tissue was inserted into a safety-locked eppendorf tube with two glass beads (diameter: 5-10 mm), frozen immediately in liquid nitrogen, ground in Tissue Lyser  to a fine powder and then kept at -80°C for further use. Until addition of the first solution, tissue samples were kept frozen. Afterward, samples were kept on ice.The extraction protocol was as described by Logemann et al.  with somg for 45 s. The liquid flow-through was appropriately discarded. The column was washed twice: first with 450 μl 3 M Na-acetate, to remove polysaccharides, proteins and pigments, and then with 320 μl 70% EtOH to remove salts. Between and after washes, the column was centrifuged at 8,000 g for 45 s and the liquid flow-through removed. The column was dried by centrifugation at maximum speed for 2 min. For elution of the RNA from the column, 30 to 40 μl of DEPC-treated water at 60°C were added directly to the column membrane, incubated for 2 min at room temperature and centrifuged at 8,000 g for 2 min. To increase RNA yield, this step was repeated with the liquid flow-through. RNA yield and purity, before and after DNase I treatment, were measured spectrophotometrically and by gel electrophoresis.Guanidine hydrochloride buffer  was added to 70 to 100 mg frozen and ground Arabidopsis leaf tissue in a safety-locked eppendorf tube and vortexed for 5 to 15 s. The liquid phase (without glass beads) was transferred to a new microcentrifuge tube and centrifuged at 4°C, at maximum speed, for 20 min to sediment the ground leaf tissue as described above. The clear supernatant was then transferred to a new microcentrifuge tube containing 1:1 v/v (~250 μl) 96% EtOH, quickly vortexed and then loaded onto the plasmid DNA extraction column . The plasmid DNA extraction column was assembled in a microcentrifuge tube, and centrifuged at 8,000 260, OD260/280 and OD260/230 and analyzed by gel electrophoresis at 100 V in a 1% agarose gel in 1× TAE buffer . Gels were stained by incubation in ethidium bromide for 20 min, followed by washing in TAE buffer for 20 min, and exposure to UV light. RNA integrity was determined with an RNA 6000 nano chip run on an Agilent 2100 Bioanalyzer, with ~250 ng RNA loaded on the chip.RNA yield and purity were measured by absorbance at ODAct1 and Tub2 primers  was treated with DNase I  and 1000 ng of DNA-free RNA was reverse-transcribed to cDNA . The products of the reverse transcription (cDNA) were detected by PCR analysis with AtPR1 gene normalized to AtPTB1F on samples from infected versus uninfected Arabidopsis leaves was calculated by the 2-ΔΔCt method. The primer sequences are listed in Additional file qRT-PCR was performed with the SYBR master mix and StepOne real-time PCR machine . The thermal cycling program was as follows: 95°C for 20 s; 40 cycles of 95°C for 3 s and 60°C for 30 s. Relative fold change of P < 0.05 and is noted in the text or table captions. All experiments shown here are representative of at least three independent experiments with the same pattern of results.T-tests were performed only when data was normally distributed and the sample variances were equal. Otherwise Mann-Whitney Rank Sum Test was performed. Significance was accepted at LogSpin: Logemann protocol coupled with spin columns; RT-PCR: Reverse transcription polymerase chain reaction; qRT-PCR: Quantitative real-time RT-PCR; EtOH: Ethanol; rRNA: Ribosomal RNA; PDA: Potato dextrose agar; DEPC: Diethyl pyrocarbonate.The authors declare that they have no competing interests.LM, MM and LF designed the experiments. HY, KB IS, EGS and MMBZ performed the experiments. ML and KB wrote the manuscript. All authors read and approved the final manuscript.Quantitation Report.Click here for fileTable S1. Primers used in RT-PCR.Click here for fileToxicity affects of phenol/chloroform/isoamylalcohol and of the β-mercaptoethanol.Click here for fileTable S2. Primers used in qRT-PCR.Click here for file


Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse.Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs  In recent years, small RNAs, including siRNAs , shRNAs , aptamers, and ribozymes -9 have ain vivo expression of recombinant RNAs has been described for some time /[total 3×pen aRNA]. Similarly, ImageJ was used to determine the yield of final product relative to the starting amount of chimeric aRNA per wet cell paste.The DNAzymes used for the excision of RNA fragments from the 5S rRNA scaffold are listed in Table aOH pH 7. containif ImageJ , and the2O at 65°C. The excised RNA fragments were isolated by affinity capture through hybridization to a complementary oligodeoxyribonucleotide, bioantiPEN  in 3× NTE buffer  . The DNApen aRNA was strongly expressed in E. coli  were investigated for their ability to excise small RNAs from the 5S rRNA expression scaffold. Their predicted excision sites on the 3×pen aRNA are shown in Figure pen aRNA band on the polyacrylamide gel  were tested. We confirmed that in the absence of Mn2+, essentially no cleavage was seen and that the highest total cutting percentage was observed at 15 mM Mn2+. With respect to the reaction temperature, it was found that 23°C was superior to 40°C in producing efficient RNA processing (data not shown). Introducing denaturation/renaturation cycles into the reaction did not improve the overall yield of the desired RNA products. Perhaps not surprisingly, it was also found that longer incubation time greatly improved the total cutting percentage when the same concentration of DNAzymes was added in the reaction mixture  a substrate to enzyme molar ratio ([3×pen aRNA]:[Pen17zyme1B]:[Pen17zyme2]) of 1:10:10, (ii) denaturation and annealing in 50 mM MOPS-NaOH (pH 7.2), 500 μM spermine at 90°C for 2 min with subsequent cooling to 23°C over a period of 10 min, (iii) incubation in 125 mM KCl, 500 mM NaCl, 7.5 mM MgCl2, 15 mM MnCl2, 150 μM spermine, and 50 mM MOPS-NaOH (pH 7.2), at 23°C for 72 h. As suggested by Table In summary, the best conditions among those tested were as follows: , it constitutes an attractive method for the large scale preparation of small RNAs through fermentation of E. coli. Under best reaction conditions, nearly all the 3×pen aRNA was digested, and 90% of it was further processed to yield the desired 71-nt RNA product after 72 h. Depending on the requirements, the rate of cleavage can be manipulated by adjusting the DNAzyme-to-RNA ratio to either speed up the process or conserve DNAzymes. In an industrial environment, the priority might be given to reducing the costs of reagents vs. the reaction rate. Manufacturing processes usually involve continuous operations, and under such circumstances the time for individual reaction cycles may not be as important as the cost for labor, chemicals, and equipment.In addition, we demonstrated the successful excision of a 3×In a further step towards improving the economical attractiveness of the method, we have shown recovery of both the RNA product and the DNAzymes through affinity chromatography. Although DNA oligomers can be chemically synthesized, the ability to readily recycle the DNAzymes will substantially reduce the cost of the whole procedure. In the present study, affinity chromatography of streptavidin agarose beads was employed to demonstrate the possibility of recovering DNAzymes. Future studies may include covalent immobilization of DNAzymes on more durable and less expensive supports such as glass or silica, which would be more compatible with industrial applications. In addition, it was shown that a longer incubation time greatly improved the overall yield of desired RNA product with equal concentration of DNAzymes in the reaction mixtures. Therefore, the cost of DNAzymes can be further reduced by trading off speed for cost.in vivo expression, convenient purification of chimeric RNAs and cost-efficient excision of insert RNA from the chimera and therefore offers a promising alternative for large scale RNA manufacturing.In summary, a fermentation-based approach to large scale RNA production of functional RNAs using the 5S rRNA scaffold strategy is described. The approach combines high levels of RNA: ribonucleic acid; DNA: deoxyribonucleic acid; 5S rRNA: 5S ribosomal RNA; mRNA: messenger RNA; tRNA: transfer RNA; DNAzyme: deoxyribozyme; RNAi: RNA interference; PEG: polyethylene glycol; PAGE: polyacrylamide gel electrophoresis; UV: ultraviolet; TLC: thin-layer chromatography; EDTA: ethylenediaminetetraacetic acid; HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; MOPS: 3-(N-morpholino)propanesulfonic acid.GEF, VGS, GWJ, and YL have one or more pending patent applications on the described technology.  This intellectual property has been licensed to BioTex, Inc.GEF, VGS and GWJ conceived the study. YL and VGS designed and performed the experiments. US, RCW, and GWJ participated in data analysis. All authors contributed to drafting the manuscript. All authors read and approved the final manuscript.


FILIA is a member of the recently identified oocyte/embryo expressed gene family in eutherian mammals, which is characterized by containing an N-terminal atypical KH domain. Here we report the structure of the N-terminal fragment of FILIA (FILIA-N), which represents the first reported three-dimensional structure of a KH domain in the oocyte/embryo expressed gene family of proteins. The structure of FILIA-N revealed a unique N-terminal extension beyond the canonical KH region, which plays important roles in interaction with RNA. By co-incubation with the lysates of mice ovaries, FILIA and FILIA-N could sequester specific RNA components, supporting the critical roles of FILIA in regulation of RNA transcripts during mouse oogenesis and early embryogenesis. RNA binding proteins (RBPs) play critical roles in germline and early embryonic development in model organisms by regulating RNA splicing, RNA subcellular localization, mRNA stability and translation in vitro assay systems, KH-containing proteins have been shown to typically bind poly-pyrimidine RNA The hnRNP K homology (KH) domain was first identified in the human heterogeneous nuclear ribonucleoprotein K (hnRNP K) filiatm/tm female mice are not completely sterile, and the early embryos in filiatm/tm female mice exhibit significant delays in their development. FILIA is important in regulation of mitotic kinase activity and spindle assembly checkpoint activation, and contributes to chromosome stability Recently, we have identified a subcortical maternal complex (SCMC) in mouse oocytes and early embryos essential for cleavage-stage mouse embryogenesis Filia is located in Chromosome 9 in mouse, and is encoded by a single-copy gene Filia is transcribed as two types of transcripts with respective lengths of 1.2 k and 1.6 k base pairs that co-exist in oocytes, but only the former transcript is translated into a functional protein with 346 residues. The FILIA protein is unique in primary sequence without any clearly identifiable domains. The C-terminus of FILIA is composed of 10 tandem repeats with each repeat comprising 23 residues. This arrangement of repeats was identified for the first time in FILIA and no homology to other known proteins has been observed Filia has been classified into a new oocyte/embryo expressed gene family in eutherian mammals, along with khdc1, dppa5, and flopedIn order to study the structural basis for its function, we expressed and crystallized the N-terminal domain of FILIA ranging from 1 to 124 amino acids (named FILIA-N hereafter). The structure of FILIA-N shows several features unique to FILIA and the probable function of the N-terminal extension prior to the KH domain was studied with regard to RNA binding. Furthermore, FILIA-N forms a stable dimer in both crystals and solution with high affinity, which is distinct from canonical KH domains.The crystal structure of FILIA-N (1–124 AA) was solved by the single wavelength anomalous diffraction (SAD) method see . The rooOn the surface of Nova2-KH3, a negatively charged groove was employed for interaction with RNA. By superposition of FILIA-N with the Nova2-KH3/RNA complex (PDB code 1EC6), a similar negatively charged patch on the surface of FILIA-N, involving the N-terminal extension, was also identified. Interestingly, a positively charged area adjacent to this negatively charged patch formed mainly by β0 and α0 was also observed, the function of which is unknown.2 compared to 930 Å2 for the Nova2-KH3 dimer. This data lead us to speculate that FILIA functions as a dimer in vivo. On the dimerization surface, most of the interactions involve the helices α2 and α3. Hydrophobic interactions contribute most of the stabilizing forces, including residues Met68, Met101, Leu65, Leu69, and Leu105. Three histidines, His61, His64, and His70, form hydrogen bonds with a neighboring molecule at the edge of the buried surface. The loop between β3 and α3 from monomer2 protrudes into a shallow pocket on the surface of monomer1 (monomer 1 and 2 are named according to their chain name), with the benzyl ring of Phe94 playing an anchoring role  extension. Both FILIA and FLOPED have a much longer N-terminal extension compared to Dppa5 and Khdc1, but no sequence similarity could be identified between these two proteins prior to the α0 helix, reminiscent of their divergent roles in embryogenesis based on filia and floped knockout mice The N-terminal 39 residues prior to the KH region form a unique substructure in FILIA-N. This substructure is composed of a loop at the beginning (residues 2-12), followed by two anti-parallel β-strands (β-1 and β0) and an α-helix (α0). Interestingly, in the newly identified oocyte/embryo specific gene family which includes E. coli or sf9 insect cells (data not shown), implying the importance of these residues in maintaining the overall structure of FILIA-N. In our FILIA-N crystal structure, Phe7 protrudes into a hydrophobic pocket surrounded by Leu51, Phe55, Ile62, Val75 and Val77, contributing to the stabilization of the local conformation. Thr9 forms a stable hydrogen bond with Glu66, and the conservation of Glu or Gln at position 66 in various species reiterates the importance of this interaction. Leu10 and the following conserved Val11 are also important for structural stabilization by hydrophobic interaction with surrounding residues  protein lacking the N-terminal extension still possessed a KH domain, it completely lost the ability to bind RNA. Other constructs with varying lengths of sequence at the N-terminus, i.e., FILIA 13–124 (FILIA-NΔ12) lacking only an unstructured stretch at the N-terminus, and FILIA 29–124 (FILIA-NΔ28) retaining the conserved α0, also lost their ability to bind RNA . These rThe folding of these mutants, i.e. FILIA 13–124, FILIA 29–124, FILIA 40–124, were detected by circular dichroism to exclude the possibility of misfolding and/or aggregation leading to the loss of RNA binding. All of these mutants shared similar spectra to the wild-type protein, indicating the correct folding of these proteins. Further denaturing by high temperature of 85°C without/with 4 M guanidine distorted the spectrum either partially or completely, which further strengthens our conclusion .The authentic RNA substrate that binds to FILIA is unknown to date. Taking advantage of the availability of the structure of the Nova2-KH3/RNA complex (PDB code 1EC6), we superimposed FILIA-N onto the Nova2-KH3/RNA complex. Clearly the sequence and/or conformation of the RNA fragment from the Nova2-KH3/RNA complex were not suitable for binding to FILIA-N. Most of the conflicts arose from residues 2–12, which clashed with the RNA derived from the Nova2-KH3/RNA complex. The other clash arose from the variable loop between β2 and β3 . On the in vitro, we tried to determine if FILIA could bind intrinsic RNA in vivo. FILIA was specifically expressed in oocytes and early embryos. Due to the scarcity of intrinsic FILIA in oocytes, we used recombinant FILIA proteins purified from E. coli to pull-down the total RNA from mouse ovaries  and was shown to bind oligonucleotides using an in vitro assay system in vitro. Furthermore, the identification of a unique N-terminal extension preceding the canonical KH region in FILIA-N structurally distinguishes FILIA from other KH domain containing proteins. Overall, the results described above indicate this atypical KH domain in this oocyte/embryo expressed gene family binds RNA like other canonical KH-domain proteins, but might have unique consequences that are restricted to oocytes and early embryonic development.FILIA is a member of a recently identified oocyte/embryo expressed family whose members are characterized by an atypical KH domain. This protein family, including Khdc1a, Khdc1b, Khdc1c, Dppa5a, Dppa5b, Dppa5c, FILIA (Ecat1) and FLOPED (OOEP/MOEP19), are specifically expressed by oocytes and play important roles in oogenesis, folliculogenesis, or early embryo development in eutherian mammals Compared to other canonical KH-containing proteins, such as Nova-1, Nova-2 in vivo, in RNA degradation during oocyte maturation or early embryogenesis. Further studies to identify the specific RNA substrate of this novel atypical KH domain or SCMC will help to elucidate the mechanism of RNA degradation in oogenesis and early embryogenesis in mammals.As previously described, the KH domain generally functions in a multiple-KH manner in KH domain containing proteins. In the current study, we demonstrated that FILIA-N forms a stable dimer in solution and in the crystal structure. Furthermore, our previous study showed that FILIA and FLOPED, which both contain KH domains, co-exist in the SCMC 2, a required area for a stable dimer suggested by Janin et al. 2, PDB code 2AXY) and hFMRP KH1-KH2 . Nevertheless, in PCBP2, no solution data to support the existence of a stable dimer is available The homodimerization of KH domains and the relationship to their function has been proposed for a long time, but conclusive evidence for the existence of homo-dimers formed by KH domains is lacking. Most of the buried dimerization area identified in KH domain crystal structures was less than 1200 ÅStructural extensions are present in some KH domains. For example, QUA1 and QUA2 extensions prior to and following the KH domain in STAR/GSG-containing proteins, such as sam68, QK1 and GLD-1, have been identified for a long time Considering the significance of the interaction between oocyte-specific factors and RNA transcripts, and the importance of this new eutherian oocyte/embryo expressed gene family in mammalian oogenesis and early embryogenesis, our results not only reveal the unique features of these oocyte/embryo specific proteins in RNA binding, but also provide a structural basis for possible intervention of abnormal embryonic development such as habitual abortion.All research involving animals in this study follow the guidelines and byelaws governing experiments on animals, and have been approved by the Ethics and Experimental Animal Committee of the Institute of Zoology, Chinese Academy of Sciences. The Animal Research Committee does not issue a number to a specific study. But each study requires the permit to use animals from the Committee, and this research was under the supervision of the Committee for all procedures. The animal facility at Institute of Zoology gets licensing from the experimental animal committee of Beijing city and the animal handling staff (including each post-doc and doctor student) must be trained before using animals. The mice were killed by cervical dislocation. The only procedure performed on the dead animals is the collection of oocytes from the oviduct. Oocytes were collected according to procedures described previously 5′-TACTTCCAATCCAATGCCATGGCCTCTCTGAAG-3′ (forward) and 5′-TTATCCACTTCCAATGCTACTCAACTCCAGCCTC-3′ (reverse) were designed to amplify the sequence coding for amino acids 1–124 of FILIA (FILIA-N). The target sequence was cloned into the pET30-TEV/LIC Vector (Novagen) downstream of a 6×His tag via the ligation independent cloning method.The gene encoding full-length filia was cloned from total RNA from mouse ovary cells. The primers Escherichia coli BL21 (DE3) strain. Cells were grown in LB Broth medium and induced with 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for protein expression. Cell lysis was performed by sonication on ice in Ni-NTA resin binding buffer . The lysate was clarified by centrifugation and supernatant was loaded onto a Ni-NTA resin column (Qiagen). The 6×His-FILIA-N protein was eluted in elution buffer (binding buffer with 500 mM imidazole). After being concentrated in an Amicon Ultra filter (Millipore), the protein was dissolved into low salt buffer . Afterwards, the 6×His tagged protein was digested by TEV protease at 289 K for 24 h. Following removal of the 6×His tag, the protein was purified by HiTrap Q HP column  and then loaded onto a HiLoad 16/60 Superdex-200 size-exclusion column  for further purification. A sharp peak corresponding to the target protein was pooled and concentrated to 15 mg ml−1 for further crystal screening.The plasmid carrying the target gene was transformed into g ml−1 L-methionine to an absorption value of 1.0 at OD600. After L-methionine depletion, 50 µg ml−1 seleno-methionine was provided and 0.2 mM IPTG was used to induce protein expression. The isolation and purification procedures were the same as for the native protein.For expression of a seleno-methionyl derivative protein, the pET30-FILIA-N vector was transformed into methionine auxotroph strain B834 (DE3). Cells were then cultured with M9 minimal medium supplemented with 50 µHuman Nova2-KH3 fragment (residues 406–492) was cloned, expressed and purified by a similar procedure as described above. The protein lacking the N-terminal 6×His tag was chromatographically purified prior to analytical ultracentrifugation experiments.−1) mixed with an equal amount of mother liquor was equilibrated against 500 µl reservoir solution  to yield FILIA-N crystals suitable for data collection. The seleno-methionyl derivative protein was crystallized by 0.2 M ammonium formate, 16% w/v PEG3350.FILIA-N crystals were grown by the hanging-drop vapor diffusion method at 277 K. A 2 µl droplet of protein solution , Tsukuba, Japan. Data from seleno-methionyl derivative crystals were collected at the Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China. For data collection under cryogenic conditions, crystals were soaked for a few seconds in a mixed solution of mother liquor with 20% (v/v) glycerol. Crystals were then mounted on the beam line in a nylon loop and flash-cooled in a liquid-nitrogen stream at 100 K. Native data sets were collected by rotating 180° with an increment of 1° per frame. For the derivative crystal, a single dataset was collected at the peak wavelength of 0.9795 Å over 180°. All data sets were processed using the HKL2000 package Due to the high quality of the data, we successfully located the positions of 9 out of 12 selenium atoms in an asymmetric unit from the peak data set, and the preliminary model was readily built following single-wavelength anomalous diffraction (SAD) M Tris pH 8.0, 200 mM NaCl and 1 mM TCEP was performed before the experiments. SV experiments were conducted at 60,000 r.p.m and 4°C using absorbance detection and double-sector cells loaded with approximately 43 µM for FILIA-N and 84 µM for Navo2-KH3. For the SE experiments, data were collected at 4°C and 15,000, 22,500 and 28,500 r.p.m with 25.5, 15.3 and 10.2 µM FILIA-N respectively. The buffer composition (density and viscosity) and protein partial specific volume (V-bar) were obtained using the program SEDNTERP (http://www.rasmb.bbri.org/). The SV and SE data were analyzed using the programs SEDFIT and SEDPHAT Sedimentation velocity (SV) and sedimentation equilibrium (SE) experiments were performed using a Beckman/Coulter XL-I analytical ultracentrifuge with double-sector or six-channel centerpieces and sapphirine windows. An additional protein purification step on a HiLoad 16/60 Superdex 200 gel-filtration column in 50 mM phosphate buffer pH 8.0, 200 mM NaCl) or denaturation buffer (CD buffer with 4 M guanidine hydrochloride) to a final concentration of 10 µM. For each sample spectrum recorded, a buffer blank was subtracted from the raw signal.This protocol was adapted from a previously published method M Tris, 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100 pH 7.4). After thorough washing, the beads were boiled in loading buffer  and loaded onto an SDS-PAGE (polyacrylamide gel electrophoresis) gel. After running for 1 hour at 180 voltages, samples were transferred to PVDF membrane and blotted by anti-6xHis antibody (Santa Cruz). HRP-labeled secondary antibody (SIGMA) was used to produce chemiluminescent signals.Full length FILIA, FILIA-N lacking the N-terminal 12 residues (FILIA-NΔ12), FILIA-N lacking the N-terminal 28 residues (FILIA-NΔ28) and FILIA-N lacking the N-terminal 39 residues (FILIA-NΔ39) were cloned and expressed using the same protocol for wild-type FILIA-N described above. Purified proteins were incubated with poly-C and poly-U agarose beads (SIGMA) in a binding buffer  and incubated with Ni-NTA Magnetic Agarose Beads (Qiagen) which were pre-sequestered with 6xHis tagged proteins  at room temperature for 2 hours. After washing 6 times with interaction buffer, the bound RNA was eluted by high salt buffer . The pulled down RNA was further purified by co-precipitation with glycogen (0.5 ug ul−1) and loaded onto 5% urea denatured PAGE. After running for 1.25 hours at 200 V, the gel was stained with SYBR Green II for 10 minutes and scanned with FLA 7000 (Fujifilm). Repeating pull-down experiments were performed by re-incubating residual RNA with FILIA-N sequestered beads and then eluting and analyzing as above.Ovary total RNA was purified from homogenized 6 week old CD1 mouse ovaries with TRIzol (Invitrogen) and digested by DNase (Promega). After phenol-chloroform extraction, the precipitated RNA was dissolved in interaction buffer . An unknown component of 2.63 KD co-purified with Nova2-KH3 impeded our trial of sedimentation equilibrium analysis of this protein.(TIF)Click here for additional data file.Figure S2Circular dichroism analysis of N-terminal mutants of FILIA-N. (a) Circular dichroism result of FILIA-N, FILIA-N F7A, FILIA-N T9A and FILIA-N L10A at 20°C. Four proteins were colored as blue, green, cyan and red, respectively. (b) Circular dichroism result of different conditions of FILIA-N. Results of 20°C, 85°C, and 85°C with 4 M guanidine hydrochloride were colored by red, green and blue, respectively. (c), (d), (e) Circular dichroism result of different conditions of FILIA-N F7A, FILIA-N T9A, and FILIA-N L10A. Results under various conditions were colored as in (b).(TIF)Click here for additional data file.Figure S3Circular dichroism analysis of N-terminal truncations of FILIA-N. (a) Circular dichroism result of FILIA-N, FILIA-NΔ12, FILIA-NΔ28 and FILIA-NΔ39 at 20°C. Four proteins were colored by blue, green, cyan and red, respectively. (b) Circular dichroism result of different conditions of FILIA-N. Results at 20°C, 85°C, and 85°C with 4 M guanidine hydrochloride were colored by red, green and blue, respectively. (c), (d), (e) Circular dichroism result of different conditions of FILIA-NΔ12, FILIA-NΔ28, and FILIA-NΔ39. Results under various conditions were colored as in (b).(TIF)Click here for additional data file.


The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca2+ entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone.The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to CaDOI:http://dx.doi.org/10.7554/eLife.01180.001 Neurons are connected to each other by junctions called synapses. When an electrical signal travelling along a neuron arrives at a synapse, it causes the release of bubble-like structures called synaptic vesicles that contain chemicals called neurotransmitters. When released by the vesicles these neurotransmitters bind to receptors on a second neuron and allow the signal to continue on its way through the nervous system.The release of synaptic vesicles from the neuron depends largely on the number of calcium ions that enter this neuron via structures called ion channels, and also on the rate at which they enter. Vesicles are released in one of three ways: they can be released quickly (within a few milliseconds) in response to the influx of calcium ions; they can be released slowly (over a period of tens or hundreds of milliseconds) in response to the influx; or they can be released at random times that are not related to the influx.It is known that the sensitivity of certain calcium sensors near the synapse influences the release of the vesicles. It had been thought that the distance between the “active zone” where the calcium ions enter the neuron and the region where the vesicles reside might also influence rate of release, but the molecular mechanism underlying this hypothesis is poorly understood.C. elegans, a nematode worm. First it was shown that the precise position of UNC-13 in the active zone depended on a domain within the protein called the C2A domain. Next it was shown that the distance between the UNC-13 protein and the calcium ion channels strongly influences the quick mode of vesicle release. Finally, Zhou et al. showed that the C2A domain also had a significant influence on the spontaneous release of vesicles, which suggests that a common fleet of vesicles might be used for both the quick and the spontaneous modes of vesicle release. Zhou et al. also generated mutant worms that mimicked a neurological disease, epileptic seizure, and showed that eliminating the C2A domain can relieve some of the symptoms associated with the disease.Zhou et al. have now shed new light on this question by performing a series of experiments that involved manipulating a protein called UNC-13 – which is known to be involved in the release of vesicles – in neurons from Many neurological diseases are caused by signals not being transmitted properly at synapses, so in addition to providing insights into the basic mechanism underlying synaptic action, these results could also assist with the development of new strategies for managing neurological diseases.DOI:http://dx.doi.org/10.7554/eLife.01180.002 These observations support an idea that spontaneous release and the fast phase of evoked release may use a common pool of SVs. We also show that reducing SV release by eliminating the function of UNC-13L C2A domain ameliorates behavioral deficits in a C. elegans model for epileptic seizure. Together, these data demonstrate that the distance between UNC-13/Munc13 to the Ca2+ entry site plays a critical role in SV release probability and release kinetics.In l region   mutation in a genetic screen for suppression of the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2 (unc-13(n2609) changes glutamine 46 to a stop codon within the C2A domain of UNC-13L  is a null allele for the entire locus, and unc-13(e1091) is a null allele for the long isoform only (unc-13(s69) and unc-13(e1091) mutants are severely paralyzed (unc-13(n2609) animals exhibited moderate slowing of locomotion (unc-13(n2609)/unc-13(s69) or unc-13(n2609)/unc-13(e1091) animals showed more movement impairment than did unc-13(n2609) homozygous mutants (data not shown). These observations indicate that unc-13(n2609) is a recessive partial loss of function mutation.The splicing   suggest that some UNC-13L proteins are produced in this mutant. Indeed, immunostaining using an antibody raised against amino acids 106–528 of the UNC-13L N-terminus (unc-13(n2609) (described later), while no immunostaining signal was detected in unc-13(s69) and unc-13(e1091) mutants  mutant must be due to translation from an ATG codon(s) downstream of the amino acid Q46 (unc-13(n2609), we next generated single-copy insertion transgenes (designated Si) expressing the full length UNC-13L or a mutant UNC-13LC2A- lacking only the C2A domain  transgene fully rescued the locomotion of unc-13(s69) null mutants, C2A-)Si(UNC-13L rescued the movement deficits of unc-13(s69) to a level similar to unc-13(n2609) mutants (unc-13(n2609) mutant is specifically deficient in the UNC-13L C2A domain, and provides a genetic background to investigate the role of the C2A domain in a physiological setting.The mild behavioral defects of terminus  revealed mutants   by electrophysiological recordings of muscle cells. SV release at these synapses occurs in a graded manner in response to membrane potential change (unc-13(s69) null animals exhibit no eEPSCs (. In unc-13(n2609) mutants the amplitude of eEPSCs was reduced to 50% of that in wild type animals (unc-13(s69) null animals expressing full-length UNC-13L or UNC-13LC2A− from the same integrated genomic locus. We observed a full rescue of the amplitude of eEPSC by Si(UNC-13L) expression in unc-13(s69) (Si(UNC-13LC2A-); unc-13(s69) animals showed significantly reduced amplitude of eEPSC, similar to unc-13(n2609). To address that the C2A domain is directly responsible for the observed physiological defect, not secondary due to reduced protein levels, we overexpressed full-length UNC-13L and UNC-13LC2A− in unc-13(s69) mutants. While both transgenes rescued the paralysis of unc-13(s69), NMJ recordings showed that simply elevating the levels of UNC-13LC2A- did not fully rescue the eEPSC amplitude, compared to overexpression of the full-length UNC-13L  . Si could be due to defective priming of SVs or a weak response of SVs to Ca2+ influx at the presynaptic terminal. A classic assay to analyze SV priming is by the application of hypertonic sucrose solution to induce vesicle exocytosis in a Ca2+-independent manner, which is often used to assess readily releasable pool (RRP) (unc-13(s69) mutants and severely inhibited in unc-13(e1091) mutants (unc-13(s69) mutants (unc-13(s69). Sucrose-induced charge transfers in the time windows of first one and 5 s were similar between wild type and unc-13(n2609) (Si(UNC-13L) and Si(UNC-13LC2A-) transgenes rescued SV priming in unc-13(s69) null mutants to the level of wild-type. These results indicate that SVs are fully competent for release in the absence of the C2A domain of UNC-13L.The reduced presynaptic release in ol (RRP) . Previou mutants . Here we mutants , compara mutants . Prolong3(n2609) . Both SiC. elegans NMJ recording cannot endure multiple stimulations, it is not feasible to record reliable responses for multiple stimulations to compare the charge transfers during eEPSC and sucrose application in the same animal. We therefore calculated the ratio of mean charge transfers during eEPSC and sucrose application for a given genotype. This ratio may not directly represent the release probability, but is positively correlated with release probability. In unc-13(n2609) mutants and unc-13(s69); Si(UNC-13LC2A−) transgenic animals, this ratio was severely reduced (unc-13(n2609) mutants display reduced evoked release but unaltered SV priming, we conclude that the C2A domain of UNC-13L regulates the release probability of SVs.The extended current evoked by sucrose stimulation under our protocol may reflect continuous release of refilled SVs to RRP . Because reduced . As unc-unc-13(s69) and unc-13(e1091) mutants, fewer docked SVs are present within 231 nm from presynaptic dense projections, while slightly more SVs are accumulated at distal regions (>330 nm from presynaptic dense projections)  showed normal ultrastructural organization (unc-13(n2609) mutant, the number of total SVs and that of total docked SVs were similar to those in wild type animals (unc-13(n2609) is less severe than those in unc-13(s69) and unc-13(e1091) mutants (unc-13(n2609) may partially account for the reduced release probability.SV priming and release probability are generally correlated with the number of docked SVs . The reqections) . To addrprotocol . Neuromunization . Consist animals . However animals . Althoug mutants , the milunc-13(n2609). To further test whether the recruitment of active zone proteins might be affected, we examined the localization of several active zone proteins, including the C. elegans RIM protein UNC-10 (2+ channels (VGCCs) UNC-2 (unc-13(n2609) animals. UNC-13L proteins, recognized by the antibodies against the N-terminus of UNC-13L, showed a punctate pattern in unc-13(n2609) mutants. However, UNC-13L puncta displayed significantly reduced co-localization with UNC-10/RIM (unc-13(n2609), compared to wild type (unc-13(s69); Si(UNC-13LC2A−) animals, comparing to unc-13(s69); Si(UNC-13L) (unc-13(n2609) mutants, these data indicate that lacking the C2A domain causes UNC-13L to be shifted away from the active zone where Ca2+ entry sites reside.UNC-13/Munc13 proteins are core components of the presynaptic active zone and interact with multiple active zone proteins . The ultn UNC-10 , ELKS-1 n UNC-10 , and thes) UNC-2 . We founs) UNC-2  and of Us) UNC-2  were indC-10/RIM . The disild type . We obseUNC-13L) . As UNC-unc-10/rim mutants (2A domain (N1–157) displayed a diffuse pattern throughout the axon . Quantitative analysis of locomotion speed showed a poor rescuing activity of unc-13(s69) paralysis by Si(UNC-13LN−), compared to Si(UNC-13L) and Si(UNC-13LC2A−) transgenes  null animals expressing each UNC-13L variant. The time constants in unc-13(s69); Si(UNC-13L) were faster than those in wild type (unc-13(s69); Si(UNC-13LC2A−) were both significantly changed, compared to those in unc-13(s69); Si(UNC-13L) (unc-13(s69); Si(UNC-13LN−) animals showed a more prolonged SV release. The time constants and the decay time of eEPSC were further affected, compared to unc-13(s69); Si(UNC-13LC2A−) , which represent spontaneous release of individual SVs, are general short  . unc-13(13LC2A−) . In C. eal short . MoreoveSi(UNC-13LN−) transgene rescued the amplitude of eEPSCs in unc-13(s69) to the wild type level, while Si(UNC-13LC2A−) showed a partial rescue (Si(UNC-13LN−) showed significantly increased sucrose evoked SV release, comparing to Si(UNC-13L) and Si(UNC-13LC2A−)  likely reflects recruitment of SVs located distally from active zones. Together, with the enhanced slow release in unc-13(s69); Si(UNC-13LN−), these observations indicate that these SVs are competent for release, but with lower release probability and slower release kinetics. unc-13(s69); Si(UNC-13LN−) animals showed significantly slower locomotion than unc-13(s69) expressing either full-length UNC-13L or UNC-13L−C2A  . Since U-13LC2A− , suggestunc-13(n2609) in 5 mM extracellular Ca2+ concentration. While high [Ca2+]ex resulted in an increase in eEPSC amplitudes in both wild type and unc-13(n2609) mutants, 5 mM [Ca2+]ex had a stronger effect in unc-13(n2609) than in wild type animals (unc-13(n2609)  showed release kinetics defects similar to unc-13(s69); Si(UNC-13LC2A−). 5 mM [Ca2+]ex increased the fraction of fast component in unc-13(n2609), compared to wild type animals, although the time constant of fast component in unc-13(n2609) remains slower than that in wild type. Nonetheless, this result shows that higher [Ca2+]ex can compensate for the lengthened distance to UNC-13L to calcium microdomain.Lastly, as a further test to our conclusion that the distance of UNC-13L to calcium entry site directly influences SV release property, we performed recordings of  animals . Further3(n2609) . In normunc-13(n2609) mutants showed a strong reduction in tEPSC frequency, compared to wild type (unc-13(s69); Si(UNC-13LC2A−), comparing to unc-13(s69); Si(UNC-13L). Overexpression of UNC-13LC2A− in unc-13(s69) did not fully rescue the defects of tEPSC frequency, while overexpression of UNC-13L displayed an enhanced tonic release (unc-13(s69); Si(UNC-13LN−) animals, in which UNC-13 proteins are diffuse throughout the axon, and observed reduced tEPSC frequency to a level similar to that in unc-13(s69); Si(UNC-13LC2A−)  and cpx-1/complexin. CPX-1/complexin is a key regulator of SV release by acting as a clamp on SNARE (cpx-1(ok1552) unc-13(n2609) double mutants was significantly reduced, compared to cpx-1(ok1552) mutants (2A domain. We next recorded evoked release in cpx-1(ok1552) unc-13(n2609) double mutants. cpx-1(ok1552) single mutant showed dramatically reduced eEPSCs (cpx-1(ok1552) unc-13(n2609) double mutants was significantly reduced, compared to wild type or unc-13(n2609), but was moderately increased, compared to cpx-1 single mutants. Analysis of charge transfer further showed a noticeable increase primarily in the fast phase of release, within 20 ms after electrical stimulation (cpx-1(ok1552) unc-13(n2609) mutants, were converted to account for the fast phase of evoked release.UNC-13 is also essential for spontaneous release . To analild type . The ampild type . Similar release . We also13LC2A−) , indicative zone  and SVs ive zone , these ron SNARE . Loss ofon SNARE , but it  mutants , indicatd eEPSCs , in partd eEPSCs . The ampmulation . Based oN−, which is diffuse in axons (unc-13(s69), both UNC-13L-miniSOG and UNC-13LN−-miniSOG rescued the paralysis to different degrees  technique . This mein axons . In unc- degrees , indicat degrees , indicatf eEPSCs . CALI byf eEPSCs .10.7554/N−-miniSOG could cause specific inhibition of synapse transmission in wild type animals. Our assumption is that transgenically over-expressed UNC-13L-miniSOG or UNC-13LN−-miniSOG would interact with native protein interacting partners by competing with endogenous UNC-13. Wild type animals carrying UNC-13L-miniSOG transgenes showed rapid movement impairment upon CALI by blue light  mutants, UNC-13L-miniSOG fully rescued tEPSC frequency, while UNC-13LN−-miniSOG partially rescued it  allele as a genetic suppressor of the behavior deficits caused by acr-2(n2420gf), which causes over-excitation in the locomotion circuit and exhibits spontaneous and frequent whole body muscle contractions (unc-13(n2609) strongly suppresses acr-2(gf)-induced convulsions (unc-13(n2609); acr-2(gf) double mutants showed reduced tEPSCs compared to acr-2(gf) (unc-13(n2813), which contains a missense mutation in the C-terminal MUN domain and reduces SV priming to less than half of that in wild type (acr-2(gf) animals  animals.We isolated ractions   . Interesild type , showed  animals . These oacr-2(gf)-induced convulsions, we expressed UNC-13L variants in acr-2(gf) animals. Interestingly, overexpression of UNC-13L or genomic unc-13 in acr-2(gf) animals exacerbated convulsions (N− in acr-2(gf) animals had no augmentative effect on convulsions. Lastly, to address whether the suppression of acr-2(gf)-induced convulsions by unc-13 mutant is due to an acute effect to inhibit over-excitation, we introduced UNC-13L-miniSOG and UNC-13LN−-miniSOG into acr-2(gf) animals. Upon blue light illumination, UNC-13L-miniSOG expressing animals showed a strong suppression of convulsions, while UNC-13LN−-miniSOG expressing animals continued to convulse similarly to acr-2(gf) animals (acr-2(gf) animals expressing UNC-13L-miniSOG gradually restored convulsions, showing a full recovery after 16 hr  mutation as well as single-copy expression of UNC-13L variants in unc-13 null mutants, we have uncovered specific roles of the C2A domain in SV release probability and spontaneous release. The precise active zone localization depends on the C2A-containing N-terminal region unique to UNC-13L, and directly contributes to SV release kinetics. Our data support a conclusion that the proximity of UNC-13/Munc13 to the Ca2+ entry site plays a critical role in SV release probability and release kinetics, and also suggest that spontaneous release and the fast phase of evoked release may share a common pool of synaptic vesicles at the active zone.Differential expression and function of UNC-13/Munc13 isoforms endow synapses with distinct release properties . Severaltein RIM . In this2A domain is necessary for their localization at the active zone , we think that this effect is likely due to both mispositioning of the remaining UNC-13L in the active zone as well as a mild effect on SV docking at the proximal region to active zones.Previous studies, largely based on overexpression of mutant Munc13/UNC-13 proteins in cultured neurons or transgenic animals, have suggested that N-terminal Cive zone . Here, wng state . A monom release . Consist2+ sensitivity modulated by distinct Ca2+ sensor proteins . Overex release . We finde of SVs . Togetheecording .C. elegans has traditionally relied on the use of genetic mutations that perturb gene function at birth. Comparing to the previously reported synapse CALI methods  animals, specifically reduces spontaneous release and the fast phase of evoked release to about 50% of wild type animals, and suppresses the increased spontaneous release in cpx-1(null) mutants. The reduced tonic release in unc-13(n2609) cpx-1(null) is accompanied with a noticeably enhanced fast phase of evoked release. CALI of UNC-13L shows a strong effect of the active zone localized UNC-13L in spontaneous release and the fast phase of evoked release, but not slow phase. In contrast, UNC-13LN− shows diffuse distribution and accounts for an enhanced slow phase of evoked release, and rescues the spontaneous release defect of unc-13(null) to a similar level to that of UNC-13LC2A−. These results suggest that SVs associated with diffused UNC-13LN−, the majority of which may be positioned distally from the active zone, mainly undergo evoked release with slow kinetics, and may not contribute to spontaneous release. Our data are generally consistent with the idea that spontaneous release and evoked release use the same pool of SVs, and further suggest that at the C. elegans cholinergic NMJs SV populations involved in spontaneous release and the fast phase of evoked release may likely reside at regions proximal to the active zone  plates at room temperature (20–22°C) as described (escribed . Double n2609 was isolated as a suppressor of the convulsion behavior caused by acr-2(gf), in a previously described EMS mutagenesis screen .Locomotion speeds were measured using Worm Tracker 2.0  , and ani2 was poured and swirled on the rim of the plate to form a ‘copper ring’ to keep worms away from the edge of plates, and excessive CuCl2 solution was removed. Plates containing worms were illuminated with blue LED light source . The diameter of illumination area is 5.8 cm to cover the entire surface of plates. The light intensity was measured to be 2.07 mW/mm2 with a power sensor D10MM connected to an Optical Power Meter PM50 . Animals were exposed to 1 Hz pulsed blue light for 12 min to avoid high heat accumulation on plates  within each such set was divided by the number of profiles in the set, resulting in a number of docked vesicles per profile for that set. The mean and SEM of all data points within each genotype was determined and used to calculate p values in two-tailed Student’s Whole-mount staining was conducted using 1% paraformaldehyde fixation as previously described . PrimaryNeuromuscular dissection methods were adapted from previous studies . Adult w3, 1.25 NaH2PO4, 2 CaCl2, 4 MgCl2, 10 glucose, and sucrose to 340 mOsm, bubbled with 5% CO2, 95% O2 at 20°C. The pipette solution containing (in mM): 120 CH3O3SCs, 4 CsCl, 15 CsF, 4 MgCl2, 5 EGTA, 0.25 CaCl2, 10 HEPES and 4 Na2ATP, adjusted to pH 7.2 with CsOH. The extracellular calcium concentration is otherwise indicated if it is not 2 mM. Conventional whole-cell recordings from muscle cells were performed at 20°C with 2–3 MΩ pipettes. An EPC-10 patch-clamp amplifier was used together with the Patchmaster software package . Tonic EPSCs were recorded at −60 mV. For record evoked EPSCs, a second glass pipet filled with bath solutions was put on the ventral nerve cord as stimulating electrode. The stimulating electrode gently touched the anterior region of ventral nerve cord to form loose patch configuration, which is around 1 muscle distance from recording pipets. A 0.5 ms, 85 μA square current pulse was generated by the isolated stimulator  as stimulus to obtain the maximal responses. For RRP depletion, the 0.5 M sucrose in bath solution was applied to ventral nerve cord near the recorded muscles by Picospritzer with 8 psi for 7 s. Under this prolonged stimulation protocol, we could observe the current decay over the stimulation window. The sucrose-evoked responses have been compensated for the basal activities by subtracting basal line current level prior to sucrose application. For miniSOG mediated CALI, illumination (15 or 30 mW/mm2) was provided with a Sutter Instrument Lambda LS fitted with a Lamda 10–2 filter wheel for shuttering . The excitation light was filtered with an eGFP filter set with 480 nm excitation  and focused on the specimen with a 63 × water immersion objective . Light intensity was measured with a calibrated photometer with an integrating sphere detector . A glass slide with a semi-spherical lens containing a water drop was placed under the objective and was used to direct the light into the integrating sphere. The area of illumination was measured with a stage micrometer for the calculation of illumination intensity. The 2–3 min continuous blue light was illuminated on the prep including the ventral nerve cord and recorded muscle for miniSOG-mediated CALI. All current traces were imported to IGOR Pro  for further analysis. The cumulative transferred charge of eEPSC was integrated over 50 ms after the electrical stimulation. The charge trace was fitted with a following double-exponential function to derive the time constant (τ) and size (A) of each component:t0 is the time of electrical stimulation. The size of slow component (Aslow) was not directly used. Instead, we subtracted the Afast from the total amount of transferred charge within 50 ms.The bath solution containing (in mM): 127 NaCl, 5 KCl, 26 NaHCOt test. For comparisons involving multiple groups, we used one-way ANOVA and Newman-Keuls post hoc test. ***p<0.001; **p<0.01; *p<0.05.We used Graphpad Prism 5  to test significance. For comparisons of two groups, we used a two-tailed Student’s  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  of UNC-13 and test the hypothesis that the C2A domain helps localize synaptic vesicles near calcium channels at synaptic active zones. The authors show a mild change in the distribution of UNC-13 proteins that either lack the C2A domain or have an early stop in the C2A domain, and argue that an increased distance from calcium channels, and thus a lower concentration in intracellular calcium, changes release probability. This is a reasonable conclusion given the non-linear dependence of vesicle fusion on intracellular concentration of calcium, but the authors need to more firmly rule out alternative possibilities. Three reviewers agree that this work is suitable for publication in eLife and that the recent acceptance of related work will not preclude publication of this work at eLife since the two studies were independently conceived and submitted for publication in a similar time frame. The following issues should be addressed:In the paper by Zhou and colleagues, the authors address how the well-studied protein synaptic protein UNC-13 (Munc13) contributes to synaptic vesicle fusion. Specifically, they evaluate the contribution of a specific domain (C1) Sucrose responses, as shown, are not a measure of pool size, since there is clearly no major pool depletion. Nevertheless, the fact, that sucrose can elicit a multiple of the eEPSC-charge and that the mutants show similar responses, can be used to argue that priming is intact and that the effect of the mutations is most likely on the Ca-triggering of the release. However, the authors should avoid calling their measured quantities ‘release probabilities’ and ‘pool sizes’. This can be addressed in the text.2) The interpretation of changes in EPSC-decays as changes in release time course is only valid if these decays are markedly slower than those of spontaneous EPSCs (tEPSCs). Unfortunately the authors analyze only tEPSC frequencies and -amplitudes. An assertion, that tEPSCs are short (in case they are) would be helpful for the reader to understand that slowness is a result of desynchronisation.2A-variant). If these fully rescue null mutants, then protein levels are a possible explanation for the partial loss of function in n2609 mutants.3) Decreased protein level of UNC-13 is an alternative explanation for the reduced current observed in the partial loss of function n2609 allele. An early stop makes message sensitive to nonsense-mediated decay or is less efficiently translated. Alternatively, the modified protein might be less stable. An indirect test is to study transgenic worms that overexpress the mutant protein or GFP-tagged variants  Greater care should be taken in the conclusions when the authors attempt to explain the observed phenotypes based on redistribution of vesicles, which seems very minor.C. elegans.5) It should be made clear that release is graded in s69 null mutants when using this protocol.6) The prolonged sucrose stimulation experiments are difficult to evaluate without control experiments showing the extent of release in the  1) Sucrose responses, as shown, are not a measure of pool size, since there is clearly no major pool depletion. Nevertheless, the fact, that sucrose can elicit a multiple of the eEPSC-charge and that the mutants show similar responses, can be used to argue that priming is intact and that the effect of the mutations is most likely on the Ca-triggering of the release. However, the authors should avoid calling their measured quantities ‘release probabilities’ and ‘pool sizes’. This can be addressed in the text.unc-13(s69) (addressing point 6), which shows little release over the extended sucrose stimulation. However, we agree that this analysis alone is not sufficiently quantitative to measure pool size. In the revised manuscript, we have used “SV priming”, instead of SV “pool size”.We apologize for not including the full trace of sucrose response in the original submission. These traces are now shown in the revised 2A domain in SV priming to: “Sucrose-induced charge transfers in the time windows of first one and five seconds were similar between wild type and unc-13(n2609) (Si(UNC-13L) and Si(UNC-13LC2A-) transgenes rescued SV priming in unc-13(s69) null mutants to the level of wild-type. These results indicate that SVs are fully competent for release in the absence of the C2A domain of UNC-13L.”We have revised the conclusion regarding the function of C3(n2609) . Both SiIn the revised manuscript we have included a statement that the ratio of mean charge transfers during eEPSC and under sucrose application “may not directly represent the release probability, but it is positively correlated with release probability”.2) The interpretation of changes in EPSC-decays as changes in release time course is only valid if these decays are markedly slower than those of spontaneous EPSCs (tEPSCs). Unfortunately the authors analyze only tEPSC frequencies and -amplitudes. An assertion, that tEPSCs are short (in case they are) would be helpful for the reader to understand that slowness is a result of desynchronisation.C. elegans NMJs, tEPSCs are short (around 1 ms for 75–25% decay time), whereas the 90–10% decay times of eEPSCs are larger than 10 ms. The decay times of tEPSC among all the recorded genotypes in our study are indistinguishable, indicating that postsynaptic receptor response kinetics is unaltered even for synapses with slower release kinetics.We appreciate this comment. We have provided 75–25% decay time of tEPSC in a figure supplement for C. elegans NMJs, the decays of tonic excitatory postsynaptic current (tEPSC)…”We have made the recommended assertion (text starting): “In 3) Decreased protein level of UNC-13 is an alternative explanation for the reduced current observed in the partial loss of function n2609 allele. An early stop makes message sensitive to nonsense-mediated decay or is less efficiently translated. Alternatively, the modified protein might be less stable. An indirect test is to study transgenic worms that overexpress the mutant protein or GFP-tagged variants . If these fully rescue null mutants, then protein levels are a possible explanation for the partial loss of function in n2609 mutants.C2A- overexpression  rescued the paralysis of unc-13(s69) mutants, UNC-13LC2A- OE did not fully rescue eEPSC amplitude or tEPSC frequency, whereas UNC-13L OE fully rescued synaptic transmission and in fact caused increased tEPSC frequency. These results thus provide further support for our conclusion that the observed synaptic defects of unc-13(n2609) is due to specific loss of functional C2A domain of UNC-13L.We have provided such electrophysiological recording data in new figures  showing 4) Greater care should be taken in the conclusions when the authors attempt to explain the observed phenotypes based on redistribution of vesicles, which seems very minor.unc-13(n2609) may partially account for the reduced release probability”.We appreciate this suggestion and have made revision to the conclusion: “the mild reduction in the centrally docked SV in 5) It should be made clear that release is graded in C. elegans.We have now included such a statement: “SV release at these synapses occurs in a graded manner in response to membrane potential change .”6) The prolonged sucrose stimulation experiments are difficult to evaluate without control experiments showing the extent of release in the s69 null mutants when using this protocol.unc-13(s69) in the first one or five seconds of our prolonged sucrose stimulation protocol. The effect within the first second of sucrose treatment is not different from previous reports using shorter sucrose stimulation (In the revised mulation .


Affiliation number 1 for the first author is incorrect. The correct affiliation is: Department of Cardiovascular Diseases, The 6th People’s Hospital (Southern) affiliated to Shanghai Jiaotong University, Shanghai, China.


Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE  approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.Definitive secondary structural mapping of RNAs  Recently, a group of compounds have been reported that acylate the ribose 2′OH where the backbone is flexible, which preferentially occurs within single-stranded regions  and kethoxal has been reported  designated the dimerization initiation site DIS, 8). The process is crucial to viral replication and is a prerequisite for successful packaging of the diploid genome into the assembling particle to form an infectious virion . In HIV,. The proUsing in-gel probing of the HIV-1 packaging signal monomer and dimer, we show significant differences across much of the structure, but consistent low acylation sensitivity at the DIS. This implies a structural switch mechanism where the palindromic DIS is base-paired intramolecularly to another sequence in monomeric RNA and where it interacts with its counterpart intermolecularly in the dimer. When our data are compared with previous studies, they most closely fit a model where, in the monomer, the DIS base pairs intramolecularly form a pseudo-knot with part of the U5 region .in vitro transcription from DNA templates containing a T7 RNA polymerase promoter at their 5′ ends. Templates were prepared by polymerase chain reaction using 1× Biomix red (Bioline), 0.5 µg of plasmid DNA  according to the manufacturer’s instructions. Each 20-μL reaction contained 7.5 mM deoxyribonucleotide triphosphates (dNTPs), 1× reaction buffer, 2 μg of template DNA and 2 μL of T7 RNA polymerase for 3 h at 37°C. DNA templates were degraded for 60 min at 37°C with 4 U of DNase . RNAs were purified on MEGAclear columns (Applied Biosystems) according to the manufacturer’s instructions, eluted in water and stored at −20°C.RNA was prepared by  pSVC21,  and 2 µM2), 130 mM KCl and incubated at 37°C for 30 min. One-fifth volume of the native loading dye (40% (v/v) glycerol, 44 mM Tris-borate pH 7 and 0.25% orange G dye) was added, and 12 equal samples were loaded onto lanes of a 20 cm × 18.5-cm 4% acrylamide non-denaturing gel prepared with 1× tris-borate magnesium (TBM)  and fractionated at 100 V for 6 h at 4°C. A gel fragment corresponding to an RNA ladder  and one RNA sample was excised and stained for 10 min with 1.3 µM ethidium bromide in 1× TBM and visualized under ultraviolet. The ethidium bromide-stained gel slice and unstained gel were aligned, and specific unstained bands were removed using a scalpel. Each gel piece was divided into two equal parts, with one fragment soaked in 1× TBM containing 10% dimethyl sulphoxide, and one containing 10% 100 mM N-methylisatoic anhydride  and placed at 37°C for 45 min. Gel slices were washed three times in 1× tris-acetate EDTA (TAE) (40 mM Tris-acetate and 1 mM EDTA pH 8.3), crushed and placed into an Elutrap (Whatman) channel containing 1× TAE. RNA was electroeluted overnight at 100 V into a chamber bordered by Whatman BT-1 and BT-2 membranes, then precipitated overnight at −20°C with 300 mM sodium acetate and 2.5 volume of ethanol. RNA was recovered by centrifugation, washed with 70% ethanol and resuspended in 10 µl of water. RNA concentration was determined by spectrophotometry.Thirty micrograms of RNA was resuspended in 200 µl of renaturation buffer  and 0.1 mM EDTA), heated at 85°C for 5 min and slow-cooled to room temperature. Fifty microliters of refolding buffer was added to adjust the buffer to 40 mM Tris pH 8, 4 mM magnesium chloride (MgClTM-labelled primer (Applied Biosystems) for NMIA-modified samples and VIC®-labelled primer (Applied Biosystems) for negative control samples. Primers were annealed to the RNA at 85°C for 1 min, 60°C for 5 min, 35°C for 5 min and extended for 50 min at 55°C following addition of 8 µl of extension mix . Complementary DNAs were combined and RNA was degraded with 200 mM sodium hydroxide (NaOH) at 95°C for 3 min, followed by cooling on ice and the addition of two sequencing ladders. Sequencing ladders were prepared using the same primers, labelled with NEDTM and PET® (Applied Biosystems), and cycle sequencing was performed using a DNA sequencing kit . Each reaction contained 50 ng of DNA template, 1.3 nmol NEDTM- or PET®-labelled primer, and 1× sequencing buffer, extension mix and enzyme as per manufacturer’s instructions. Cycle sequencing was performed at 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 37°C for 30 s and 70°C for 1 min in a Multigene thermocycler (Labnet). Samples were resuspended in 30 µl of formamide and fractionated by capillary electrophoresis . SHAPEfinder software was used to align the sequences and calculate the relative areas of each peak as in  element, previously characterized by NMR, X-ray crystallography . RNA was probed in situ with NMIA, recovered from the gel, reverse transcribed and analysed as described in Materials and Methods. Seven independent experiments were performed, and on average, the SHAPE reactivity category  was the same at each nucleotide in more than five of these, showing the results are highly reproducible. NMIA reactivity profiles of the monomeric TAR hairpin are shown in Supplementary Figure S2. Reactivities were used as pseudo-free energy constraints in the RNAstructure prediction algorithm to examine the TAR hairpin . When examining 100–500-nt RNAs, the yield remained consistent at ∼40%, giving sufficient recovery for multiple primer extension reactions (which require ∼4 pmol of RNA) from each gel. However, recovery yields dropped when eluting larger molecules, to ∼3% of an 851-nt RNA . Increasing electroelution time did not recover significantly more RNA, with 16 h being the optimal period . Probing with 1M7 for 3 min in place of NMIA also yielded accurate results when TAR structure was examined as before . Data shown were from RNA probed with 10 mM 1M7 and are an average of four replicates; probing with 5 mM 1M7 also yielded similar results, but data were more variable between replicates (not shown); hence we recommend probing with 10 mM 1M7.The conditions we used for in-gel SHAPE were chosen to closely resemble those previously optimized for solution ,9. Thererdiamide , in placin vitro transcription, was renatured and fractionated by native gel electrophoresis. The stained portion of the gel is shown in Supplementary Figure S5 and illustrated for nts 100–354 in As in-gel SHAPE can probe individual RNAs of different electrophoretic mobility within a mixed population, it follows that it could be used to examine conformational switches or dimerization processes. The HIV-1 RNA genome dimerizes via palindromic sequences  within Comparing the SHAPE profiles of the monomer and dimer reveals that the DIS, corresponding to nts 257–262, is unreactive in both C. There Many of the differences in reactivity between the monomer and dimer are statistically significant D. The laSupplementary Figure S5) is also proposed to be within the stable R-Gag duplex in the LDI model, whereas it is adjacent to a bulge in the Lu et al. model  switch proposed by Abbink and Berkhout  are genel. model . For comet al. . Where RNAs of interest migrate differently enough to be separated on a gel, it allows probing of two or more individual structures within the same mixture. Its uses could extend to the examination of structural switches, RNA multimerization or RNA structure within RNA–protein complexes.We hypothesized that in-gel SHAPE could facilitate resolving the native structures of the HIV-1 packaging signal monomer and dimer without the need for mutagenesis to stabilize either conformation. The structure of the HIV-1 packaging signal RNA dimer, investigated by in-gel SHAPE, is almost identical to previously published structures of the monomeric RNA, with base pairing between the two molecules across the DIS . Reactivity profiles correspond closely with the proposed model, and the higher reactivities of some nucleotides in the dimer refolded under conditions that promote both monomer and dimer formation, compared with conditions that strongly favour formation of the dimer, illustrate nucleotides that may adopt different structures in the folding pathway. C219 is reactive, yet within a helix. Some non–A-form backbone angles have been shown to increase reactivity to SHAPE reagents , which mSLs1–3 are designated the major packaging signal region and contain the majority of high-affinity Gag binding sites. SL3 in particular has been shown to bind Gag specifically, and this interaction is thought to be an initial step of the packaging process . HoweverSupplementary Data are available at NAR online.Biomedical Research Centre and the Medical Research Council [G0800142 to A.L.] and the Intramural Research Program of the National Institutes of Health, National Cancer Institute [to S.Le G.]. Funding for open access charge: Medical Research Council [G0800142].Conflict of interest statement. None declared.


O,4′-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery.“Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2′- Modified nucleic acids have great potential for applications in oligonucleotide-based drug design. As natural RNA and DNA molecules are highly sensitive towards nuclease digestion and often possess low thermal stability, great effort has been made to design nucleic acid modifications that stabilize RNA or DNA while simultaneously maintaining the overall Watson-Crick base pairing ability. Modified nucleic acids are indispensable for future applications comprising diagnostic and clinical approaches like the use of aptamers or the siRNA technology.O-methyl-RNAs [O,4′-C-methylene-β-D-ribofuranose (LNA) modification, a significant increase in thermostability can be observed in accordingly substituted RNAs. For example, the melting temperature of modified RNA helices can be increased by +2 to +10°C per LNA monomer substitution.Extensive and challenging experiments and investigations have been undertaken to develop nucleotide analogues that maintain the overall A-RNA-type conformation and N-type sugar puckering, as such modifications are likely to allow the substitution of RNA without large changes in functionality. Considerable effort has been made in the synthesis and characterization of 2′-hyl-RNAs , 2′-F-RNhyl-RNAs , and thehyl-RNAs . By usinO,4′-C-methylene-β-D-ribofuranose LNA-RNA mix-mer duplexes maintain mainly the overall A-type nucleic acid conformation [O,4′-C-α-L-ribofuranose LNA modification is used in DNA substitution, as this modification preserves the overall B-type nucleic acid geometry of DNA [O,4′-C-methylene-β-D-ribofuranose nucleotides (LNA) for RNA substitution and the 2′-O,4′-C-methylene-α-L-ribofuranose nucleotides to modify DNA.To understand the stabilizing effects of LNA-substituted RNAs, numerous structural investigations have been performed during the past years to investigate their conformation in detail. These studies provided insights in the local geometric parameters of mix-mer LNA-RNA helices and of LNA-RNA heteroduplexes. The 2′-ormation . On the y of DNA . Thus, tO,4′-C-methylene-β-D-ribofuranose LNA strand to RNA, whereas a mixed N- and S-type sugar puckering is induced by hybridizing a 2′-O,4′-C-methylene-β-D-ribofuranose LNA to DNA [O,4′-C-methylene-α-L-ribofuranose LNA strand targeted to DNA [2′-O,4′-C-methylene-β-D-ribofuranose “locks” the LNA in the C3′-endo conformation. This approach is used to direct the geometry of the phosphate backbone in a manner to orient the duplex towards a more efficient base stacking.The structure of heteroduplexes, consisting of one fully modified LNA strand hybridized to either RNA or to DNA, revealed the following: the RNA conformation is maintained upon hybridizing a 2′-A to DNA . The B-td to DNA . It is gO,4′-C-methylene-β-D-ribofuranose LNA-RNA mix-mer duplexes maintain the overall A-type nucleic acid conformation, molecular dynamics simulations [O,4′-C-methylene-β-D-ribofuranose building blocks, yielded insights into a novel nucleic acid geometry. An “all LNA” duplex shows alterations in the local and overall helical parameters as compared to natural RNA and can rather be compared to other modified nucleic acids, like glycol nucleic acids (GNAs) [Even though the 2′-ulations  and a crulations  of “all”s (GNAs) , peptides (GNAs) , or homos (GNAs) . An LNA s (GNAs)  is a geoE. coli tRNASer isoacceptor with the data base “Compilation of tRNA sequences and sequences of tRNA genes” ID RS 1661 [Ser aminoacyl stem microhelix that has been crystallized previously possessing the sequence 5′-(G-G-U-G-A-G-G-)-3′ and 5′-(C-C-U-C-A-C-C-)-3′ [O,4′-C-methylene-β-D-ribofuranose building blocks. The base sequence of the RNA was maintained for further comparative studies, except for the U to T and the C to m5C exchange used in standard LNA synthesis. The chemically synthesized single strands with the sequences 5′-(G-G-T-G-A-G-G)L-3′ and 5′-(m5C-m5C-T-m5C-A-m5C-m5C)L-3′ were purchased from IBA  with HPLC purification grade. Crystals were grown within 3-4 days using 40 mM sodium cacodylate, pH 5.5, 20 mM cobalt hexamine, 80 mM sodium chloride, 20 mM magnesium chloride, and 10% (v/v) MPD with equilibration against 1 mL 33–41% (v/v) MPD at 21°C using the hanging drop vapour diffusion technique [The 7mer LNA helix was derived from the  RS 1661  and reprC-C-)-3′ . The LNASer-microhelix was measured at the DESY synchrotron  at a wavelength of 0.8123 Å, 100 K temperature, and diffracted up to 1.2 Å [Ser microhelix structure but exchanging the riboses by 2′-O,4′-C-methylene-β-D-ribofuranose residues [5C substitutions in LNA as compared to RNA. Refinement calculations were done applying the program REFMAC [Data collection of the LNA crystals was performed at the ELETTRA synchrotron  beam line XRD-1 at a wavelength of 1.0 Å and a temperature of 100 K. The crystal diffracted up to 1.9 Å resolution . The corto 1.2 Å . All datto 1.2 Å . Moleculto 1.2 Å  within tto 1.2 Å . The RNAm REFMAC , and elem REFMAC , as implm REFMAC . Data anm REFMAC  was usedm REFMAC .O,4′-C-methylene-β-D-ribofuranose nucleotides (We analysed the crystal structure of a “locked” nucleic acid duplex , which cleotides , in compThe LNA helix structure reveals a nucleic acid duplex geometry that significantly differs from the canonical A-type RNA structure  and 3. T−, ap+, sc+, sc+ ap, sc−, and ap+ conformation for the α, β, γ, δ, ε, ζ, and χ angles with the sugar puckering being in the 2′-exo conformation. The phosphate-phosphate distances are in the region of 5.6 Å as compared to 6.0 Å for RNA. It is conceivable that the altered helical parameters in LNA duplexes provide an enhancement in nucleotide stacking, leading towards stronger Π-Π interactions of the base pairs.In the LNA helix, the low twist angle of 26° and the large pitch of 14 base pairs per turn lead to an unwinding of the duplex, as compared to RNA, which possesses a twist of 32° and a pitch of 11 base pairs per turn. The helical rise of LNA falls into a range of 2.8–3.0 Å, whereas the helical rise in RNA is 2.6 Å. Due to the increased rise and the unwinding of the helix, the LNA possessed an enlarged helical pitch of 39 Å, as compared to 29 Å in RNA helices. The backbone torsion angles resembled the scO,4′-C-methylene-β-D-ribofuranose in LNA allows a comparable hydration as described for RNAs. Therefore, we focussed our investigations on analyzing the arrangement of the solvent molecules surrounding the LNA duplex. We observed that the distribution of water molecules in the LNA minor groove follows the general pattern known for RNA hydration, as the bridged 2′ oxygen atoms in the 2′-O,4′-C-methylene-β-D-ribofuranose moieties serve as hydrogen bond acceptors similar to the 2′-hydroxyl residues in RNA. An example for the LNA hydration is shown in It is well accepted that the extensive hydration of the RNA minor groove plays an important role in the structure/function relationship . As the Interestingly, the structure of an RNA/LNA hybrid helix represents a geometric intermediate between RNA and “all LNA” helices. In Ser microhelix for this study, exhibits a melting temperature of above 90°C, whereas the corresponding RNA has a Tm value of 45.0°C [Tm value of 84.3°C, whereas its corresponding RNA helix melts at 22.4°C. The drastic shift in thermostability in both LNAs as compared to the RNAs corresponds to an average of 4.5°C per nucleotide building block, which is consistent with the reviewed observations [As has been reviewed , an incrf 45.0°C . SimilarTm values by substituting natural nucleic acids with single or multiple nucleotides by LNA residues seems to be a summative property, the challenge of using LNAs as tools in nucleic acid stabilization becomes obvious. Nearly any natural nucleic acid can be modified by introducing single and multiple LNA building blocks or even complete LNA duplexes, thereby stepwise increasing the thermostability depending on the number of introduced LNA residues. Depending on the particular requirements, any nucleic acid can thus be stabilized at will with little or no loss of function. In this respect, LNA substitution may serve as a reliable method to stabilize nucleic acids, in particular aptamers, for clinical applications.Considering that the increase in An upcoming challenge is to stabilize aptamer stem regions by introducing LNA portions without affecting the loop regions that are usually essential for target binding and specificity. Several reports in the literature highlight a forthcoming application of LNA-substituted aptamers with retained or even improved ligand-binding capacity. An exciting example is the use of LNA modifications within hammerhead ribozymes that improve the overall cleaving capacity . In addi