Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A Specifically a protein nanopore, with sub 2 nm diameter, is inserted in a planar lipid bilayer membrane that separates two electrolyte filled compartments. Applying a voltage across the two compartments leads to a constant flow of electrolyte ions via the nanopore (i-t). This flow is reduced by the passage of a single molecule through the pore. In one approach, alpha-Hemolysin (α-HL) conjugated to a polymerase enzyme at the entry of the pore, directs the synthesis of the complementary strand of a target DNA. Upon elongation with polymer-conjugated mononucleotides, the cleaved polymer goes through and is detected by the pore12. Using a unique polymer tag for each nucleobase leads to sequencing-by-synthesis (SBS)12. A second approach, using α-HL16, Mycobacterium smegmatis porin A (MspA)19, or the proprietary nanopore of the MinION20, achieves direct sequencing of the target strand. Here a single stranded (ss) DNA/RNA strand moves through the pore assisted by an enzyme that acts as a motor to move the strand one nucleobase at a time slowing down the translocation to a rate amenable to measurement23.Nanopores made critical strides the last 30 years due to the efforts of scientists and technologists who spearheaded nanopore-based DNA sequencing8. Solid-state nanopores were used to fingerprint nucleic acid nanoparticles24, and for protein detection and quantitation25. α-HL was successfully used for polymer sizing26, for metallic nanoparticle characterization27, and to measure the kinetics of enzymatic phosphodiester bond formation28. A modified MspA was shown to measure the movement of enzymes along DNA in real time29, wt aerolysin to resolve a series of deoxyriboadenylates30, and an engineered Fragaceatoxin C to discriminate among peptides31. Cytolysin A nanopore was used to report the concentration of glucose and asparagine from samples of blood, and other bodily fluids32, introducing nanopores to the arena of non-invasive diagnostic assays.In addition to sequencing, several nanopore platforms are successfully employed for nanoparticle or single molecule analyses5. In 2014 ONT commercialized a hand-held, relatively inexpensive device, the MinION. This device is equipped with an array consisting of over 2000 preassembled nanopores from a bioengineered pore-forming lipoprotein, CsgG33, and achieves DNA/RNA sequencing in conjunction with a laptop and the internet. The software of the device, MinKNOW, selects 512 nanopores with optimal properties, and records simultaneously 512 i-t traces that decode to nucleic acid sequence. Of these, a typical run initially has less than 500 pores and this number decreases over time as the pores degrade or become blocked. The i-t traces are also reported as raw data in fast-5 file format, and can be visualized using MatLab from Mathworks. The availability and convenience of this device led us to evaluate it as a simple analytical tool for RNA oligos, and to compare it to traditional analytical tools such as high-performance liquid-chromatography (HPLC).Using a nanopore experimental platform is a complex process. Specifically, the monomeric unit of the nanopore protein needs to be manufactured, purified, assembled as a pore, and inserted in a premade stable lipid bilayer. This is followed by addition of the nucleic acid in a small reaction vessel without disrupting the lipid bilayer/nanopore construct5. The MinION exhibits a 3.012 kHz sampling rate, equivalent to reporting 3 data points per 1 ms. This slow acquisition rate implies that DNA/RNAs, let us say 100 nucleotides (nt) or shorter, may not be detected/recorded by this device. In addition to our own experiments (see below), multiple reports confirm this expectation. In one study, attempts to sequence RNAs shorter than 200 nt following ONT protocol were unsuccessful34. In another study 100 nt deoxyoligos were successfully sequenced by the MinION, albeit only after circularization and rolling circle amplification35. Several experimental nanopore platforms have been successfully exploited for detection of short RNAs by using a target probe complementary to the desired RNA sequence38. Current technologies for “small RNA” identification/quantitation, such as microarray, sequencing ), and qRT-PCR-based methods, are complex and have extensive resource demands40. All these different methods exploit base-pairing or SBS, and therefore misinterpret any post-transcriptionally modified bases41 that may be present in the target RNA. Short RNAs are found in biological fluids, are relatively stable, are likely to contain modified bases, and are shown to exhibit important regulatory functions (see discussion later). Hence it is important to find ways to properly characterize them, and establish the presence, identity, and abundance of any modified bases within.Voltage-driven translocation of RNA via a nanopore is very fast and measures 5 to 10 μs per base43. Assuming the label has high specificity, selectivity, and exhibits negligible side-reactions, the bulkiness alone is expected to slow down translocation to measurable i-t levels. The presence of a bulky tag should lead to a markedly larger discrimination between tagged base and native base, compared to the discrimination between two native nucleobases. In turn, better discrimination may reduce i-t dependence from the reported 5 nt sequence34 to, perhaps, a single labeled base. The selectivity/reactivity of the label for one base over another –see below, for example the selectivity of OsBp for Thymine (T) over Cytosine (C) - might enable base identification for some among the known 150 post-transcriptionally modified bases41. Our approach in passing the tagged nucleic acid via a nanopore is different from the one described above (SBS), where only the tag(s) are passing via the nanopore12, which is intrinsically blind to post-transcriptional modifications.To enable short RNA sequencing we proposed to leverage the physicochemical properties (differences) of the bases and selectively tag one or more with a bulky label47. Recent work showed that OsBp addition to DNA, coined here osmylation, using Yenos’ proprietary protocol is a remarkably clean reaction. Osmylation yields pyrimidine conjugates in practically 100% yield with no detectable reactivity towards purines, and no phosphodiester bond cleavage48. The selectivity of osmylation for deoxythymidine (dT) over deoxycytosine (dC), is 30-fold, and leads to labeling of either mostly dT, or both dT + dC using low or high OsBp concentration, respectively42. Using a training set of deoxyoligos a UV-Vis assay was developed and validated to measure the extent of osmylation42 (see later). Monitoring the osmylation kinetics by HPLC or capillary electrophoresis (CE) resolved all the intermediate products, and led to the conclusion that the process is kinetically independent of sequence, length, and composition. As shown with M13mp18, a circular 7249 nt ssDNA, secondary structure does not affect the efficiency of osmylation, most likely due to a denaturing effect exhibited by the OsBp reagent43. The same protocol yields practically 100% pyrimidine osmylation in both short and long ssDNA43.The reactivity of OsBp, known to add to the C5-C6 double bond of the pyrimidines, was established in the 1970s49. Experiments with dA10dPydA9 via wt α-HL showed readably slow and distinct translocation features for different deoxypyrimidines (dPy)50. Specifically, when dPy = dA, dT(OsBp), dC(OsBp), dU(OsBp), or 5-MedC(OsBp) the distribution of the fractional residual ion current, Ir/Io, where Io is open pore ion current and Ir is the residual ion current of the blockade or event has a maximum at 0.14, 0.08, 0.11, 0.12 and 0.12 (with STD ± 0.01), respectively. Similarly the observed duration or dwell times (τ) measured at −120 mV are 50, 150, 310, 360 and 470μs, respectively50. The effects are small with respect to residual ion current differences, but substantial with respect to translocation duration for oligos with a single dPy(OsBp)50. The collective data in two distinct nanopore platforms illustrate a remarkable slow down and visible discrimination between intact and osmylated DNA oligos.The outstanding labeling features of OsBp led us to undertake nanopore-based single molecule translocation experiments in a number of collaborating laboratories both in industry and academia. Pore size suitability using solid-state silicon nitride (SiN) nanopores showed that 1.6 nm wide pores permit translocation of 80 nt long osmylated deoxyoligos, and demonstrated dramatic tranlocation slowdown with increasing osmylation53. microRNA (miRNA) is a group of short RNAs, 17 to 25 nt long, with universal regulatory functions51 and with applications in personalized medicine as biomarkers and potential therapeutics56. Due to the interest in short RNA characterization we extend here our osmylation protocol to RNA. Here we report that due to the mildness and selectivity of our osmylation protocol OsBp exhibits excellent labeling properties with RNA pyrimidines, comparable to that described above for DNA pyrimidines. Using the MinION and high quality synthetic RNA oligos . Within the stochastic nature of the translocation process, a small number of long translocations yielded a rudimentary sequence pattern in the form of pyrimidine-purine (Py-Pu), that may be sufficient for short RNA characterization in a mixture. These observations serve as proof-of-principle for using commercially available nanopore device(s) as auxiliary analytical tools for short RNAs and miRNAs enhanced with an Osmium tag.The above findings, namely that DNA oligos can be selectively tagged on the pyrimidines and that these tagged oligos exhibit pyrimidine-dependent translocation features, led us to extend the work to RNA oligos. In addition, we now make use of a commercially available nanopore-device, so that the experiments can be reproduced by other scientists less familiar with the nanopore field. RNA oligos in the range of 20 to 300 nt include coding and non-coding nucleic acids with known functions in mRNA translation and in regulation of most biological processes43; these conditions were reevaluated, and optimized for RNAs. Briefly the RNA osmylation protocol requires 3 hour incubation in glass vials at room temperature in water with no buffer in the presence of 12 to 14 mM OsBp . Osmylation kinetics were monitored automatically every 15 min using CE, and the CE profiles are included in the corresponding figure of the oligo in the Supplementary Information. With the exception of the oligoriboadenylate with 5-MeU substitution, which is subject to very fast osmylation, just as with the deoxy derivative42, all other tested oligos exhibit comparable reactivity. Observed relative reactivity towards osmylation obtained from the kinetics of T11 though T16 using 5.2 mM OsBp at 26 °C in water are U/C = 4.7, 5-MeC/U = 0.9, 5-MeC/C = 4.1 and 5-MeU/C = 44. This trend is in excellent agreement with the relative reactivity observed in deoxy oligos50. Osmylation reactivity depends on the electrophilicity of the C5-C6 double bond, and this is the feature that will enable selectivity of one vs. another nucleobase among some of the post-transcriptionally modified RNA bases41. Typically the reactivity of a base in a sequence mirrors the reactivity observed with the mononucleotide.Osmylation kinetics at the above manufacturing conditions are independent of RNA length, composition, and secondary structure, as seen in Fig. 57 . Comparison between the HPLC profiles after 3 h and 24 h of osmylation , because OsBp migrates (CE) or elutes (HPLC) well ahead of the oligo and of the osmylated product. However UV-Vis spectrophotometric measurements will not differentiate between label and labeled oligo, and therefore removal of the label is necessary. HPLC/CE profiles for each tested intact oligo and its fully osmylated derivative are included in the corresponding figure in the Supplementary Information. Osmylation does not lead to side-reactions, as can be seen in these profiles. In addition, the stability of the RNA in the presence of OsBp was evaluated with an extra pure 74 nt long RNA and with a 4500 nt long mRNA Cas9 using a stability-indicating HPLC method43. Using the ratio R instead of the absorbance at 312 nm serves to normalize the measurement, and minimize instrument sampling variation. When experimental value R(312/272) is equal to 2 × , osmylation is practically 100% complete43. The wavelengths 312 nm and 272 nm were chosen in order to maximize the effect and to equalize contributions by osmylated dT, dC or dU, assuring that nucleic acid composition doesn’t affect the assay’s accuracy which stands at ±3%. The R(312/272) data obtained in this study  creates a new chromophore in the wavelength range of 300 to 320 nm, where nucleic acids exhibit negligible absorbance. We exploited this observation and used a deoxyoligo training set to show that extent of osmylation can be measured using the equation R312/272) = 2 × . R(312/272) is the ratio of the observed absorbance at 312 nm over the observed absorbance at 272 nm2/272 = 257. Typically RNA oligos up to the 60-mer can be resolved by ion exchange (IEX)58 or ion-pair reversed phase (IP-RP)59 chromatography. Both chromatographies were exploited here for analysis of the RNAs used in this study. As HPLC analytical columns  we used the ones that in our experience give the best possible resolution, but alternative columns/methods may be put to the test. The 31 nt oligos in this study vary minimally from each other, and their separation and identification by HPLC presents a challenge. Despite method development efforts, complete separation was not achieved. Figure HPLC analysis is routinely used for identification, resolution from impurities, stability evaluation, and quality control of pharmaceuticals. RNAs, such as single guide RNA (sgRNA), mRNA, or miRNA, are considered for therapeutic applications, and validated analytical methods to characterize them are being sought60. Due to the strand directionality these two isomers are detectable by either HPLC or CE . Each of the 31 nt osmylated oligos tested here exhibited two peaks with a product ratio not far from unity. It is known that voltage-driven translocation of nucleic acids via nanopores yields different ion current levels depending on the direction of the strand, 3′-entry vs 5′-entry61. Since RNA(OsBp) exists in two isomeric forms, it is plausible that a nanopore will sense and discriminate them by exhibiting two different Ir/Io levels. Depending on the strand entry in the pore each isomer may yield two Ir/Io levels for a total of four. In the absence of experiments with immobilized RNAs within the pore63, it is difficult to assess this proposition. Nevertheless the Ir/Io histogram of T11(ACA) presents four distinct (Ir/Io)max, supporting the postulate of four Ir/Io levels  and T32(UU)  equipped with a proprietary glass nanopore membrane (GNM) . In this platform (NanoPatch/wt α-HL) the discrimination of the nanopore for RNA compared to DNA appeared to be much stronger  discrimination in deoxyoligos observed with α-HLIr/Io data determined for each can be found in the corresponding figure in the Supplementary Information. It should be emphasized that the ONT protocol was not followed, no library was created, and the processing enzyme was not included in this study. Hence all the reported translocations are unassisted, driven by the voltage drop, and conducted with a biased voltage in the range −140 mV to −220 mV.The sequences of the tested RNA oligos 22 nt to 100 nt long are listed in Table i-t traces from three experiments each conducted at −180 mV with a A15PyA15 where Py is 5-MeU, U, and 4-SU. Visual inspection, as seen by the position of the boxes, suggests that the observed translocations with the oligo carrying 5-MeU(OsBp) yield substantially less residual ion current (Ir) compared to the translocations with the oligo carrying U(OsBp), and the later yields dramatically less residual ion current compared to the one with the 4-SU(OsBp). The “Ir” value at the middle of each box is around 20, 40, and 100 pA for 5-MeU, U, and 4-SU, respectively, spanning a range of 80 pA, somewhat larger than what currently is used for sequencing RNA with the MinION34. Figure Figure i-t trace from the 512 channels, and by graphing the translocation events (see discussion below) separately for a number of channels. Judging from obtaining superimposable histograms from different channels, channels are comparable. Open pore ion current (Io) from different channels may vary by up to ±15 pA within a single experiment, but data are normalized by reporting the fractional residual ion current (Ir/Io) as seen in the histograms in the Supplementary Information. Typically two experiments were conducted with the same oligo using different flow cells, and data are reported from four or more channels for each experiment. It was practically established that 300 Ir/Io values are sufficient to yield a valid/reproducible fingerprint, and 500 to 1400 Ir/Io values are reported per oligo.To the best of our knowledge, the MinION is exclusively used for enzyme-assisted sequencing. Hence our work regarding voltage-driven RNA characterization is a novel application for this platform. In this context, we evaluated MinION’s suitability as an analytical tool for short RNAs. Three basic questions were addressed: (a) Do all working nanopore channels provide comparable results, (b) Are intact RNA oligos detected by the MinION, and (c) Is the MinION’s pore protein a size-suitable nanopore for osmylated RNA. Briefly the answers to these questions are: (a) and (c) Yes; (b) No for shorter than 31 nt and Yes for longer than 74 nt RNA. The first question was answered by visually inspecting every recorded i-t recording of ONT proprietary buffer. It is noticeable that the open pore current is interrupted by “instrument lines” only, and not by any translocation events. These “instrument lines” traverse the i-t trace, and typically exhibit a mirrored image of lines extending vertically up and down. Figure i-t recording from an experiment conducted with an intact 31 nt RNA (T11(ACA)). Figure 5 that estimates τ = 0.7 ms for the 31 nt RNAs in our study, and MinION’s specifications of 3 data points per 1 ms. In contrast, Fig. i-t recording from an intact 74 nt RNA  and poly(U) translocate via the MinION and exhibit substantially lower Ir levels , compared to the one observed with T6  moieties. A large number of events is observed; some exhibit high residual current Ir, attributed to “bumping events”, and some exhibit low Ir, attributed to true RNA(OsBp) translocations. The latter are grouped into two Ir levels, as highlighted by the blue transparent blocks. It is noteworthy that the Ir levels considered as true translocations with the osmylated oligo are more ion current obstructing compared to the Ir levels considered as true translocations of the intact. This visible distinction is attributed to the presence of the bulky OsBp tag within the confined space of the nanopore.Figure i-t trace from an experiment conducted with a fully osmylated 100 nt RNA  whether relatively long oligos, such as the 100 nt sg RNA(OsBp) can traverse the pore, and (b) whether the presence of multiple consecutive Py(OsBp) prohibits translocation. Figure Io ≈ 270 pA, considered sufficient to induce numerous translocations  two 32 nt oligos were designed with 9 or 13 pyrimidines, lined in groups with up to 5 adjacent pyrimidines (see T7(13) and T8(9) in Table A fourth question that was not fully investigated is whether the MinION may be used for quantitation of osmylated RNAs. The experiments reported here are conducted with a formal oligo concentration about 1.5 μM in the ONT buffer, obtained by dilution in the ONT buffer from a 20 μM RNA(OsBp) stock solution. Considering that the pyrimidine osmylation is practically 100% and that purification leads to practically full recovery with no dilution, it will be of interest to quantify RNA(OsBp) by nanopore and deduce the concentration of a certain RNA in an unknown sample. Limited experiments with dilutions in the range of 3- to 10-fold from the typical concentration illustrate proportionality between concentration and number of actual translocations and supports quantitation. To further probe this issue the software, buffer, and pore temperature, which is currently at 34 °C, of this platform need to be optimized for unassisted translocation of osmylated RNA. Over a period of 10 months a total of 8 different flow cells was used, primarily due to our inexperience with this platform and the novelty of the application. Over 70 experiments were conducted lasting an hour each.i-t recordings in fast-5 file format and direct visualization with MatLab software on a i-t plot that includes a units grid, which allows one to precisely determine Io, Ir and τ for any event at an apparent accuracy much better than the device delivers. This feature permits two people, one reading and one reporting, to estimate and report lowest Ir values for every event at ±(1 to 2) pA at a rate of about 700 data per hour. Concatenated i-t traces, e.g. the ones reported in Fig. fast-5 files in txt format readable by open source software QuB  and manually “cleaning up” the file to remove instrument lines, and other events that can’t be attributed to single molecule translocations, as will be described next.The software of the MinION, MinKNOW, is currently set up for sequencing experiments, and not for analysis of single molecule translocation data. A recent MinKNOW update includes acquisition of raw Ir, and duration in the range of a few seconds that seem to increase with voltage, oligo concentration, and flow cell use. These events were attributed to noise and ignored. (ii) Relatively shallow and short, τ ≤ 2 ms, events that measure 0.4 < Ir/Io < 0.9, presumably resulting from molecules bumping at the pore; these were also ignored. (iii) Long events with Ir/Io < 0.4 that appear to be the result of multiple translocations, one molecule following the other without reaching open pore current between events; these were also ignored. (iv) Last, but not least, events were observed of low noise with Ir/Io < 0.4 and durations in the range of 2 to 200 milliseconds. We measured 150 to 250 events of group iv (at about 1.5μM oligo concentration) per half an hour of i-t recording. We believe that the count of group iv should approach 1000, if one were to count in molecules that translocate as part of group iii, or if the system is optimized so that group iii is suppressed, and replaced by group iv. The observed Ir for group iii is likely not comparable to the corresponding Ir for single molecule translocation due to occupation of the pore by more than one molecule at the time. We attribute group (iv) to single molecule translocations and reported the lowest observed Ir (pA) value for each translocation. Initially we also reported duration (τ) for each translocation  against each other by normalizing the counts of each bin for the total number of translocations as seen in Fig. Values 2 moiety on C4, whereas U contains an Oxygen (O) on C4, hence the mass unit difference between these two RNAs is 1/10,500, where 1 mass unit is the difference between NH2 and O and about 10,500 is the molecular weight of a singly osmylated 31 nt RNA. Figure Ir/Io profile (fingerprint) of T11(ACA) has, perhaps, up to four maxima with (Ir/Io)max = 0.08, 0.15, 0.19 and 0.23, whereas the Ir/Io profile of T12(AUA) has two maxima at (Ir/Io)max = 0.12 and 0.17. It is presumed that the translocation(s) with the least residual ion current corresponds to a 3′-entry and the one with the higher residual ion current corresponds to 5′-entry, in analogy to the observations with intact nucleic acids61. The four (Ir/Io)max values with T11(ACA) may be attributed to the four different configurations of interaction between a singly osmylated RNA and the nanopore max values observed with most of the 31 nt oligos.Each figure in Fig. Ir/Io)max values from the unassisted translocations should correspond closely to the ion current levels in the presence of the enzyme. Figure Ir/Io)max values) that, for a typical Io = 200 pA, translates to 8 pA. A difference of 8 pA suggests that the ACA subsequence is well discriminated from AUA, and that the presence of an enzyme-motor would result to a highly accurate base calling for C(OsBp) vs. U(OsBp). Assuming that the nucleotide ahead of the tagged one (A here), may not contribute much to the observed discrimination, we propose that this platform senses a two nucleotide subsequence, when the first nucleotide is tagged.For the purpose of the following discussion the proposition is made that, if a motor enzyme could be engineered to process osmylated RNA one base at a time, then the observed (Ir/Io from the experiments with T11(ACA) and T14(GCG), and illustrates the effect of replacing the adenosine with guanosine. It appears that (Ir/Io)max = 0.08 and 0.19 with ACA are now shifted to more residual ion current (Ir/Io)max = 0.13 and 0.21 with GCG. This is a major shift and supports the above proposition of sensing a two nucleotide subsequence, when the tagged nucleotide is Cytosine. In contrast to the effect observed with tagged C, tagged U doesn’t result to such large effect, i.e., yielding comparable Ir/Io profiles between T12(AUA) and T15(GUG) max = 0.12 and (Ir/Io)max = 0.13 may yield a 2 pA (from (0.13–0.12)*200 pA) distinction only. Enhanced discrimination in this case may be achieved in the presence of a second tag to react selectively with Guanosine and leave the other bases intact.Figure UG) Fig. ; hence tIr/Io)max = 0.03), that the difference in the pyrimidine moiety becomes “silent”. Whether or not replacing A with G adjacent to the 5-Me-pyrimidine will make a difference, remains to be tested. It is noticeable that replacing a canonical pyrimidine with its 5-Me derivative yields 10 pA for C (from (0.08–0.03)*200 pA) and 18 pA for U (from (0.012–0.03)*200 pA) lower maximal residual ion current leading to, perhaps, the most accurate base calling envisioned for 5-Me modifications compared to the unmethylated base. Since the specific nanopore platform does not discriminate 5-MeC from 5-MeU, one could use the higher reactivity of OsBp for 5-MeU over 5-MeC, C, U, and 4-SU max in the range of 0.03 ≤ (Ir/Io)max ≤ 0.13. This range falls below the (Ir/Io)max = 0.15 reported for the intact 74 nt T6, and far below the (Ir/Io)max = 0.31 observed with poly(I) max = 0.27 max = 0.27 is the result of shorter oligos, due to phosphodiester cleavage at the 4-SU base, is excluded based on IEX HPLC analysis57 of the osmylated T17 that reveals no detectable shorter oligos. The osmylation mechanism in the presence of tertiary nitrogen donor ligands, including 2,2′-bipyridine, is well documented , which would have been the product of this transformation, translocates with (Ir/Io)max = 0.08 and not 0.27 max data in Table 15 tails were to be replaced by A25 tails, then HPLC resolution is unlikely. In contrast, a nanopore interrogates the molecule as it passes through, and length is practically a non-issue. Hence nanopore-based characterization of RNA(OsBp) is, in certain cases, superior to intact RNA analysis by HPLC.Due to sequence similarity, discrimination among the seven 31 nt RNAs presents an analytical challenge. Osmylation results in HPLC peak broadening, or formation of two peaks, due to the presence of two topoisomers. Hence HPLC analysis of osmylated oligos will not yield better resolution. A comparison can be made between resolution of the intact oligos by HPLC and discrimination of the osmylated oligos by nanopore. In this context only T17 is easily discriminated from the others by both HPLC and the MinION  and T32(AUUA) and illustrates their different fingerprints. The observed (Ir/Io)max = 0.03 and 0.06 are listed in Table Ir/Io)max values between T11 (with one C(OsBp)) and T31 (with two adjacent C(OsBp) clearly shows less residual ion current for the latter. The same observation is made between T12 (with one U(OsBp)) and T32 (with two adjacent U(OsBp)). These results support a two-base discrimination in this platform. In addition, T31 exhibits lower (Ir/Io)max compared to T32, just as T11 exhibits lower (Ir/Io)max compared to T12, indicating internal data consistency. If any, the shorter sequences (T31 and T32) should have produced more, not less, residual ion current, but the length appears to play a smaller role here. Shorter sequences, 22 nt instead of 31 nt, were chosen for T31 and T32 in order to achieve better resolution of the anticipated three diastereomeric products max = 0.03 for both, only that the longer oligo exhibits relatively more counts towards less residual ion current compared to the shorter oligo. This feature can be rationalized on statistical grounds considering that molecules with two U(OsBp) in OL configurations max with T3(UU-UU) and T6(UU-UU-UU) measures 0.03, whereas (Ir/Io)max with T32(UU) measures 0.06, indicating that the residual ion current inside the pore for unassisted translocations is modulated by a sequence of, at least, 4 + 19 = 23 nucleotides (from UU + UU + 19As). This conclusion is strictly valid for an unassisted translocation that yields a single event, and not applicable to either motor-enzyme assisted translocations or slow translocations that yield sequence information (see later). Figure Ir/Io)max = 0.01 for both. The similarity suggests that during unassisted translocation a certain number of OsBp moieties, perhaps 9 out of 32 nt, is responsible for modulating ion current and additional OsBp do not add to obstruction and do not prevent translocation. The extreme case of osmylated poly(C) or poly(U) remains to be tested. In addition to the 100 nt sgRNA (T1), T7 and T8 were specifically designed with five adjacent pyrimidines at the 3′-end to support the expectation that t-RNAs and sgRNAs will translocate via the MinION. Figure 66 (see more references in the corresponding figures in the Supplementary Information). Figure Ir/Io)max = 0.04. To rationalize this difference more miRNAs need to be tested. These two miRNAs could, in principle, be discriminated based on their Ir/Io different histogram profiles, but not within a mixture.Figure ons Fig.  are stat37, but we believe to be the first to propose a nanopore-based assay to determine purity and impurities in a sgRNA preparation. The first question to ask is whether the MinION/OsBp platform has such potential. On average i-t recordings include 500 single molecule translocations per channel per hour, as shown above. Selecting only the ones with low Ir attributed to OL and PA configurations max listed in Table Ir level is consistent with OL configuration and the other two with PA configuration  subsequence. Notably a 10-fold lower concentration (0.15 μM) used for this experiment led to about a 10-fold decrease in translocations. Figure Ir level that correspond to the three subsequences with consecutive pyrimidines and the higher Ir level that corresponds to the purines .We present here a practical approach of how to obtain sequence-bearing translocations, and show such examples in Figs Ir level that corresponds to single Py(OsBp) interspersed among purines, and exit from the 3′-end consistent with low Ir level that corresponds to the three consecutive Py. The left figure highlights the dips (pink blocks) that may be attributed to the six single Py(OsBp) separated by purines, all six at a higher Ir level compared to the Ir level of the last dip that is attributed to the Py-Py-Py at the 3′end. Figure Ir levels: the high Ir level is attributed to Pu, and the low Ir level attributed to the two subsequences each with four consecutive Py(OsBp). The translocation with the longer duration (right) appears to match better the sequence of miRNA140, in the form of Py-Pu. Figure Figure 2 and experiments reported here  of any functional group within the pore will remain intact67, unless it is replaced by a new component in the system. In this context higher solvation requirements can rationalize the typically lower Ir of ribooligos compared to deoxyoligos, the typically lower Ir of pyrimidines  compared to adenine , and the reported here higher Ir level of 4-SU compared to U, consistent with the almost double atomic radius of S vs. O and its resulting easier desolvation. Reduced solvation requirements may also rationalize the apparently lessened effect of OsBp’s size on the ion current reduction as follows: OsBp moiety, with its almost parallel to the strand axis configuration, serves as a “shield”, and by steric hindrance replaces water molecules that otherwise accompany/solvate the nucleobases. The observed extra low residual ion current levels observed in this study, illustrate “touching” proximity between tagged-RNA and nanopore, leading to desolvation and reorganization that enforces superior recognition and discrimination.When we proposed to combine nucleic acid labeling with nanopore-based analysis, we expected that the bulkiness of OsBp will limit nanopore selection. The first surprise came by observing translocations of osmylated oligos via α-HL, which has been the prototype protein pore for intact nucleic acids. How is it possible that the increased bulkiness of RNA(OsBp) fits via the same pore size as intact RNA? The second surprise was to observe translocations of T7 and T8 (both with a five consecutive Py(OsBp) subsequence) via the MinION pore protein, albeit at the higher biased voltage of −220 mV. How is it possible to use a platform suitable for intact RNA and detect translocations of overlapping OsBp moieties? The unexpected observations we made with the bulky OsBp fitting pores suitable for intact nucleic acids are not in disconnect with earlier reportsere Fig.  that purEarlier nanopore studies with intact DNA/RNA exploited an immobilized strand in order to show base-to-base discrimination. Osmylation of nucleic acids adds a bulky tag to all pyrimidines, but does not prevent the modified strand from successfully translocating via protein or solid-state nanopores. Unassisted voltage-driven translocation of osmylated oligos is dramatically slow, and residual ion current is markedly reduced in several nanopore platforms. Here we reported on the translocation properties of osmylated RNA oligos and miRNAs via the MinION device from ONT. We showed that this platform discriminates oligos with structural differences so small that even traditional analytical tools like HPLC may not resolve. For example, conductance measurements visibly discriminate among 31 nt oligoadenylates carrying a single OsBp-tagged pyrimidine. We proposed that this discrimination is the result of slow translocation and close proximity at the narrowest point of the nanopore. The proximity reduces the number of solvating water molecules and enhances interaction/recognition of the chemical components. We demonstrate that histograms of residual ion current from the lowest 25% range serve as the RNA fingerprint, and may be used to confirm/reject proposed sequences in short RNAs, and miRNAs.The MinION/OsBp platform exhibits substantially reduced residual ion current in the presence of a Py(OsBp)-Py(OsBp), compared to a Py(OsBp)-Pu, indicating a 2 nt sensing, in contrast to the 5 nt sensing observed with intact nucleic acids. Our study illustrates the contribution of the labeling approach for improved recognition in nanopore-based analysis. The MinION and the newer Flongle devices are commercially available, easy to use, relatively inexpensive, and could be further optimized for use with labeled nucleic acids. This proposition is supported by examples of extra slow single molecule translocations bearing multiple ion current levels that mirror the Py-Pu sequence of the tested RNA. RNAs in the range of 20 to 300 nt have important regulatory functions and are currently promoted as biomarkers and/or pharmaceuticals. A simple assay to identify and quantify short RNAs and miRNAs via their Py/Pu sequence may be easy to develop, implement, and commercialize.in-vitro transcription was purchased from tRNA probes, Texas. Homopolymers poly(C), poly(U) and poly(I) were purchased from Sigma-Aldrich. Purity of oligos was tested by HPLC in-house, typically about 85%, but varies depending on the purification level that was requested from the manufacturer. RNAs were diluted with Ambion Nuclease-free water, not DEPC treated, from Thermo Fisher Scientific typically to 200 or 400 μM stock solutions and stored at −20 °C.Custom-made RNA oligos were purchased from Trilink Biotechnologies or Dharmacon (Horizon Discovery Group); their sequences and the properties of their osmylated derivatives are listed in Table 4 in ampules at 2 mL each) was purchased from Electron Microscopy Sciences. 2,2′-Bipyridine 99 + % (bipy) was purchased from Acros Organics.Buffers DNase- and RNAse-free TRIS.HCl 1.0 M pH 8.0 Ultrapure was purchased from Invitrogen and TRIS.HCl 1.0 M pH 7 from Sigma. Triethylamine acetate buffer 2.0 M pH 7 and Sodium hydroxide 1.0 M bioreagent were purchased from Sigma-Aldrich; KCl and NaCl crystalline ACS min 99.0% from Alfa Aesar. 50 mM sodium tetraborate buffer pH 9.3, HPCE quality, from Agilent Technologies. Monomeric protein of the wt α-HL was purchased from List Biologicals, Mountain View, CA. Distilled water from ArrowHead or Alhambra was used for preparation of HPLC mobile phase. A 4% aqueous osmium tetroxide solution  according to the manufacturer’s instructions which takes about 8 min. Close to 100% recovery of RNA is achieved with minor volume/concentration changes, and OsBp reagent is reduced to undetectable amounts after 2-fold purification.OsBp reagent was prepared by preweighing the equivalent of 15.7 mM of 2,2′-bipyridine (bipy) in 18 mL of water in a scintillation vial and adding the full content (2 mL of a 4% OsO4) supplied in an ampule in order to prepare a 20 mL 15.7 mM OsBp stock solution, 1:1 in OsOAnalyses targeting purity and resolution of oligos of similar sequence were conducted by gradient HPLC; both IEX and IP-RP modes were exploited. Kinetic measurements were primarily conducted by CE, that requires less analysis time compared to gradient HPLC. Analyses were conducted automatically using thermostatted autosamplers. Both CE and HPLC peaks were detected and identified using a diode array detector (DAD) in the UV–vis region 200–450 nm. The electropherograms or chromatograms were recorded at 260, 272 and 312 nm and reported here selectively. Samples were prepared with RNAse free water, but buffers were not. No RNA degradation has been observed in our Laboratory.57. An IP-RP HPLC method was also employed to test RNA resolution using HPLC column DNAPac RP from ThermoFisher Scientific (Dionex) in 2 × 100 mm configuration and flow at 0.35 mL/min. Method IP-RP is exploiting a 25%v/v acetonitrile-water gradient in a 0.1 M TEAA buffer pH 7 at 30 °C or higher column compartment temperature57.For HPLC analysis we used an Agilent 1100/1200 LC HPLC equipped with a binary pump, Diode Array Detector (DAD), a 1290 Infinity Autosampler/Thermostat, and Chemstation software Rev.B.04.01 SP1 for data acquisition and processing. As IEX HPLC column DNAPac PA200 from ThermoFisher Scientific (Dionex) was used in 2 × 250 mm or 4 × 250 mm configurations. The performance of the instrument and the column was qualified using standards every time ahead as well as after analysis of research samples. Two IEX HPLC methods were routinely used and have been validated for purity determination and RNA stability. IEX method at pH 8 is exploiting a 1.5 M NaCl gradient in a 25 mM TRIS.HCl pH 8 buffer, with 30 °C column compartment; typical gradient 85%A-15%B to 45%A-55%B in 12 min where A is 25 mM TRIS.HCl and B is 1.5 M NaCl in A. IEX method at pH 12 is exploiting a 1.5 M NaCl gradient in a 0.01 N NaOH solution (no other buffer needed) with 10 °C column compartment; typical gradient is 100%A-0%B to 5%A-95%B in 16 min where A is 0.01 M NaOH and B is 1.5 M NaCl in A. IEX method pH 12 is validated and recommended for longer RNAs in order to suppress secondary structure that broadens peaks and yields low and misleading resolutionCE measurements were conducted with an Agilent G1600 Capillary Electrophoresis (CE) instrument equipped with DAD and Chemstation software Rev.B.04.03(16) for data acquisition and processing; the CE was used in conjunction with a circulating bath to control the autosampler’s temperature. The capillary’s temperature was controlled by the instrument’s software. CE analyses were conducted with an untreated fused silica capillary (50 μm × 40 cm), coined “short” capillary in pH 9.3 50 mM sodium tetraborate buffer using 20 kV or 25 kV. Same buffer was used with an untreated fused silica capillary (50 μm × 104 cm), coined “long” capillary to resolve the topoisomers from the osmylated oligos Figs , Part C.68, and summarized here. A lipid bilayer was formed across a glass nanopore membrane (GNM) and wt α-HL, purchased from List Biological Laboratories in the monomer form of lyophilized power, was dissolved in water at 1 mg/mL and used in 1–3 μL aliquots to form a single nanopore within a lipid bilayer. The above KCl buffered electrolyte was used to fill the solution reservoir and the GNM capillary. A voltage of 120 mV (trans vs cis) or higher was applied across the GNM between two Ag/AgCl electrodes placed inside and outside of the capillary. The i-t traces were filtered typically at 10 kHz and sampled at 50 kHz. Events were extracted using QuB (version 1.5.0.31), and histograms were analyzed by Origin 9.1. Heat plots were plotted using data analysis programs provided by EBS.A limited number of experiments was conducted with the wt α-HL/EBS membrane (EBS) platform  2D format. Ir, Io, and τ values for each event were visually estimated from the grid at an apparent accuracy better than the one of the device. Microsoft Excel was used to obtain the histograms as shown in the figures (part D) in the Supplementary Information and plot the normalized counts vs Ir/Io bin in Fig. Fast-5 files were transferred to txt format, loaded to QuB open source software  and concatenated , add sample, clean the device after use, and store it. Added samples were either intact oligos or Yenos proprietary osmylated RNA oligos; all the samples were added as is, typically at 1.5 μM concentration in ONT buffer. No library was prepared, and no processing enzyme was added to the sample or buffer, so that all the translocations reported here are unassisted and voltage-driven. Raw data files were acquired in see Fig. .Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag.


RNA molecules generated by ribonuclease cleavage sometimes harbor a 2′,3′-cyclic phosphate (cP) at their 3′-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP’s prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles. With the advent and evolution of next-generation sequencing technology, efforts to identify and catalog the expressed RNA molecules have greatly advanced our understanding of RNA biology. However, the current standard RNA-seq methods, particularly those targeting short ncRNAs, do not fully capture all of the RNAs expressed but allow for some “escapers” to slip. RNAs generated by ribonuclease cleavage sometimes harbor a 2′,3′-cyclic phosphate (cP) at their 3′-ends, and those cP-containing RNAs (cP-RNAs) are one such escaper that are not ligated to a 3′-adapter and thus uncaptured by standard RNA-seq. Although an increasing number of studies has been suggesting their functional significances, cP-RNAs remained a hidden component in the transcriptome, infrequently recognized and characterized. In this study, we provide the first genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. By using cP-RNA-seq technique that can specifically sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from tRNAs, mRNAs, and rRNAs. cP-RNAs are generated by previously-uncharacterized, highly-specific RNA cleavage events between cytidine and adenosine, which is regulated through aging. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles. Cells express immensely diverse species of RNA molecules that play essential roles in numerous biological processes. With the advent of next-generation sequencing (NGS) technology, efforts to identify the expressed RNA molecules have greatly advanced our understanding of RNA biology . AlthougGiven that cP-RNAs are expressed as functional molecules, capturing the entire repertoire of cP-RNAs would broaden the catalog of functional ncRNAs and reveal significant biological events that have been eluding standard RNA-seq. We recently developed “cP-RNA-seq,” which is able to specifically sequence cP-RNAs ,12, and LysCUU and tRNAAspGUC, both of which abundantly produce tRNA halves in human breast cancer cells [Figs S1A). Although the levels of mature tRNAs were comparable across all the examined tissues, tRNA halves showed differential expression patterns among the tissues, implying a regulated biogenesis of tRNA halves. The abundant accumulation of 5′-tRNA halves found in the spleen and less abundant accumulations in other tissues, such as the brain, heart, liver, and testis, are consistent with previous reports [Fig 1B, the band of the 5′-tRNA half shifted up by CIP treatment, suggesting the presence of a P in the 5′-tRNA half. T4 PNK treatment also shifted the band up, and further up-shift was observed by the acid plus CIP treatments, indicating the additional presence of a cP in the 5′-tRNA half. These results validated that, as observed for the 5′-tRNA halves in human cell lines [Because ANG-generated 5′-tRNA halves contain a cP, exploration of the accumulation of cP-RNAs in mouse tissues began with analyses of tRNA halves. Northern blots for cytoplasmic (cyto) tRNAer cells , reveale reports ,14. To c reports ,12. As sll lines , the 5′-Fig 1A). The four tissues commonly showed accumulations of short RNAs that were expected to include 5′-tRNA halves , whereas we did not observe such short RNA accumulation in HEK293T and HeLa cells , implying that the constitutive accumulation of short RNAs might be tissue specific. We chose to focus on the 20 to 45-nucleotide (nt) RNAs which were gel-purified and subjected to cP-RNA-seq [Fig 1C). Illumina sequencing of the amplified bands from the respective four tissues yielded approximately 38–47 million raw reads, of which >90%–97% were extracted as the cP-RNA reads with a length of 20–45 nt (S1 Table). Read lengths of the extracted cP-RNAs similarly showed broad distribution patterns in different tissues . Mapping the cP-RNA reads to the mouse ncRNAs and genome unexpectedly revealed that not only tRNAs but also the other RNA species serve as abundant substrates for cP-RNA production . Although tRNA-derived reads were the most abundant cP-RNA species and occupied a substantial proportion of cP-RNAs, ~55%–80% reads are mapped to other regions of the genome, of which messenger RNAs (mRNAs) and ribosomal RNAs (rRNAs) were particularly rich sources of cP-RNAs (S1 Table).To capture an entire picture of short cP-RNAs, we selected four tissues  that exhibited an abundant expression of 5′-tRNA halves . tRNA-derived cP-RNAs were mostly 30–40 nt long  and the majority were 5′-halves  as expected. Considering that the mouse genome encodes 55 cyto tRNA isoacceptors with different anticodon sequences [ValCAC, tRNAGlyGCC, tRNALysCUU, and tRNAGluCUC, which are in aggregates the sources of 75%–84% of the identified 5′-halves . The 3′-terminal nucleotides of the 5′-halves had a strongly biased composition in being pyrimidines (C > U), which was a stable property in all four examined tissues . The anticodon-loop nucleotides that are one position downstream of the 3′-end showed biases of A and U, allowing us to predict that the anticodon-loop cleavage mainly occurs between C and A/U to produce cP-containing 5′-halves .tRNA-derived ncRNAs can be classified into 5′-tRNA half, 3′-tRNA half, 5′-tRF, 3′-tRF, and internal-tRF (i-tRF) ,16,17,18equences , the ideHisGUG is unique in that it contains an additional nucleotide at nucleotide position  were also substantially detected, suggesting that the presence of U–1 as well as G–1 is a conserved feature in mouse cyto tRNAHisGUG, which is retained in the identified 5′-halves.Among all species of tRNAs, tRNAof tRNAs ) –1 of icleotide . As showS3A Fig), nearly all (89%–98%) of the identified 3′-halves were 3′-CC-halves (S2 Table), suggesting that the 3′-terminal cP formation resulted from the cleavage between C75 and A76 of tRNAs. The in vitro ANG-catalyzed cleavage between C75 and A76 was previously reported [in vivo. The 3′-CC- and 3′-C-halves were identified as cP-RNAs also in human brain [Figs S3B). Therefore, specific tRNAs are likely to be selected for cP-RNA production and at least some of their 5′- and 3′-half pairs could be concurrently produced by endonucleolytic cleavage at the anticodon-loops.It was unexpected that a substantial proportion (9%–31%) of tRNA-derived cP-RNAs were occupied by 3′-halves. All three tRFs  were identified as minor cP-containing species compared to the tRNA halves. When subclassified into the four subgroups . The lengths of the mRNA-derived cP-RNAs were broadly distributed between 20–45 nt . Both coding and untranslated regions of mRNAs became substrates for cP-RNAs . Astonishing nucleotide composition biases were observed at both the 5′- and 3′-ends of mRNA-derived cP-RNAs in all four tissues . The 5′-ends were mostly A (78%–89%), whereas the 3′-ends were mostly pyrimidines, and C (70%–81%) was especially enriched compared to U (17%–29%). None of the biases were observed in the nucleotides from the second position to the position preceding the 3′-ends. The nucleotides that are one position upstream of the 5′-ends and one position downstream of the 3′-ends showed clear A and C biases , strongly suggesting that the mRNA-derived cP-RNAs are produced by an endoribonuclease that specifically recognizes and cleaves mRNAs between C and A residues.Although not anticipated, not only tRNAs but also other RNA species were identified as rich sources of cP-RNAs. mRNA-derived cP-RNAs were enriched in cP-RNA libraries from all four examined tissues, accounting for 15%–20% of all cP-RNA species (S3 Table), and the cP-RNAs were derived from many different regions throughout the mRNAs . In the spleen, many abundant cP-RNAs were derived from hemoglobin mRNAs, which might be correlated to the role of the spleen in red blood cell turnover that requires the degradation of hemoglobin [S4C Fig), showed very focal production of specific cP-RNAs, suggesting that the cP-RNA production and accumulation are not caused by random mRNA degradation. The most prominent example is Ncbp3 mRNA which commonly produces the most abundant mRNA-derived cP-RNA in all four tissues (S3 Table). Although there are 406 “CA” sequences  throughout Ncbp3 mRNAs, almost all cP-RNAs are produced from only one site  in the 3′-UTR , where single-stranded loop regions are expected to be formed . The common and specific production of cP-RNAs might suggest that these RNAs are produced as functional molecules.Major mRNA species producing cP-RNAs are not very consistent among different tissues , were enriched at similar levels  with mRNA-derived cP-RNAs. All four rRNA species  produce cP-RNAs , and the most abundant cP-RNA species were derived from 28S rRNA (S4 Table). Nucleotide biases observed in mRNA-derived cP-RNAs were retained mostly in rRNA-derived cP-RNAs; the 3′-end and a nucleotide upstream of the 5′-end were strongly biased to C and U (C > U) . The 5′-end and a nucleotide downstream of the 3′-end were not only biased to A, but also to G, and sometimes to U. Alignment visualization showed production of specific cP-RNA species from rRNAs , suggesting their controlled biogenesis mechanisms. In 28S rRNA, the positions 186–217 was the most abundant cP-RNA-producing region , and the representative cP-RNA from the region, termed cPR-28S, is produced from the cleavages between C185 and G186, and C217 and A218 in domain 1 . Cleavage positions reside in the loop regions of 28S rRNA, possibly because the regions are highly accessible to endoribonucleases.rRNA-derived cP-RNAs, whose read lengths were broadly distributed between 20–45 nt . Moreover, not only the cP-RNAs derived from the nuclear genome but also the cP-RNAs derived from the mitochondrial genome showed similar nucleotide biases . Although those cP-RNAs were not abundant, the consistent nucleotide biases suggest that cP-RNAs are produced from a wide variety of RNA species through identical RNA processing events mainly between cytidine and adenosine.cP-RNAs from small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), and long ncRNAs (lncRNAs) showed terminal nucleotide bias to A and C for their 5′- and 3′-end, respectively, in all four tissues (GlyGCC (5′-GlyGCC), cPR-Ncbp3, and cPR-28S , which are one of the most abundantly sequenced species among tRNA-, mRNA-, and rRNA-derived cP-RNAs, respectively. To analyze 3′-terminal structures of the three species, total RNA from mouse skeletal muscle was treated with CIP or T4 PNK. A mutant T4 PNK, which lacks 3′-dephosphorylation activity [Fig 4C, all three target RNAs exhibited the highest amplification signals upon T4 PNK treatment, indicating that the major 3′-terminal form of the identified sequences are a cP. CIP treatment showed certain signal levels, suggesting co-expression of 3′-P-containing RNAs as a minor form or occurrence of hydrolysis reaction converting a cP to a P during experimental procedures.To biochemically validate that the identified sequences are expressed as cP-RNAs, we focused on the three representative cP-RNAs, 5′-half of tRNAactivity , was alsactivity . As showS9 Fig). The determined abundances of the three cP-RNAs per 1 μg of total RNA were summarized in Fig 4D. Whereas the amounts of 5′-GlyGCC and cPR-Ncbp3 were comparable between lung and muscle, the amount of cPR-28S in lung is ~14-fold higher than that in muscle. Considering previous reports estimating that 1 μg of total RNA from mouse liver contains 0.005–8.8 fmol of miRNA [Using their specific quantification by TaqMan RT-qPCR, we explored absolute amounts of the three cP-RNAs expressed in mouse lung and skeletal muscle. The calculation of the amounts was based on the standard curve from synthetic RNAs which showed excellent linearity between input amounts and amplification signals . The cP-RNA levels in 12- and 24-week old mice, both of which are considered as adult mice, did not show a significant change between them and were in-between those of 2- and 84-week old mice. To ask whether the phenomenon is conserved in different tissues, we further investigated lung of mice with different ages, showing the reduction of the expression levels of all three cP-RNAs through aging . Total RNA staining pattern further suggested global reduction of the expression of 30–45-nt short ncRNAs, which are expected to contain cP-RNAs, through aging .To explore physiological relevance of cP-RNA expression, TaqMan RT-qPCR quantification of the three cP-RNAs were performed for skeletal muscle of mice with four different ages, 2, 12, 24, and 84 weeks. While a control U6 snRNA showed consistent expression levels in all the examined mice, the expression levels of all three cP-RNAs were reduced along with aging, and the differences between 2- and 84-week old mice were especially apparent , cP-RNA-seq amplified less abundant cDNAs from the 84-week old mice compared to the 2-week old mice . Illumina sequencing of the cDNAs yielded ~23–29 million raw reads, of which ~3–6.5 million reads were extracted as the reads of mRNA-derived cP-RNAs (S5 Table). After normalization based on the read number information of a spike-in cP-RNA, fold changes of cP-RNA read numbers of 84 week samples, with respect to those of 2 week samples, were compared with fold changes of mRNA-seq read numbers for each mRNA in a scatter plot . Strong aggregations of the spots appeared at the point where the levels of cP-RNAs were 4-fold reduced in 84-week samples compared to 2-week samples without changes in the levels of their substrate mRNAs. Consistent with the results of TaqMan RT-qPCR , reduction of cPR-Ncbp3 was confirmed in the scatter plot . As representative results, normalized alignment patterns of cP-RNAs in the three selected mRNAs, Ncbp3, Cd93, and Tsc22d1, are shown in Fig 5F. Specific productions of cP-RNAs through cleavage between C and A residues and their reduction in 84-week samples were observed. Together with the decreased C and A nucleotide biases in 84-week samples , these results suggest that the expression of cP-RNAs is decreased through aging, which is independent of the levels of substrate RNAs but is caused by reduced C-A cleavage activity for cP-RNA productions or by reduced stability of cP-RNAs.To investigate potential relationship between cP-RNAs and their substrate RNAs in expressional regulation through aging, we focused on mRNA-derived cP-RNAs. Total RNAs from lung of 2- and 84-week old mice were subjected to cP-RNA-seq and mRNA-seq for identification of mRNA-derived cP-RNAs and their substrate mRNAs, respectively. Consistent with the downregulated expression of cP-RNAs in older mice . The endoribonuclease generating the identified cP-RNAs is unknown but should possess highly specific recognition and cleavage activity for the 5′-CA-3′ sequence. ANG might be the candidate enzyme because ANG produces cP-RNAs [In this study, we unraveled a previously hidden layer of transcriptome by comprehensively identifying short cP-RNA species expressed in mouse tissues. Each cP-RNA library, obtained by cP-RNA-seq, commonly comprised previously uninterrogated cP-RNA species that are expected to be generated by specific RNA cleavage between cytidine and adenosine  and were approved by the Institutional Animal Care and Use Committee at Thomas Jefferson University. Mice were euthanized by COFor cP-RNA-seq, tissues of thymus, lung, spleen, and skeletal muscle from hindlimbs were dissected from male C57BL/6J (B6) mice at approximately 3-months ages. The dissected tissues were homogenized in TRIsure (Bioline) using a glass homogenizer on ice, after which total RNA was extracted according to the manufacturer’s instruction. For TaqMan RT-qPCR, frozen tissues were crushed using a frozen mortar and a pestle, followed by total RNA extraction by TRIsure. To investigate the terminal structures of the 5′-tRNA half, total RNA was treated with CIP (New England Biolabs), T4 PNK (New England Biolabs), or acid (incubation in 10 mM HCl at 4°C for 3 h) followed by CIP treatment as previously described .LysCUU half, 5′- GTCTCATGCTCTACCGACTG-3′; 3′-tRNALysCUU half, 5′-GCTCGAACCCACGACCCTGAGA-3′; 5′-tRNAAspGUC half, 5′-GGGATACTCACCACTATACTAACGAGGA-3′; 3′-tRNAAspGUC half, 5′- GAATCGAACCCCGGTCTCC-3; and miR-16, 5′-GCCAATATTTACGTGCTGCTA-3′.Northern blot was performed as previously described  with theFor four tissue samples , 20 to 45-nt RNAs were gel-purified from total RNA and subjected to cP-RNA-seq procedure as previously described ,12. The For comparison between 2- and 84-week old mice, lung 20–45-nt RNAs were gel-purified together with a synthetic spike-in cP-RNA . cDNAs amplified by cP-RNA-seq procedure were sequenced using Illumina NextSeq 500 at the MetaOmics Core of Sidney Kimmel Cancer Center in Thomas Jefferson University.) and are publically available at NCBI’s Sequence Read Archive . Before mapping, we used the cutadapt tool (DOI: http://dx.doi.org/10.14806/ej.17.1.200) to remove the 3′-adapter sequence. After selecting 20–45-nt reads, we used Rbowtie (1.15.1) [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For the nucleotide composition analyses of the sequences which are upstream or downstream of cP-RNA producing regions on the genome, 4-nt upstream/downstream sequences were retrieved by referring fasta files and subjected to FastQC analyses. The Integrative Genomics Viewer (IGV) [The cP-RNA-seq libraries contain ~22–47 million raw reads ((1.15.1)  for the (1.15.1) , and theer (IGV)  was usedFor comparison between 2- and 84-week samples, the extracted 20–45-nt cP-RNA reads were first mapped to the sequence of the spike-in cP-RNA, followed by the sequential mappings as described above. The read numbers of the spike-in cP-RNA were used to calculate the normalized RPKM of each cP-RNA-producing mRNA. mRNA-seq was performed by Novogene and RPKM of each mRNA was also obtained. These RPKM values were used for calculations of fold-changes of 84-week samples, with respect to 2-week samples, and the values derived from cP-RNA-seq and mRNA-seq were compared in the scatter plot. The experiments of cP-RNA-seq and mRNA-seq were performed using biological duplicates and triplicates, respectively.S6 Table. For the quantification of absolute amounts of cP-RNAs, each synthetic RNA was mixed with 1 μg of E. coli total RNA and the mixtures were used as the substrates to obtain standard curves shown in S9 Fig. In Fig 5A and 5B, Student’s t-tests were used to evaluate the differences between 2- and 84-week samples.TaqMan RT-qPCR for specific quantification of cP-RNAs was performed as previously described . BrieflyS1 Fig(EPS)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.S6 Fig(EPS)Click here for additional data file.S7 Fig(EPS)Click here for additional data file.S8 Fig(EPS)Click here for additional data file.S9 Fig(EPS)Click here for additional data file.S10 Fig(EPS)Click here for additional data file.S11 Fig(EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA.Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages.A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes. Understanding the function of genes requires knowledge about the localization and timing of their expression. Consequently, the analysis of expression pattern of genes during plant development, in different mutant backgrounds, after exposure to stressors, in changing growth conditions etc. has been utilized to unravel novel gene functions and to attribute genes to genetic networks.Several methods were developed over the years to provide quantitative or semi-quantitative information on gene expression, such as Northern Blotting, where total RNA is fixed to a membrane and the target gene sequence is labelled and hybridized to the immobilized RNA. More indirect methods to detect RNA abundance include RT-PCR and qRT-PCR as RNA quantification is calculated from increase of PCR amplification products. The method offering the highest spatial and temporal resolution of gene expression analysis is RNA in situ hybridization for which a, e.g. digoxigenin-labelled antisense probe is hybridized to either whole-mount tissue or tissue sections but it is unsuited for transcript quantification .These methods all have in common that single or few genes at the time can be analysed without the need for genetic manipulations. In contrast, reporter gene studies depend on genetic manipulation of the target species as the promoter:reporter gene combination requires transfer into the plant. This method also allows a high spatial and temporal resolution and, in addition, live-cell imaging and has become invaluable to follow gene expression over time. However, many species are recalcitrant to genetic transformation and thus this method is not applicable for a large fraction of plants .Microarray experiments allow the simultaneous quantification of thousands of genes’ expression but require at least partially sequenced genomes or transcriptomes from which the costly gene chips are generated. In the past years, RNAseq has become the most popular method to analyse gene expression changes across the entire transcriptomes in a large variety of species. However, this method also requires tissue collection for RNA extraction, with high quality requirements for the RNA to be sequenced, whereas high spatial and temporal resolution is as challenging as for the other methods .A. thaliana, Citrus clementina (clementine) and Solanum lycopersicum (tomato)  was calibrated to optimize target fragment dissection and collection. The films were fully dried before starting the laser-assisted cutting, because moisture in the films may lead to warping of the membrane while cutting or fragments adhering to the film. Two sample collection tubes were attached to the sample collection tray: one for the samples and another for discarding unwanted material. Before starting the cut, the collection tube cap was filled with 20 μl of extraction buffer [PicoPure™ RNA Isolation Kit ]. The samples were located and the elements of the desired tissue were marked. Samples were cut with slow speed and the collection cap was inspected to make sure that the target tissues had fallen into the collection tube. The collection tube was detached from the tray, closed and vigorously vortexed for one minute. The tubes were then submerged in liquid nitrogen for further break down of protective cell structures.RNA extraction for RNAseq was carried out using the PicoPure™ RNA Isolation Kit according to the manufacturer’s instructions and the samples were stored at − 80 °C. Multiple early stage samples were pooled together for a single extraction to increase the RNA concentration of the eluate. The quantity and the quality of the samples was measured using the Agilent bioanalyzer with Agilent RNA 6000 Pico Kit .RNA samples were pre-amplified using Nugen Ovation® RNA‑Seq System (PART NO. 7102)  suitable for an input of 500 pg–100 ng of RNA and the cDNA generated was used for library preparation. Amplification and cDNA generation was conducted according to the manufacturer’s instructions. Library preparation was done with the Illumina Nextera XT kit  suitable for 1 ng DNA. The cDNA was measured before the preparation with Qubit and normalized to 0.2 ng/µl. 5 µl of dilution was used for the library preparation. Libraries were prepared according to the manufacturer’s instructions. Generated libraries were sequenced with Illumina HiSeq 4000. 9 samples were sequenced per lane generating approximately 33 million reads per sample. Paired-end sequencing was used for each sample with read length of approximately 76 bp.A. thaliana [O. sativa were selected and their expression in TPM values within the transcriptomes were analyzed. O. sativa orthologs to A. thaliana chromatin remodelers were identified by using ProteinOrtho [Raw reads of paired end sequencing data were imported to CLC genomics workbench version 11.0.1. CLC Genomics workbench was used for quality test, trimming, RNA-seq and PCA analysis using the default parameters using information deposited in Additional file thaliana  and O. seinOrtho . Their eeinOrtho  utilizineinOrtho , NumPy [einOrtho , SeaborneinOrtho  and MatpeinOrtho . To evaleinOrtho .Arabidopsis thaliana Col-0 was grown in conditions described above. RNA was extracted with the NucleoSpin® RNA -kit  from complete buds at growth stages 9 and 12 [9 and 12  and cDNAAdditional file 1: Additional figures and tables.Additional file 2: Appendix protocol plant methods.


Symbiodinium plays important roles in the adaptation of coral to environmental changes. However, coral-Symbiodinium symbiotic associations are not well-understood in the South China Sea (SCS) whilst considering environmental factors and host taxa. In this study, next-generation sequencing of the internal transcribed spacer region 2 (ITS2) marker gene was used to explore the symbiotic associations between Symbiodinium and five typical coral species across tropical and subtropical reef regions of the SCS. The results showed that Acropora sp., Galaxea fascicularis, Platygyra lamellina, and Sarcophyton glaucum exhibited distinct Symbiodinium compositions between tropical and subtropical reef regions, whereas Porites lutea had stable Symbiodinium compositions. More heterogeneous Symbiodinium compositions among different coral species were observed in the tropical region, but there were no statistically significant differences in Symbiodinium compositions among different coral species in subtropical reef regions. There was a correlation between the Symbiodinium compositions and environmental factors, except for the composition of P. lutea. Symbiodinium subclades D1, D2, C71, C71a, C21, C3b, and C161 were primarily explained by the seawater temperature, nitrate, ammonia, and phosphate. Several host-specific Symbiodinium subclades  were observed in P. lutea as well. The findings of this study demonstrate the relationship of Symbiodinium diversity with coral hosts and the environment are helpful for elucidating the adaptation of corals to global climate change and anthropogenic disturbance.The coral symbiont  Symbiodinium, a photosynthetic algae that is a dinoflagellate, plays a central role in the adaptation of corals and the maintenance of coral reefs  have been identified  profiles of the less-conserved Symbiodinium internal transcribed spacer region 2 (ITS2) sequence  remain common on devastated reefs, even though they fail to change their symbiont communities.At present, nine entified . Clades n corals . Within sequence . Previouorldwide . In contantation . Therefoantation . For exa2, including at least 571 coral species), and coral reefs mainly develop in the Nansha Islands, Zhongsha Islands, Xisha Islands, Hainan Island, Leizhou Peninsula, and Dongsha Islands  and Montipora spp. (scleractinian) in tropical (Hainan Island and Xisha Island) and subtropical (Hong Kong) reef regions of the SCS by next-generation sequencing of Symbiodinium ITS2 region, suggesting that temperature is the main factor that drives coral-Symbiodinium symbiotic associations. A similar result was also reported by G. fascicularis in the SCS. However, knowledge gaps still remain. At present, little is known regarding the diversity of Symbiodinium and their symbiotic associations with abundant corals (such as Acropora and Porites) in the SCS whilst considering environmental factors and host taxa.The South China Sea (SCS) has extensive an coral reef development  and Porites lutea (scleractinian) as well as the three common coral species of G. fascicularis, Platygyra lamellina (scleractinian), and Sarcophyton glaucum  were sampled from different reef sites across tropical  and subtropical  reef regions of the SCS. The Symbiodinium compositions in these coral samples in the SY, XW, and DY reef regions were analyzed by next-generation sequencing of Symbiodinium ITS2 region, aiming to elucidate coral-Symbiodinium symbiotic associations across these regions. Furthermore, the relationship of Symbiodinium compositions with environmental factors and host taxa were analyzed.In this study, the dominant coral species of Sampling regions of SY , XW , and DY  are located in the northern SCS Figure . The XW Acropora sp., G. fascicularis, P. lamellina, P. lutea, and S. glaucum were obtained from the SY, XW, and DY reef regions in August and September 2015  at least three times.Coral samples of r corals , and it ® at 4°C in the field and stored at -80°C in the lab until DNA extraction was conducted.The seawater samples from at least five reef sites in each reef region were collected and filtered using 0.22-μm polycarbonate membranes . Next, the filtered seawater from each of the different reef sites, that is, SY, XW, and DY reef regions, were mixed. All samples were preserved in RNAlaterThe mean annual SSTs of the SY, XW, and DY reef regions were calculated based on the SST of these reef regions in spring, summer, autumn, and winter of 2015  were analyzed using a Lachat QC8500 Flow Injection Autoanalyzer   were measured by OCEAN SEVEN 320 Plus CTD . Dissolved oxygen (DO) was detected using a YSI 6600V2-02 multi-parameter instrument . Dissolved nutrients . The purity of the DNA was determined by NanoDropTM 2000 Spectrophotometer . All DNA samples were stored at -20°C for further use.Total DNA of each coral sample was extracted using the Qiagen DNeasy Plant Mini Kit  according to the manufacturer’s protocol. The integrity of the DNA was monitored by 0.8% (w/v) agarose gel electrophoresis stained with 1 × SYBR Safe . The amount of DNA was determined by a QubitSymbiodinium was PCR amplified using a pair of barcoded primers: ITSintfor2 (5′-GAATTGCAGAACTCCGTG-3′) and ITS2-reverse (5′-GGGATCCATATGCTTAAGTTCAGCGGGT-3′) . All qualified amplification products were mixed in equal amounts followed by sequencing on an Illumina MiSeq instrument  according to the manufacturer’s instructions using a 300 × 2 paired-end configuration. The raw data were submitted to the NCBI Sequence Read Archive (SUB2393447).The ITS2 region of the ribosomal RNA gene of GGGT-3′) . The PCRSymbiodinium subclade against a custom reference database of Symbiodinium subclades  diversity index was calculated in the R software environment (R 3.1.2) to assess the alpha-diversity of Symbiodinium subclades associated with coral samples. The differences in the Shannon–Wiener (H′) diversity index of Symbiodinium subclades among different samples was tested with the multiple comparison test (Tukey’s HSD) using R software (R 3.1.2). A rarefaction curve was plotted using “make_rarefaction_plots.py” script in the QIIME package based on the merged count table  of Symbiodinium subclade compositions was performed by the vegan package in R  was conducted in R using the vegan package  and maximum likelihood (ML) methods. Analyses were performed with the Kimura 2-parameter model, and the evolutionary models that fit best for the datasets were calculated using the program JModelTest version 2.1.7  were retained after quality control. Thirty-two Symbiodinium subclades were classified  were selected for further analysis, while the rest (19 Symbiodinium subclades) were designated as others   index values of Symbiodinium subclades were not statistically significantly different across the three reef regions (P > 0.05).The same coral species or genus in the SY reef region showed higher Shannon–Wiener (H′) index values of ) Figure . One excSymbiodinium subclade counts of different coral samples in the SY, XW, and DY reef regions revealed a segregation of Symbiodinium subclade compositions .A PCoA based on s Figure . The SymSymbiodinium subclade compositions in explored corals living between tropical (SY) and subtropical (XW and DY) reef regions, except those in P. lutea, were probably explained by the different environmental factors of the different reef regions  and subtropical (XW and DY) reef regions. The Symbiodinium subclades D1, D2, C71, C71a, C21, and C3b mainly occurred in the SY reef region and were positively correlated with the SST and NO3-, NH4+, and PO43-. Conversely, the Symbiodinium subclade C161 occurred predominantly in the XW and DY reef regions and was negatively correlated with those environmental factors. However, there was no clear correlation between Symbiodinium subclades C15, C15.6, C91, and C15i (mainly associated with P. lutea) and all of the measured environmental factors.The distinct  Figures , 6. AccoSymbiodinium subclades used for phylogenetic tree construction were less than 97%, supporting the fact that the 13 Symbiodinium subclades from explored coral samples were distinct  could shape coral-Symbiodinium symbiotic associations  to D1a (stress tolerant) or to a mixture of Symbiodinium subclades C1/C3/C21a/C15 and D1a. Corals from some extremely hot regions with high SST were found to host heat tolerant Symbiodinium, that is, subclades within clade D  with high SST, NO3-, NH4+, and PO43- were distinct from those from the subtropical reef regions (XW + DY), suggesting that nutrient inflow might also affect coral-Symbiodinium symbiotic associations in addition to SST. These results are consistent with Symbiodinium community structures associated with Pocillopora verrucosa in the Red Sea.It has been suggested that corals may adapt to environmental changes by regulating their hosted iodinium ,b. For e clade D . Extremerecovery . The recPorites-Symbiodinium symbiotic associations were stable over broad geographical scales and temperature ranges as well as through bleaching events or after transplantation (Symbiodinium subclade compositions of P. lutea (Porites) are not statistically and significantly different across subtropical and tropical reef regions in the SCS. In addition, there are no clear correlations of the Symbiodinium subclades C15, C15.6, and C91 mainly detected in P. lutea with all detected environmental factors. All of these results reveal that the host could also affect Symbiodinium subclade compositions, and the Symbiodinium subclades C15, C15.6, and C91 seem to be specific to P. lutea.In the Indo-Pacific Ocean, previous studies have shown that antation . SimilarSymbiodinium. In the SCS, temperature is one of the measured environmental factors that could shape coral-Symbiodinium symbiotic associations. Therefore, the rise of SST is a potential threat to corals in the SCS. Previous studies have shown that heat-tolerant corals contain a greater number of clade D Symbiodinium. Similar to G. fascicularis in the SCS appears to host more heat-tolerant Symbiodinium subclades within clade D, which may lead to their dominance during future climate change. In addition, the present study shows that other environmental factors  can also serve as major factors affecting coral-Symbiodinium symbiotic associations. Increases in coastal nutrients are frequently linked to human activities   . Based oSymbiodinium community with both the coral host and the environment. Corals might adapt to different environmental conditions by changing their Symbiodinium communities . Conversely, certain corals, such as P. lutea, might be able to adapt to different environmental conditions  via host-specific Symbiodinium subclades . Elucidating detailed mechanisms of corals’ adaptation to different environments warrants future investigation in both field and lab settings.This study demonstrates the multiple relationships of the SUB2393447.The raw sequence information in this paper has been deposited in the GenBank Sequence Read Archive with accession number SG and ZL conceived and designed the experiments. SG performed the experiments and analyzed the experimental data. SG, LX, JL, FL, HC, and BL contributed to reagents and materials. SG, ZL, GC, YX, FZ, and KY wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer CA-G and handling Editor declared their shared affiliation at time of review.


Nano-objects made of nucleic acids are becoming promising materials in the biomedical field. This is, in part, due to DNA and RNA self-assembly properties that can be accurately computed to fabricate various complex nanoarchitectures of 2D and 3D shapes. The nanoparticles can be assembled from DNA, RNA, and chemically modified oligonucleotide mixtures which, in turn, influence their chemical and biophysical properties. Solid-phase synthesis allows large-scale production of individual oligonucleotide strands with batch-to-batch consistency and exceptional purity. All of these advantageous characteristics of nucleic-acid-based nanoparticles were known to be exceptionally useful as a nanoplatform for drug delivery purposes. Recently, several important discoveries have been achieved, demonstrating that nucleic acid nanoparticles (NANPs) can also be used to modulate the immune response of host cells. The purpose of this review is to briefly overview studies demonstrating architectural design principles of NANPs, as well as the ability of NANPs to control immune responses. The human immune response can be classified as innate, adaptive, or both. The innate response is nonspecific, and the response rate is usually rapid, thus making it the body’s ‘first line of defense’. On the other hand, the adaptive response is an acquired response that is more specific and involves memory. With adaptive immunity, each successive exposure to the foreign substance increases the defensive response of the immune system, but innate immunity is present prior to any exposure to the foreign body. Both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are known to induce immune responses, and they have been extensively studied for medicinal applications ,2. In PAThe ability to control the immune system response to foreign substances is extremely challenging due to the broad and diverse chemical identities of molecular patterns. The nano-objects made of nucleic acids are highly attractive candidates in the developmental immunology field. Immunomodulation therapy using nucleic acid material is an emerging field ,9,10,11 in vitro.This review focusses on discussion of geometrical properties of naturally occurring RNA structural motifs and artificial DNA and RNA building blocks that are commonly used to fabricate various intricate nano-objects of 2D and 3D shapes of no more than 50 nm. Furthermore, their application as immunomodulators is explained with emphasis on immunostimulatory cytosine-phosphate-guanosine (CpG) oligonucleotides, as well as other approaches, including NANPs with specific shapes, sequences, and nucleic acid composition affecting immune cells Hypothetically, any naturally occurring RNA motifs can be implemented to construct an RNA complex of a specific shape. This is becoming more evident as a broad class of different RNA motifs was implemented in nanoparticle design and now play a critical role in the assembly of RNA nano-architectures. A few of those examples include kissing hairpin loops , cognateAnother well-known RNA structural motif routinely used to fabricate various shapes is the RNA three-way junction (3WJ), which consists of three interconnected RNA strands . AccordiVarious geometrical 3WJ motifs can be extracted from the RNA junction database developed in Shapiro’s group, and this software allows particular 3WJ family characteristics to be searched and organized . One of H. marismortui was demonstrated by Jaeger’s group, where the tetragonal shape RNA nanoparticle named tectosquares was assembled [Application of another naturally occurring 3WJ motif from 23S rRNA of ssembled . More imssembled .Haloarcula marismortui large ribosomal subunit [H.marismortui 23S rRNA, and they are also prominent in the structure of Thermus thermophiles 16S rRNA [The kink-turn (k-turn) motif is an architectural motif that was shown to be applicable to design and assemble various shapes, including triangular DNA and RNA structures. The kink turn motif is schematically demonstrated in  subunit ,45. This subunit . The mot16S rRNA . These K16S rRNA . The K-t16S rRNA . The int16S rRNA . The str16S rRNA . One of 16S rRNA . To demo16S rRNA ,51,52. B16S rRNA   or internal loop/loop–receptor interaction motifs that can be combined in pairs for high affinity. Such interacting motifs are important components and often used to combine multiple building blocks into multimeric RNA nanoparticles of globular or planar shapes. For example, the previously mentioned pRNA from the phage phi29 DNA packaging motor have been shown to assemble into multimeric pRNA nanoparticles, as well as 3D RNA triangular and tetragonal nanoprisms by utilizing a bottom-up self-assembly principle ,54,55 Fa,b. Suchvia canonical Watson–Crick base-pairing. The design relies on the energy minima driven by the formation of the RNA/RNA duplex with the assumption that any potentially formed 3D configuration supported by non-canonical interactions will have unfavorable free-energy formation. Overall, RNA complexation is driven by helical folding and the helix can be treated as a rigid module. This design principle is known as a helix-centric approach [mfold can be effectively engaged [With the previously mentioned design principles, several variations and modifications to the nanoparticle shapes can be employed using computationally developed RNA building blocks. In this approach, the design principles of RNA nanoparticles folding into pre-programmed configurations is dictated by the formation of A-form RNA helices approach  and has approach ,9,24. In engaged . This alRNA 3D nanocubes were one of the earliest examples of nanoparticles designed using the helix-centric approach ,15. Due All previously mentioned examples of nucleic acid nanoparticles have demonstrated tremendous potential in nanomedicine and have shown to be potent nanovehicles to carry RNA functional moieties such as aptamers, proteins, riboswitches, ribozymes, or short interfering RNAs (siRNAs) to target and treat cancer cells ,59. HoweIn the field of therapeutic nucleic acids, unmethylated cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODNs) have received tremendous attention ,65,66,67Various studies have attempted to use CpG conjugated into NANPs to evaluate immunostimulatory efficacy as a function of nanoparticle size and shape ,16,76,77For example, Guo’s group utilized planar nanoparticles of triangular (~10 nm), tetragonal (~12 nm), and pentagonal ~13 nm) shapes  nm shape. ImmunosThe study has demonstrated an enhanced and advantageous immunostimulatory effect of the hairpin CpG compared to linear forms. Assembly of multiple CpG motifs on the most branched dendrimer is highly effective in promoting the immunostimulatory activity of CpGs. Fan’s group studied application of a DNA tetrahedron as a CpG carrier to produce an immunostimulatory response. A DNA tetrahedron containing CpG can noninvasively and efficiently enter macrophages, such as RAW 264.7 cells, without the aid of polyatomic transfection agents. The combined studies—utilizing either 2′f-modified RNA or DNA nanoparticles—illustrate the importance of the size and shape of NANPs for the improvement of activity of CpG-based immune responses by innate immune systems. However, the NANP-based delivery of the CpG system required further optimization for controllable immunostimulatory effects.Recent studies employed by Afonin’s group have shown that NANPs alone (without carrying imunostimulatory agents like CpG) can be used to induce an innate immune response ,10. CellThe success of internalization of NANPs in immune cells and subsequent magnitude of IFN response correlates with physicochemical properties of NANPs. To study internalization, RNA cubes were labeled with a fluorophore dye and exposed to PBMCs overnight. The NANP-associated fluorescence was localized to the interior of the cell, suggesting NANPs are associated with phagocytic monocytes. In correlation with earlier observations, RNA cubes were also detected on the surface of smaller lymphocytes. Lyso-ID Red was the stain source of acidic vesicles involved in the endolysosomal pathway. This confirmed that NANPs are taken up via the endolysosomal pathway associated with scavenger receptors. The initiation of the IFN response occurs by endosomal TLRs. This finding also suggests that plasmacytoid dendritic cells are the main source of IFNs.Collectively, it has been demonstrated that physiochemical characteristics including, size, 3D structure, composition and connectivity of the NANPs significantly affect immunological recognition. By using globular RNA structures, the desired induction of IFNs can be enhanced, while DNA-based NANPs, planar, and fibrous RNA structures can reduce unwanted immune responses. NANPs require delivery carriers to exhibit immunological activity irrespective of size, shape, composition, and connectivity. Through endocytosis, the NANPs enter the cells and endosomal TLRs initiate the release of IFNs primarily sourced from plasmacytoid dendritic cells . These fTo address questions regarding specificity of TLRs to different NANPs structures, a mechanistic study involving a human peripheral blood mononuclear cell (primary immune cells) was recently explored by Hong et al. . It was At this point, it is becoming certain that immune response can be controlled to some extent depending on the type of NANPs, and there is clearly a large library of RNA structures which can be utilized. Further understanding of mechanisms of the immune-response triggered by NANPs is critical to achieve immune induction in a controllable fashion. Although programmable NANPs offer a novel platform for controlled immune therapy, more experimental studies are necessary for progressing toward further applications in a clinical setting. Nonetheless, the first steps toward understanding recognition mechanisms of artificial NANPs by immune cells have already been taken, but there is still much to be discovered.