The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches  far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs  and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RNases based on the catalytic peptide [(ArgLeu) The idea of sequence-specific inactivation of pathogenic RNA with the use of antisense oligonucleotides was first proposed several decades ago ,2,3 and This review contains consecutive descriptions of different aspects of ss-aRNase development from chemical issues such as design and synthesis to the essential biological characteristics including specificity, selectivity and efficiency of action in vitro and in vivo. With this in mind, the manuscript may be considered as a single text or as individual chapters drawing attention of the broad audience: from specialists in organic synthesis to molecular biologists and biochemists. For the convenience, the main characteristics of reviewed ss-aRNases are summarized in Table 1. Antisense oligonucleotides silence RNA targets either by acting as steric blocks of functionally significant regions, or as guide sequences for recruited RNase H. The attachment of molecular scissors of various chemical natures to the oligonucleotide allows its gene-silencing properties to be improved via the irreversible destruction of RNA chains. Such approaches can also offer an opportunity for the functional manipulation of RNA. The realisation of this concept resulted in the appearance of a variety of aRNases.An inherent feature of site-selective artificial ribonucleases (ss-aRNases), which are conjugates of an oligonucleotide and a catalytic moiety, is their capability of RNA sequence recognition, which is provided by the oligonucleotide domain, and cleavage of phosphodiester linkages, which is mediated by the catalytic domain. Nonspecific aRNases lacking the RNA-recognising motifs are beyond the scope of this review. Since 1994, when the first ss-aRNases were created ,23,24, a2+ and Zn2+ chelates. Although the pioneering studies demonstrated a higher efficiency of metal-ion dependent aRNases  peptide as a cleaving construct during the initial stage of ss-aRNases development while targeting tRNALys 2 2) dissolved in DMSO was added to the oligonucleotide solution for condensation. The synthesis of “dual” conjugates, which consisted of two oligonucleotide motifs connected by a catalytic peptide, required a more complex synthetic scheme. The conjugation of two separate oligonucleotide recognition motifs to the catalytic peptide was carried out in two consequent stages: first via coupling of the first oligonucleotide at the N-terminus, and then via attachment of the second oligonucleotide at the C-terminus of the same peptide. This could be achieved by implementing two different methods which normally require different types of 3′- and 5′-terminal oligonucleotide modifications. Method 1 is based on the formation of a phosphoramidate bond between the 5′-terminal phosphate group of the first oligonucleotide and the peptide N-terminal amine -1H-1,2,3-triazolopyridinium 3-oxide hexafluorophosphate (HATU) activating agent, followed by deprotection, when the free amino group of the growing chain was used to synthesise PNA oligomer by incorporating Fmoc–PNA–(Bhoc)–OH building blocks. This was followed by the incorporation of the second Fmoc–PEG2–COOH fragment at the N-terminus of PNA to give the binding domain PEG–PNA–PEG of the ss-aRNases A and B. The synthesis of ss-aRNase A was finalised by manual coupling with Fmoc–His(Trt)–OH and Fmoc–Gly–OH amino acids to produce the HGG domain at the N terminus of PNA chain. In the case of ss-aRNase B, following deprotection of the PEG linker, the Fmoc–Lys–(MTT)–OH amino acid was first incorporated to provide a suitable attachment point for conjugation with the catalytic domain. After deprotection of the ε-amino group of the incorporated lysine, the resin was subjected to treatment with (Boc)2–DETA–Succ–OH from solution to generate the desired conjugate.An interesting design of ss-aRNases was proposed in . This sspeptide-oligonucleotide assembly can follow one of the two patterns, either “oligo-first-peptide-next” or “peptide-first-oligo-next”. Predominantly, peptides are synthesised first using a Boc or Fmoc synthetic strategy, followed by oligonucleotide conjugation via the phosphoramidite method 2 and [(LeuArg)4Gly] proved to be the most efficient cleavers as part of the ss-aRNase subject to the certain rules of conjugate design. For example, “bulge-inducing” POCs (see Peptidyl-oligonucleotide conjugates (POCs) represent another large class of ss-aRNases, which are based on incorporation of the catalytic peptide into the oligonucleotide recognition motif(s) at different positions using a broad variety of attachment methods ,42,47,50POCs see  require 2Gly]2 generally exhibited increased [4Gly].The aminohexyl linker seems to provide a sufficient flexibility to catalytic groups, thus facilitating phosphodiester bond cleavage. Another way to enhance the overall flexibility of a catalytic peptide could be the insertion of an additional glycine residue, which serves as a flexible joint between two Leu-Arg-Leu-Arg blocks. Thus, the probability of achieving an ‘in-line attack’ conformation can be increased to promote catalytic activity. Indeed, the conjugates incorporating the peptide 2 cleaved up to 100% of target tRNAPhe within 4 h thin 4 h . This coThe essential component of ss-aRNAses, which provides specific binding with a target, is the oligonucleotide-based RNA recognition motif (ON). According to the structure of ON, established ss-aRNAses can be divided into four major groups: (1) “linear”, (2) “dual”, (3) “bulge-inducing” and (4) “hairpin” .2Gly]2, [His(Gly)2] (Phe) or biologically relevant RNAs, for instance, miRNAs, were used.Most of the ss-aRNases belong to the group with a “linear” RNA recognition motif. In these conjugates, the oligonucleotide of 11–20 nts in length forms a perfect complementary duplex with the target RNA sequence . As a res(Gly)2]  26,33,3,33,32GlyPhe or oncogenic miRNAs, the RNA recognition motif is split into two oligonucleotide shoulders complementary to the 5′- and 3′-extremeties of RNA targets, while the catalytic group is placed between them opposite a short (3–5 nts) single-stranded gap, which is formed upon binding with the RNA target  of such RNAs significantly complicated the design of ss-aRNases because such ss-aRNAses should leave a single-stranded region of RNA substrate accessible for cleavage upon binding with the target. At the same time, it should maintain the balance between effective duplex formation and the dissociation of ss-aRNase from a complex with RNA after cleavage to maintaining the catalytic mode of action.Nowadays, several research teams have successfully developed ss-aRNases targeted to short RNAs. For instance, the group of Lönnberg designed Zn-dependent ribonucleases based on 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane (azacrown) and “linear” 18-mer 2′-OMe-oligonucleotides. Such conjugates targeted to 36-mer synthetic chimeric 2′-OMe-RNA substrate promote 90% target cleavage within 120 h .M. Göbel et al. designed “linear” ss-aRNAses consisting of tris(2-aminobenzimidazole) and 15-mer DNA, PNA or DNA-LNA mixmer oligonucleotides targeted to 22–29-mer fragments of 3′-UTR PIM1 mRNA ,30,32,722+ ions with Cu+ leads to a manifold increase in the cleavage rate by neocuproin-based conjugates. Such PNAzymes forming a 4-nt bulge-loop are characterised by a τ½ equal to 1.5–3 h 2 developed by our group quantitatively cleave the miRNA target with τ½ 15.1 h ½ 15.1 h  35]. Cr. Cr2Gly]ectively  64]. mi. mi2Gly]ectively  30]..2Gly]2 dThe second group of miRNA-targeted ss-aRNases are “dual” conjugates targeted to oncogenic miR-17, miR-21, miR-18a and miR-155 . These c4 is attached to oligonucleotide via a thymidylate bridge [2Gly]2 and DNA oligonucleotides with a 14-mer fragment, complementary to miR-21 e bridge . Such coe bridge . The higo miR-21 . Such deSpeaking of ss-aRNases, we always remember that they can be multifunctional instruments, making subtle manipulation with targeted RNA possible; however, their main objective is to cleave selected RNA in vivo, influencing various biological processes. Thus, all studies devoted to ss-aRNases imply the targeting of biologically relevant RNAs in the long-term. The cleavage of long mRNA transcripts causes additional difficulties because the secondary and tertiary RNA structures would prevent the binding of ss-aRNases with it. Since the review of Lonnberg , when thLys 2 attached to the hairpin oligonucleotide via an aminohexyl linker cleaved miR-21 exclusively at G-X bonds, representing mimics of RNase T1 2. Bearing this in mind, the main question is whether the utilised ss-aRNase cleaves off-target RNAs with an efficiency comparable with that of target RNA cleavage. To date, the only work to have studied this issue argues that the effect of non-specific cleavage can be neglected [40 while the level of several other miRNAs  remained unaltered [Another important issue concerning ss-aRNases is the cleavage of non-complementary substrates. Although ss-aRNases are intended to cleave target RNA only upon forming a complementary complex, the opportunity for non-specific cleavage cannot be ruled out. Obviously, the cleavage of non-complementary targets may cause undesirable side effects, so it is therefore necessary to try to avoid them. Sometimes it is possible to achieve this goal ,34; howeeglected . The stunaltered .It is noteworthy that the nature of the recognition domain of an ss-aRNase can play a crucial role in its clinical performance. Favourable properties increasing the therapeutic potential of aRNases are the enhanced resistance of the oligonucleotide component to nucleases, high affinity to the target RNA and increased penetrating ability. These capabilities may be provided by substitution of the DNA scaffold by DNA/LNA, and 2-OMe and PNA-modified oligonucleotides used as the binding domains in the developed conjugates .In contrast to DNA oligonucleotides, which undergo rapid digestion by intracellular nucleases, the special advantages of 2′-OMe, LNA and PNA oligonucleotides are enhanced nuclease resistance and a high affinity for the target RNA. Since even a small change in the conjugate structure may significantly impair its catalytic performance, one of the main issues in the design of ss-aRNases is the influence of chemical modifications on their ribonuclease activity.The catalytic properties of tris(2-aminobenzimidazole)-based ss-aRNases, which contain different patterns of LNA modifications of oligonucleotide domain, were also investigated . It was Our studies showed that “hairpin” ss-aRNases containing a fully 2′-OMe-modified RNA recognition domain led to a substantial decrease in ribonuclease activity compared to unmodified analogues: the maximum cleavage efficiency did not exceed 81% in 72 h . In contAn investigation by Danneberg et al. demonstrated that the structure of modified oligonucleotides may also influence the specificity of cleavage. It was found that elongation from 10- to 15-mer of PNA oligonucleotides in tris(2-aminobenzimidazole)-based ss-aRNase results in the loss of site-selectivity of cleavage, leading to the cleavage of target RNA, not only at specific linkages but at all available sites in its single-stranded region. Moreover, lengthening of the PNA oligonucleotide contributes to the formation of aggregates leading to the decrease in effective conjugate concentration in solution .Thus, chemical modifications of the RNA recognition domain, indeed, influence the efficiency and specificity of RNA cleavage by ss-aRNases. To enhance the performance of chemically modified ss-aRNAses, the screening for optimal lengths of oligonucleotide parts should be conducted. Moreover, modified analogues may replace not all of the nucleotide backbone, but should be introduced in certain positions alongside the catalytic group attachment point. Such a strategy seems to be promising since it does not impede the catalytic turnover of conjugates as a result of the less robust binding with the RNA target compared to fully modified analogues .The fruitful work of the last decade has led to the development of highly potent ss-aRNases that are capable of the sequence-specific multi-turnover destruction of target RNA. Developed synthetic enzymes with desired biocatalytic properties are an indispensable tool for in vitro manipulation with RNA. However, a more vital application of ss-aRNases is their use for therapeutic purposes as highly selective inhibitors of disease-relevant RNAs. The multiple catalytic turnover, underlying the functioning of ss-aRNases, offers potential therapeutic opportunities for using a substoichiometric amount of a drug-enzyme in relation to a target RNA, allowing the irreversible elimination of multiple copies of disease-associated target molecules.O-(2-methoxyethyl) oligonucleotide with the lanthanide macrocyclic complex Eu(THED)3+ leads to a significant decrease in the level of ICAM-1 protein in endothelial cells [Despite the high efficiency and prospects of ss-aRNases application, data on their activity in eukaryotic cells are extremely sparse. At the beginning of the century, a few reports provided evidence of the successful use of ss-aRNases in cell cultures. It was shown that the conjugate of 2′-al cells , and theal cells . These sThe key condition for therapeutic efficiency of ss-aRNases is the preservation of their stability and activity in the biological medium, including temperature, pH and concentrations of various cations. Huge hopes are inspired by ss-aRNases developed in the past fifteen years , since sDespite the outstanding achievements in the development of ss-aRNases working in vitro, only conjugates carrying short peptides as a catalyst have been tested as inhibitors of pathogenic RNA in vivo. Our research group has recently designed peptide-based ss-aRNases aimed at inhibiting oncogenic miRNAs in tumour cells, which were termed miRNases ,49,50. TAs mentioned above, in addition to its own ribonuclease activity, the presence of DNA targeting oligonucleotide increases the efficiency of these inhibitors by activating intracellular RNase H. A key discovery was the detection of the synergistic action of miRNases with intracellular RNase H towards miRNA . The mulO-Methylphosphonate (MEPNA) [N-(methanesulfonyl)-phosphoramide (mesyl or µ) [These results are of global importance, as they open up new possibilities for the use of ss-aRNases in vivo. RNase H can hold a great service even in the case of using ss-aRNases that do not exhibit a multi-turnover regime of action in vitro. We have shown that the efficiency of RNA degradation by the conjugates that do not function in a multi-turnover catalytic mode multiplies in the presence of RNase H ,48. When (MEPNA)  or mesylyl or µ) , or gapmyl or µ) ,90.Analysis of the published data showed that highly efficient constructs of ss-aRNases have already been created see , many ofThe success of ss-aRNases developed to date is encouraging; however, models employed to test the activity of ss-aRNases in vitro are maximally simplified low-component systems, using predominantly short unstructured RNAs. Biologically active RNA generally forms secondary and tertiary structures and is assembled in nucleoprotein complexes, which turn most types of intracellular RNA molecules into inaccessible to intermolecular base pairing interactions. Thus, the bioavailability of the target RNA molecule is the major challenge on the way to the development of an efficient sequence-specific enzyme. When developing therapeutic ss-aRNases, the following parameters inherent in the selected RNA target should be addressed: (1) the structural availability of RNA, namely the formation of a strong and stable secondary or tertiary structure, (2) interactions with RNA-binding proteins, and (3) localisation in an accessible and therapeutically functional cellular compartment.The design concepts of ss-aRNases strictly depend on the structure of the target RNA, in particular, the length of the molecule, its sequence, and the secondary or tertiary structure. Similar to antisense oligonucleotides and the siRNA design, the identification of target sites in stretched structured RNA-targets for the invasion of an antisense recognition domain of ss-aRNAse is crucial for their successful application. To approach this problem, a number of sophisticated theoretical and practical methods have been proposed in order to determine effective target sites for antisense oligonucleotide cellular application, including ‘sequence-walking’, the application of random oligonucleotide libraries or genomic libraries, and microarrays ,92. The When targeting long RNA molecules, a favourable thermodynamic stability of DNA/RNA duplex over intramolecular RNA/RNA helices needs to be obtained. At this point, the binding affinity of the RNA recognition domain is also an important issue. Widely used chemically modified oligonucleotides analogues, such as 2-OMe, LNA and PNA may increase the affinity of aRNases to the RNA target. Efficient binding of the oligonucleotide domain of an ss-aRNAse induces the refolding of RNA into an alternative conformation, providing an opportunity for the catalytic group to attack the RNA-target.Searching for the optimal length and structure of the recognition domain is of crucial importance, since the balance between the specificity and efficiency should be achieved. A reduction in the length of the binding domain to a minimum (less than 8–10 nts) can lead to (1) a decrease in binding efficiency, (2) a significant loss of specificity, and (3) an increase in off-target interactions. On the other hand, the excessive elongation of the targeting fragment can significantly reduce the efficiency of the ss-aRNase due to (1) the probability of the formation of a stable secondary structure, (2) the reduction of available complementary sites in the target molecule, and (3) the formation of an extra stable complex with the target, which cancels out the multi-cyclic catalytic mode of ss-aRNase function.It should be noted here that the development of ss-aRNases is also associated with additional conditions. The application of these synthetic enzymes is limited, not only by the accessibility of the target site, but also by sequence requirements for their catalytic function. The motifs of sequences adjacent to ss-aRNase binding sites intended for degradation by the catalytic group should be carefully correlated with their nucleotide base specificity. The catalyst should be spatially located in close proximity to regions that are abundant in linkages that are sensitive to the corresponding type of ss-aRNase. For instance, conjugates of trisbenzimidazole and dimethylphenanthroline are efficient for adenine-rich motifs, while ss-aRNases based on short catalytic peptides predominantly cleave G-X and pyrimidine-A sites . InteresThe design of ss-aRNases targeted to short types of intracellular RNA is a special challenge as it is tightly restricted by the target sequence. The short length of RNA does not allow the position and composition of the RNase binding domain to be varied. Of particular importance is the length of the targeting oligonucleotides, which must inevitably be shorter than the target by at least several nucleotides. The introduction of additional structural elements and modifications of the oligonucleotide domain can be necessary satellites in the construction of ss-aRNases targeted to short RNAs. The implementation of a hairpin structure contributes to a significant increase in the efficiency of hybridisation due to stacking interactions ,93. The It should be noted that the introduction of modifications can significantly change the conformational structure of ss-aRNase, leading to its deactivation. The most rational is (1) the introduction of modifications at nuclease-sensitive sites, (2) the use of modifications recruiting RNase H activity or gapmeric variant of oligonucleotides, and in some cases, and (3) the preservation of nucleotides close to the point of attachment of the catalytic group without modifications. The use of hairpin structures and chemically modified substitutions can also contribute to the nuclease resistance of conjugates, which is critical for the drug lifetime in vivo. The addition of the hairpin to the 3′-end of the oligonucleotide and attachment of the peptide to the 5′-end significantly enhances the stability of conjugates in biological media . “Dual” Among the currently known long types of cellular RNA, disease-associated protein coding sequences, such as mRNAs, and regulatory non-coding RNAs, such as lncRNAs, circRNAs and pre-miRNAs that are located in the cytoplasm, may be attractive targets for ss-aRNases. Malfunctioning of these molecules is involved in many pathological processes during the development of a wide range of human diseases ,97,98,99In addition, ss-aRNases may show great potential as therapeutic antibacterial agents, which are efficient in combating multidrug resistance. In addition to the direct sequence-specific degradation of bacterial coding-RNA fragments, responsible for its viability, tRNA-targeted ss-aRNases developed to date can be used to reduce the level of tmRNA in bacteria, RNA molecules that combine tRNA and mRNA elements in their structure and which are responsible for the stress resistance of bacteria and maintaining their viability . In clinThe biomolecular platform for ss-aRNase design may also serve for the development of efficient antiviral agents that could meet the current urgent need for these drugs. Successful studies on the suppression of SARS-CoV, the virus that is highly homologous to SARS-CoV-2, by siRNAs and ribozymes ,106,107,


Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3′-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3′-cleavage activity increases growth rate but is nonessential.RNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD  promotes Pol III active center-intrinsic RNA 3′-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD  of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3′-cleavage activity is essential, we show that NTD-L can provide the essential function of  RNA polymerase III (Pol III) employs termination-associated reinitiation-recycling to express high amounts of tRNAs, which involves the essential C11 subunit. Here the authors determine the functions of various C11 domains, finding that the N-terminal domain (NTD) stimulates termination, reinitiation-recycling requires an NTD-linker, and the C-terminal domain stimulates RNA 3′-cleavage. Pol II transcribes the largest and most complex gene repertoire, responsible for all mRNAs and many noncoding (nc)RNAs, Pol I transcribes a single multicopy large rRNA gene, and Pol III is specialized to produce a great number of short transcripts which occurs by high-efficiency recycling on tRNA genes, and other ncRNA genes1.RNA synthesis occurs by evolutionarily related multisubunit RNA polymerases which in bacteria and archaea are comprised generally of 5 and 12 subunits, respectively. In eukaryotes, three homologous systems utilize Pols I, II, and III, composed of 14, 12, and 17 integral subunits2. Human pathogenic mutations are linked to subunits of Pol III transcription factors (TFs), and another class of diseases arises from dysregulation of ncRNA genes that are transcribed by Pol III3. Single-allele mutations in a small number of Pol III subunits cause a conditional phenotype, the severe outcome of infection by Varicella Zoster Virus (chickenpox), an otherwise self-limited disease, reflecting deficiency of an innate immune function that requires Pol III transcription activity in the cytoplasm6. More recently, it was reported that Pol III synthesizes the RNA of an RNA:DNA hybrid that serves to protect DNA 3′ ends during homologous recombination7. A large source of mutations that cause the syndromic disorder hypomyelinating leukodystrophy (HLD) occurs in the two largest Pol III subunits, whereas POLR3K/RPC10 the highly conserved small-subunit homolog of yeast C11 has also been a target12. Another complex disorder, Wiedemann–Rautenstrauch  syndrome, and an expanding spectrum of pleiotropic phenotypes are also due to Pol III subunit mutations19.In yeast, Pol III produces tRNAs at ≥15-fold molar excess relative to ribosomes from ~275 dispersed tRNA genes22 attributable to its enzymatic properties as well as stability of the transcription initiation complex24. Much work including recent structural studies of the yeast components indicate that TFs IIIC and IIIB are required to assemble a stable complex on a tRNA gene prior to the first round of transcription by Pol III25. The two domains of IIIC, τB and τA, recognize the B- and A-box internal promoter elements25. The τA domain recruits TFIIIB  and helps it optimize binding upstream of the transcription start site (TSS) prior to Pol III recruitment25. Melting of the DNA strands occurs by allosteric action of Pol III-specific subunits assisted by Brf1 and Bdp1 resulting in bubble extension to the TSS30. Once assembled, TFIIIB remains fixed on the DNA and can direct recurrent reinitiations by Pol III24.High transcript production by the Pol III system reflects the efficient transition from termination to reinitiation1. The trimeric and dimeric subunit complexes Rpc31/34/82 and Rpc37/53 are homologs of TFIIF and TFIIE, some of which facilitate Pol II preinitiation complex formation, and act in promoter opening31. However, in contrast to TFIIF and TFIIE which disassemble as Pol II elongates and must reassociate prior to subsequent initiation, C37/53 and C31/34/82 remain integrally associated with Pol III throughout the transcription cycle31.Properties conducive of reinitiation–recycling are attributable to Pol III-specific subunits that function as intrinsic transcription initiation factors33. Second, C37/53 increases the efficiency of termination and promotes reinitiation along with C1122. Thus in addition to the two largest subunits, Rpc2 and Rpc1 which comprise the active center, C37/53 and C11 increase Pol III termination and reinitiation–recycling39. C11 is a highly conserved protein of ~110 aa whose NTD is homologous to Pol II subunit Rpb9 and its CTD is homologous to the RNA 3′-cleavage domain of Pol II elongation factor TFIIS40. The NTD resides peripherally on Pol III, in contact with C37, the Rpc2 lobe, and Rpc1 jaw41. Recent structures of human Pol III (hPol III) confirm this and the overall general architecture of the 17 subunits in the corresponding elongation complexes (ECs)45.Pol III termination is also conducive to efficient recycling. First, it occurs by an autonomous and apparent simple mechanism in which RNA release is triggered upon reaching a short run of Ts in the nontemplate DNA41. Cryo-EM structures of hPol III resolved its full-length C11-homolog, RPC10, in different conformational states of a transcription complex43. In one of these states, the NTD linker (L) turns inward and extends along the jaw into the funnel, projecting the CTD into the active center pore, positioning the tip of the acidic hairpin toward the elongating RNA 3′ end45. Conservation of sequence and structure by yeast C11 and human RPC10 is illustrated in Fig. Although the C11 CTD was not visible in the yeast Pol III EC, it was resolved in the Pol III apoenzyme complex in which it was docked peripherally, although a connection path to the NTD was unresolved because the central linker region was not visible41. Initially documented for a yeast Pol III EC, the loose RNA:DNA-binding site was confirmed as a specific feature of hPol III EC43. Structures of hPol III ECs subjected to 3D variability analysis revealed that large movements of the C11 CTD and linker from their positions concomitant with elongation-termination was associated with the opening of the Rpc1 clamp (and presumed RNA release), suggesting that C11 may integrate activities involved in different phases of Pol III recycling43.Likely facilitating termination is a loose RNA:DNA hybrid binding site that can readily release upon entering the first ≥5T tract encountered41. A potential link connecting the RNA:DNA-binding site and the dsDNA-binding site was suggested by positive selection screens for loss-of-function (LOF) rpc11-alleles. Two different termination-related activities, read-through (R–T) and RNA 3′-cleavage, differentially mapped to its NTD and CTD46. Similar screens isolated termination R–T mutants in C3738, as well as LOF and gain-of-function (GOF) mutants in Rpc147. Evidence of connections between the Pol III periphery and active center was also obtained for C37/53 by biochemical and other approaches48. High-resolution structures have shown that parts of the C31/34/82 and C37/53 complexes are juxtaposed to downstream DNA-binding sites while other parts penetrate the active center41. Germane here is that the C11 NTD is docked at the periphery in contact with C37 while its CTD can reach the active center to rescue elongation-arrested Pol III via its RNA 3′-cleavage activity40.The C31/34/82 initiation subunits associate with the Rpc1 clamp and dsDNA-binding site, whereas C11-NTD-C37/53 are at the jaw on the other side of this site49. Importantly, despite low termination efficiency at 5Ts, Pol III-core terminates with high efficiency (≥80%) in a 9T terminator, comparable to the 17-subunit holoenzyme although with more propensity to arrest than to read-through49. Studies indicate that an rU:dA hybrid of ≥8 bp spontaneously destabilizes the Pol III-core EC, presumably reflecting an innate trigger of the termination mechanism, and that C37/53/C11 can counteract the propensity to arrest39. A CTD region of C37 referred to as the initiation/termination loop50 promotes termination to occur proximally in the T-tract which appears to work in conjunction with recognition of nontemplate (NT) DNA T-residues as a pause signal33. In addition, C37/53 appears to help maintain RNA 3′-end pairing with the template during termination, preventing arrest39.It is relevant to note that purified Pol III ECs terminate in a 5T terminator with ~50% efficiency, whereas the 14-subunit Pol III-core ECs lacking C11/37/53 terminate with ~10% efficiencySaccharomyces cerevisiae, the C11 subunit is essential for cell viability22. Multiple activities have been attributed to C11, and moreover, some data led to perplexing conclusions regarding its essential function. Prior to the discovery of the physical association of C11 with C37/53, the termination deficiency of purified Pol IIIΔ lacking the (mutant) C11 indicated its importance for termination40. It was then discovered that Pol IIIΔ deficiency was due to the lack of a newly uncovered subunit, C37 which unknowingly dissociated, with its heterodimeric partner C53, along with the mutant C1122. Thus the same study concluded that although C11 was not required for nor influenced Pol III termination, it was required for, along with C37/53, facilitated recycling by Pol III22, the process of multiple cycle RNA syntheses from a stable transcription complex dependent on proper termination20.As was found for all Pol III subunits in 22 and that its cleavage activity was essential for viability40. Overexpression of cleavage-deficient mutant C11-D91A,E92A40 (hereafter DE-AA) was toxic in S. cerevisiae, and this was confirmed with cleavage-deficient C11-(D90G) in S. pombe46. Further, C11 RNA-cleavage activity was deemed essential based on C11-(DE-AA) plasmid shuffle experiments when expressed under promoter-repressive conditions40 . Yet, errors due to lack of C11 cleavage-mediated transcription fidelity51 are not expected to be consequential to short-length Pol III transcripts. Also, the RNA 3′-cleavage factor for Pol II TFIIS nor Pol I subunit Rpa12.2, are essential in S. cerevisiae54. It was unclear why C11 RNA-cleavage activity would be essential.However, confounding the results were data that showed that C11 RNA-cleavage activity was not required for Pol III facilitated recycling39, C11 cleavage activity can occur at Pol III termination with functional outcome46. C11 was shown to stimulate the transcription termination by C37/C5339. Although the CTD was not visible in the yeast Pol III (yPol III) EC41, the hPol III EC showed much of the linker and CTD in the funnel when the DNA clamp was open but was retracted to an outside the funnel position in a closed clamp structure43. Further analysis provided evidence for the important possibility that the mobile C11 linker and CTD participate in the dynamics of termination and recycling in addition to their role in providing RNA 3′-cleavage activity during transcription arrest and backtracking43.Prior studies showed that although not required for terminationS. cerevisiae C11 domains for various Pol III-dependent activities and for cell viability. The NTD alone stimulates termination by C37/53 but not reinitiation–recycling in vitro nor can it provide the essential C11 function. The NTD-L stimulates termination by C37/53 and is required for reinitiation–recycling, and contrary to current belief also provides the essential C11 function in vivo indicating vital importance of the linker. The data resolve misunderstandings about C11 function and advance insights into the mechanisms involved in termination-associated reinitiation–recycling by Pol III.We examined The collective data suggest that the NTD and CTD of C11 appear capable of independent function to stimulate termination and RNA 3′-cleavage, respectively, and when connected by the essential linker to confer the critical reinitiation–recycling activity that is unique to Pol III among the RNA polymerases.39, are shown in Fig. 2c49. Epitope-purified Pol III-core assembled as an RNA:DNA hybrid complex whose 5′-32P-RNA has two mismatched nucleotides at the 3′-end mimics of a backtracked elongation complex (BEC), is a C11 substrate49. Prior analyses had shown that C37/53 tempers the backtracking activity of C11 in these BEC assays39. Our goal was to determine which of the C11 constructs could exhibit basic cleavage activity, somewhat similar to work in which purified Pol II ECs on tailed templates were supplemented with recombinant domains of TFIIS55. Therefore, C37/53 was not included in Fig. 2, whereas C11-(DE-AA), NTD, and NTD-L were inactive.The NTD and CTD of C11 are comprised of Cys-4 zinc ribbon motifs connected by a conserved linker domain  as well as hydrophobic packing and other type interactions at the jaw that involve Rpc1 positions 1177–1180 and 1256–1258 (PDB 5FJ8)41. We also note that the linker had no apparent influence on CTD in this assay  that decreased termination, manifested as R–T past a 6T terminator37. Assays using Pol III-core ECs showed that C37/53-dependent termination is stimulated by wild-type C11 or cleavage-defective C11-(DE-AA)49. Although initially observed on a 9T terminator , characterization of a 5T terminator led to a more quantitative assay39 which we used here , and plotted in Fig. 39. Pol III-core exhibits low TE, ~10% which increases to ~20% by addition of C37/53  was active for Pol III recycling 37. Y31, F33, and I35 are in a region that interacts with a C37 extension (aa 130–140) (PDB 5FJ8)41. In addition, Tyr-22 interacts and fits between Rpc1 residues 1180 and 1275 (PDB 5FJ8). Thus C11 Y22 and F33 help anchor the C37/53/C11 trimeric complex at the jaw  failed termination at T1 and extended to T2 Fig. , lane 4.40. For this work, we developed a suppressor-tRNA allele to be dependent on RNA release in a short T tract49 in S. pombe  suppressible reporter gene along with firefly-luciferase (ffLuc) gene as a separate transcription unit as an internal control. The nLuc/ffLuc TMS assay data  DNA strand on pre-termination pausing by Pol III in promoter-independent single-round assays33. Although this region was not visible in Pol III EC structures it could be modeled next to the melted NT DNA, referred to as the termination/initiation loop . This region was resolved in Pol III initiation complexes associated with elements from TFIIIB Bdp1 and C34 during promoter opening30.C37/53 has been shown to function in termination, Pol III reinitiation–recyclingTyr transcription initiation complexes; C37*/53 in which the five amino acids are substituted with alanine, and C37D/53 in which they are deleted. These mutations had been shown to diminish termination in a single-round, promoter-independent assays, although were not compared to each other39. Lanes 1–4 of Fig. D was comparable to Pol III-core alone , and also significant for C37*. The C37D mutation would appear to render C37 nonfunctional for Pol III termination on nonrecycling transcription complexes.Heterodimers with C37 position 226–230 mutations were examined for activity on the SUP4-tRNAD/53 are active for C11-dependent Pol III-core reinitiation–recycling on the transcription complexes, although not without notable effects. The mutated C37 reactions produced less RNA than WT C37, and more so for C37D. The termination deficiencies reflected by the ratio of T2:T1 in the nonrecycling reactions were apparently suppressed with recycling in the presence of C11 (compare lanes 3–4 and 6–7). The cleavage-deficient C11 NTD-L also activated the C37* and C37D proteins for Pol III recycling  designated C11-(Lkmt)  Fig.  and exammt) Fig. . Recent We also examined the C11-(Lkmt), NTD-L, and the corresponding NTD-L-(Lkmt) with the same mutations for the ability to activate C37/53-dependent recycling by Pol III-core Fig. . In bothRPC1151. As noted, C11-(DE-AA) is toxic when overexpressed, as well as C11-(D90G) in S. pombe46. Our data above suggest that C11-(DE-AA) is not merely cleavage-deficient but that by its ability to insert into the active center it interferes with C37/53 function. This prompted re-examination of the essentiality of the C11 CTD and its stimulatory Pol III intrinsic RNA 3′-cleavage activity.Either of two cleavage-defective alleles, C11-(DE-AA) and C11-E92H tested did not support viability in the absence of 62 to ask which if any S. cerevisiae (sc)C11 constructs could support viability in the rpc11∆ heterocomplemented strain yGAKL which has been a source of Pol III-core (aka pol IIIΔ) used for in vitro assays60. Deletion of the essential RPC11 gene in S. cerevisiae yGAKL is complemented by a plasmid expressing S. pombe (sp)C11 from a GAL1-inducible promoter on a URA3 plasmid28. By the exchange strategy depicted in Fig. URA3 plasmid is counter-selected against by 5-fluoroorotic acid (FOA)63; viable cells will grow on 5FOA media only after loss of the URA3 spC11 plasmid and acquisition of a LEU2-selectable plasmid expressing a scC11 construct that can confer its essential function.We employed the plasmid shuffle techniqueLEU2-scC11 construct plasmids, multiple individual transformant colonies were streaked on plates permissive for growth with both plasmids and then onto plates containing 5FOA. The top and bottom rows of Fig. S. cerevisiae colonies  after spotting onto high expression media, manifested as small colony size relative to C11 and the other mutants , and C11-(Lkmt) transformants reproducibly exhibited slow growth on subsequent plates. Growth in liquid media demonstrated that the central linker DD-AA mutant C11-(Lkmt) was compromised relative to C11, and NTD-L was slower growing with or without these linker mutations Fig. . To examnts Fig. . By contrpc11∆ yGAKL (lane 2), as expected for unreactivity to spC1146.Antibody raised against full-length scC11 recognized a band in the pYX242-scC11 shuffle transformed yGAKL\SML1a Fig. , lane 3 S. cerevisiae.RNAs from wild-type BY4741, yGAKL, and its transformants were analyzed by northern blot with probes specific for scC11 and spC11 mRNAs Fig. . The scCS. pombe strain yYH1 produces TMS when C11 mutants are deficient for RNA 3′-cleavage activity46. The NTD-L expression construct produced TMS although less than the positive control C11-D90G,R107C46  stimulation of C37/53-dependent termination which was attributed to the NTD, (ii) C37/53-dependent Pol III reinitiation–recycling, attributed to the NTD-linker, and (iii) activation of Pol III intrinsic RNA 3′-cleavage activity which was known to map to the CTD. Similar to TFIIS, the C11-mediated RNA 3′-cleavage activity could reactivate arrested Pol III for elongationS. cerevisiae, only the one for Pol III is essential40  and RPA12.2 (RPA12/RRN4) are not54. Thus while Fig. RPC11 is essential40, it shows that its RNA 3′-cleavage activity is not. This now groups Pol III with Pol II, Pol I, and bacterial RNA polymerase, whose intrinsic RNA 3′ cleavage-stimulatory activities are dispensable64. However uncovering this commonality further distinguishes the significance of the NTD-L which confers Pol III reinitiation–recycling activity in vitro, as well as the essential function of C11 in vivo. The latter is notable with regard to the nonessentiality of what is considered the homologous Pol I subunit Rpa12.254, and the relative paucity of sequence conservation in its linker and NTD65. Therefore, these conclusions resolve important longstanding biological issues regarding the essential function of C11. The earlier report40 led to the idea that the RNA 3′-cleavage activity of C11 was an essential function. Our data provide compelling evidence that the essential function is the NTD-L-mediated termination-associated reinitiation–recycling, which is unique to Pol III among the RNA polymerases. Results of the recycling assays reconstituted with the multiple C11 domains are consistent with this and a model in which this unique activity is essential. As discussed below, the CTD-mediated RNA 3′-cleavage activity is important, it can function in termination and it promotes growth rate, but is nonessential for viability, and likewise is not required for Pol III recycling activity. Thus the collective data provide satisfying and unifying consistency.Of the three nuclear RNA polymerase-associated 3′-cleavage factors examined in l40 Fig. , whereas40. Overexpression toxicity lethality was also observed in S. pombe46. Moreover, the C11-(DE-AA) mutations were deemed lethal because the plasmid shuffle experiments used promoter-repressive expression40. However, whether the mutant C11 achieved accumulation to the same level as endogenous under those conditions was not reported. Given the collective results including the data reported here the complex issue of whether there may be a titratable level of expression at which the DE-AA mutant could provide the essential C11 function without lethal toxicity remains to be determined.It would appear that previous genetic approaches have led to two types of confounding results obtained with C11-(DE-AA) or other cleavage-deficient mutants. Describing how the present report helps resolve may also help advance understanding of Pol III transcription. Prior studies reported that C11-(DE-AA) was unable to rescue viability due to toxicity of overexpression40. Although we did not systematically compare constructs for both activities, of all analyzed no other cleavage-deficient mutant exhibited termination interference in the recycling assay nor toxicity   was not only inactive for cleavage Fig.  but inte46. This came from a genetic screen of a randomly mutated C11 library that employed a suppressor-tRNA gene architecture with a relatively long, 7T tract terminator in S. pombe. The mutants obtained had mutations clustered in each of the four cysteines as well as the hairpin of the CTD that were deficient for RNA 3′-cleavage46. (Screening of same C11 library with a different suppressor-tRNA gene architecture that selected for terminator R–T produced distinctive effects that whose C11 mutations clustered in the NTD37.) Two of the characterized CTD cleavage-deficient mutants produced prolonged 3′-oligo(U) tails on precursor-tRNAs which increased their interaction with the pre-tRNA chaperone La protein leading to high-efficiency production of suppressor-tRNA46. Those results added to other data that Pol III intrinsic RNA 3′-cleavage occurs at U residues during termination66. Moreover, they provided evidence that C11-mediated RNA 3′-cleavage is a means to modulate the functional outcome of Pol III termination46. However, nonessentiality of the CTD, slow growth of the NTD-L mutant, and termination interference by C11-(DE-AA), suggest alternative interpretations and a more integrated model of Pol III transcription.The second type of confounding results prompted the idea that C11-mediated RNA 3′-cleavage may be required for normal termination22, which was confirmed using the tailed template and single-round transcription assays on assembled DNA scaffolds49. The latter and additional studies showed that the Pol III-holoenzyme terminates proximally in a 9T tract, whereas Pol III-core lacking C37/53 and C11 is deficient for proximal termination, it extends into the distal 9T tract where it terminates efficiently49, triggered by the instability of the prolonged (rU:dA) hybrid39. The T3-T5 nucleotides in the nontemplate DNA strand were identified as part of a sequence-specific pause and release element that is recognized by Pol III-holo for termination but not by Pol III-core33. DNA scaffolds with a 5T tract limited termination to the proximal region, revealing that C11 increased C37/53-mediated termination reconstituting the holoenzyme mechanism involving a short (rU:dA) hybrid39. Moreover, this revealed that this C11 activity was independent of its role in RNA 3′-cleavage39, reminiscent of its role in the Pol III recycling process22.C37/53 was shown to stimulate termination by Pol III in the absence of C1141. The collective data are consistent with a model in which the NTD of C11 contributes a function independent of the linker-and-CTD in Pol III termination. In this model, the C11 NTD stimulates the activity of the C37/53 heterodimer to increase the efficiency by which Pol III recognizes and terminates in a 5T  terminator, in the context of an (rU:dA) hybrid. The data in Fig. 41 indicate that such a termination effect by the C11 NTD is dependent on interactions with C37/53 at the Pol III periphery but would not involve interactions transmitted to or from the active center by the C11 linker or CTD.That C11-WT, C11-(DE-AA), and the isolated NTD are comparable in the DNA scaffold termination assay reflects its NTD effect that is C37/53-dependent  hybrids in long T-tract terminators33. We propose that C11-mediated cleavage activity serves to rescue Pol III that fails to terminate in a proximal T-tract and might otherwise falter66 or arrest in a lengthening (rU:dA) hybrid39. Cleavage activity would realign the RNA 3′ end and more importantly reverse arrest to allow spontaneous release associated with (rU:dA)(n) instability39. For a terminator proximal region where the degree of (rU:dA) hybrid instability is less than when longer, a previous study suggests that C37/53 alone without the need for C11 can manage RNA 3′-end termination39. Accordingly, we suspect that the C11 CTD mutants obtained from the genetic screen that used the long terminator46 may reflect the rescue activity pathway rather than a requirement for RNA 3′-cleavage per se in normal termination.In the revised Pol III model of termination, RNA can be released in the proximal T-tract, dependent on C37/53 but independent of C11-mediated cleavage activity, whereas extension into a distal T tract comes with the risk of arrestS. pombe Rpc1 followed by screening for disruption of normal termination identified clusters of single-point mutants in four key regions, (i) the trigger loop and its connected helices and (ii) the bridge helix which participate in enzymatic and translocation mechanisms, and more relevant to the present study, (iii) the extended funnel helices, and (iv) another pair of shorter helices connected by a short loop referred to as the cleft-pore entrance loop (CPEL)34. The base of these two pairs of helices forms the entrance to the pore through which the C11-distal linker and CTD pass to the active center. Clustering of termination mutations in the Pol III funnel helices and associated tunnel elements, as well as the CPEL, now provide additional evidence for C11-distal linker and CTD involvement in termination34 , C11-Δhp (Δ81-95), NTD (1–36), NTD-L (1–70), NTD-L-(D52AD53A)-(1–70), CTD-(60–110), and CTD-L-(38–110). These same BamHI-XhoI fragments were subcloned into pYX242 for in vivo assays. These fragments of C11 were generated by PCR. The pGEX-4T-1 plasmids containing the C11 inserts were transformed, expressed in Rosetta (DE3) pLysS cells with 100 uM Isopropyl -D-1 thiogalacto pyranoside (IPTG) for 16 h at 18 °C. Cells were lysed in lysis buffer  and then centrifuged at 15,000×g at 4 °C for 30 min. The supernatant was incubated with glutathione–Sepharose , equilibrated and washed with lysis buffer, and then with wash buffer . Protein was eluted by digestion with thrombin (Biopharm laboratories) at 4 °C overnight in WB. The thrombin was inactivated by adjusting the solution to 2 mM benzamidine–HCl, and the purified proteins were stored at −70 °C.The C11 and C11(D91A-E92A), as well as additional C11-derivatives cloned in the BamHI/XhoI sites of pGEX-4T-1 plasmid, were purified as described39. Both subunits were co-expressed in bacteria. pET28-nH6TEVC53 was cotransformed with pET21-nFLAGC37 or mutated versions thereof into Rosetta (DE3) pLysS and the cells were plated on LB media containing ampicillin (100 g/ml) and kanamycin (50 g/ml) and induced with 0.5 mM IPTG. Cells were lysed in lysis buffer , collected by centrifugation at 15,000×g, the supernatant was collected and passed through Ni-NTA resin (Qiagen). The complexes were eluted with 20 mM K-HEPES, pH 8, 200 mM NaCl, 10 mM 2-mercapto- ethanol, 10% glycerol 300 mM imidazole. Peak fractions were pooled, a total of 40 units of Turbo TEV protease  was added, and dialyzed against elution buffer lacking imidazole. Samples were again passed through Ni-NTA to retain unwanted tagged fragments/undigested proteins and the flowthrough was passed through an SP Sepharose  column equilibrated with dialysis buffer using the ÄKTA purifier system (GE Amersham). Peak fractions were pooled, and buffer  was exchanged using a PD10 column  using the ÄKTA system. The C53/37 complexes were applied to a Q-Sepharose  column and eluted with 300 mM NaCl.C37/53, C37*/53 , and C37**/53 (deletion of amino acids 226–230) recombinant heterodimers were purified as describedS. cerevisiae yZN16, carrying an N-His-FLAG-tagged RPC128 (C128) gene; Pol III-core was purified from yGAKL carrying N-His6-FLAG3-tagged C128 (RET1) in which Rpc11 was deleted and complemented with S. pombe C1149. Briefly, 60 g of cells pellet was lysed by bead beater in ice-cold lysis buffer . The supernatant was collected after centrifugation at 15,000×g for 30 min and then ultracentrifugation (Beckman) at 100,000×g in a 60 Ti rotor for 1 h at 4 °C. The top layer was recovered (S100), avoiding the murky stuff above the pellet. The S100 was incubated with Ni-NTA resin (Qiagen) at 4 °C for 2 h. Pol III was eluted with 20 mM Na-HEPES, pH 8, 20% glycerol, 500 mM NaCl, 10 mM 2-mercaptoethanol, 7 mM MgCl2, 100 mM imidazole, and complete protease inhibitor (Roche). Small aliquots were stored at −70 °C. For the assays, units of activity for Pol III-holo and Pol III-core is that which give near equal amounts of total RNA products from ECs assembled on DNA scaffolds with a 9T terminator, as seen for example in Fig. 39.Pol III-holo was purified as described from 39. Pol III with a His-tag on Rpc2 was immobilized on magnetic Ni-NTA beads (Thermo-Scientific) and washed with EC buffer . Template (T) DNA (1 µM of SMO77) and 5′ 32P-labeled RNA were annealed in ECB by heating to 55 °C and rapid cooling to room temperature and incubated with immobilized Pol III and after 10 min incubation at room temperature, 2 µM nontemplate . DNA was added and incubated for 10 min followed by 3washes. Now the EC assembly is ready for transcription. For all experiments, equal amounts of Pol III-holo and Pol III-core transcription activities were used. A typical 1× reaction contained 25 µl of beads. For some experiments, a larger batch of ECs was prepared, washed then aliquoted into equal smaller volumes for subsequent analysis. For reconstitutions of Pol III, recombinant proteins were incubated with the ECs for 15 min prior to NTP addition. A total of 500 µM NTPs containing 7 mM MgCl2 was added and incubated for 10 min at room temperature followed by separation of released from bound by the magnet and immediate phenol extraction/ethanol precipitation. Samples were dissolved in formamide dye, resolved on 15% sequencing gels, dried, exposed to PI screen, and visualized using Typhoon .EC assembly on scaffolds and transcription reactions were performed as described32P 5′-labeled 10-nt RNA annealed to the DNA template (T) strand with 5 As in the termination zone (TZ) assembled with Pol III-core and a nontemplate (NT) DNA strand (5Ts in the TZ) is prepared and washed. The ECs are aliquoted to 1.5-ml tubes, components are added, reactions are initiated and incubated for 10 min and then immediately stopped upon separation of the supernatant, which represents the terminated or released (R) RNA fraction, and the bound (B) RNA fraction that remains associated with Pol III. The purified RNA products are separated on urea polyacrylamide gels which are processed, the 32P-RNA quantified, and termination efficiency at the 5T terminator is calculated39.A 2 and incubated for 15 min; the reaction was stopped and samples loaded in the gel.For cleavage assays, the EC was assembled as described above with a 2-nt 3′ overhanged RNA primer employed and used template AGO16X, RNA primer RNA6, and NT AGO 7X. And incubated with the presence or absence of WT C11/ derivatives of C11 for 15 min at room temperature followed by the addition of 7 mM MgCl39.Transcription termination efficiency (%) was calculated as follows. Transcription termination efficiency =  × 100. Total = RNA released in TZ + released RT RNA + bound at TZ + bound at RT). TZ refers to terminator zone (TZ) and RT refers to read-through. Phosphoimages were analyzed by using Multigauze V3.2 software (Fuji)2, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) and EDTA free protease inhibitor (Roche) and poured into 60-ml small chamber of bead beater cell disrupter. An equal volume of 0.445-mm diameter acid-washed glass beads (Sigma) sufficient to exclude all air from the chamber was added to the chamber. The unit was assembled and placed in an ice jacket containing an ethanol-ice slurry. Cells were lysed by homogenization for 9–10 min total time, in 30-s bursts with 4 min cooling time between bursts. The lysate was decanted from the glass beads, and 1 mM PMSF and 2 mM DTT were re-added, respectively. 0.4 M  ammonium sulfate was added in the lysate from 4 M stock ammonium sulfate (pH 7.9). The extract was incubated on ice for 30 min before centrifugation at 15,000×g for 60 min. The supernatant was collected and centrifuged at 100,000×g (rotor 60 Ti) again for 1 h. The supernatant was collected. The proteins in the supernatant were precipitated by adding very slowly ammonium sulfate (0.35 g per ml of lysate) and kept on ice for 30 min then centrifuged to precipitate the proteins. The precipitated proteins were dissolved in a minimal volume (10 ml) of Buffer C  and desalted by using a S200 column (2.5 cm diameter and 12 cm height) as described68 and elution was collected after 1.5–2× of the load volume passed from the column by gravity flow.Cells were grown in YPD media  to a density at 650 nm of 3.17 g of cells were harvested by centrifugation, washed with chilled distilled water, and resuspended into 25 ml lysis buffer (200 mM Tris-C1 (pH 8.0), 10% v/v glycerol, 10 mM MgCl2, 100 mM NaCl) containing 10 µl of Txn extract for 20 min at RT. (2) The supernatant was replaced with 25 µl TB containing 0.5 mM each nonradioactive NTP with heparin at 0.03 mg/ml, incubated for an additional 3 min at RT. (3) The complexes were washed once with 50 µl of TB containing 500 mM NaCl and twice with 50 µl of TB. (4) Complexes were then used for subsequent transcription by purified Pol III-holo or Pol III-core.Preparation of Pol III-depleted initiation complexes was as follows, prepared in batch, calculated per reaction. (1) 200 ng of biotinylated PCR-derived sup4e DNA, immobilized on 30 ul magnetic streptavidin beads, incubated with 30 µl transcription buffer . Reactions were stopped after 30 min of incubation at RT by the addition of 50 µl acidic phenol. Total RNA was purified by ethanol precipitation, resuspended into 15 µl RNA loading buffer (Invitrogen), and analyzed by 8% Urea-PAGE.The subsequent transcription assay was as follows. Pol III-holo or Pol III-core was added to the freshly prepared extract-derived Pol III-stripped complexes. After 20 min of incubation, the supernatant was replaced with 20 µl of TB, and 5 µl of S. pombe WT C11 and C11- in pRep4X were available46. C11 NTD-L(1–60) and CTD-L(36–109) were PCR amplified and cloned in pRep4X under BamHI and XhoI-restriction sites.S. cerevisiae yGAKL strain  from which the essential chromosomal RPC11 gene was deleted, is heterocomplemented by S. pombe C11 expressed from the centromeric plasmid pRS316 (URA3) under control of the GAL1 promoter. This strain is viable when galactose is the sole carbon source. WT scC11 and its derivatives cloned in pYX242, 2 μm (LEU2), were transformed into yGAKL strain and plated on SC (synthetic complete) media without Leu and Ura +galactose plates. Multiple colonies from each transformant plate were streaked on SC w/o Leu +galactose plates and these colonies were then streaked on SC −Leu +Galactose +5FOA plates. The URA3 pRS316 containing S. pombe C11 will be counter-selected against by 5FOA (see the text).S. cerevisiae strain F729  was transformed with pYX113 plasmid which has a GAL1 promoter70 driving expression of the different scC11 constructs, and plated on SC media lacking uracil. Colonies were inoculated in liquid media (SC without uracil) for overnight growth at 30 °C. From the overnight culture, cells were diluted into fresh SC media without uracil and incubated at 30 °C. Logarithmically growing cells at an OD600 of 0.6 were spotted at a cell concentration of 1.0 OD/ml and tenfold dilutions thereof; 5 µl of each was spotted onto SC + glucose w/o uracil plates and SC + galactose +raffinose w/o uracil plates and grown at 30 °C for 3 days.ade6-704 in S. pombe strains harboring integrated suppressor-tRNA genes was as previously described57 (reviewed in ref. 61). Strain ySHA24 was transformed with pRep4X containing different derivatives of C11, Rpc1 or the empty vector and other and plated onto EMM-complete media lacking uracil with limiting adenine (10 mg/L). Representative transformant colonies were streaked and subsequently transferred to liquid media, grown overnight, diluted, grown logarithmically to OD600 of 0.6. Cells were spotted onto plates as above and grown at 32 °C for color development.Expression plasmid pRep4X-mediated suppression using the red–white assay based on suppressible S. pombe Chr I between SPAC6F6.11c and SPAC6F6.1271. Each of the genes in the construct is an independent transcription unit with its own promoter, Kozak sequence, and terminator. The nano-Luc gene was mutated to replace a TCC serine codon with a TGA premature stop codon that is suppressible by the opal suppressor-tRNA and its derivatives61. The pRep4X-mediated suppression was as above. Cells were collected and used to make an extract from which dual-luciferase activities were measured.For the dual-luciferase assay, a construct with the following gene arrangement; NanoLuciferase-KanR-firefly Luciferase, was inserted by homologous recombination into S. cerevisiae strains having different scC11 constructs in pYX242 were grown for 24 h at 30 °C, 250 rpm shaker in 5 ml synthetic complete (SC) media without leucine +galactose liquid media. After 24 h, the cultures were diluted to 0.05 (OD600) in 25 ml of the same media in a 250-ml flask for each. At each time point, OD600 was measured as indicated.600 of 0.1–0.4 were harvested, washed with water, and resuspended in 300 μl TES buffer . In total, 300 μl acidic phenol (Invitrogen) was added and incubated at 65 °C for 60 min, with vortex every 10 min. To the samples, 300 μl chloroform was added and centrifuged. The supernatant was extracted twice with acid-phenol/chloroform and once with chloroform before ethanol precipitation. The precipitated pellet was resuspended into 50 μl water, quantified, and mixed with loading buffer. In all, 10 and 20 μg of RNA was resolved on 1.8% agarose-formaldehyde gel and transferred to a GeneScreen-Plus membrane (PerkinElmer). After UV cross-linking and vacuum-baking at 80 °C for 2 h, the membrane was prehybridized in 6× SSC, 2× Denhardt’s, 0.5% SDS, and 100 µg/ml yeast RNA for an hour at the hybridization incubation temperature (Ti). 5′-end-labeled 32P-oligo DNA probes complementary to scC11 mRNA, SCR1 RNA, and spC11 mRNA were incubated at the Ti overnight and washed. The blot was stripped and examined for probe removal prior to rehybridization.Total RNA was isolated using hot phenol. In short, 50-ml cultures grown from an OD600 cells were harvested, washed, and suspended into lysis buffer . In all, 40 µg protein/lane was separated by SDS-PAGE on 10–20% polyacrylamide gels and transferred onto the nitrocellulose membrane . Membranes were blocked in 1% (w/v) skimmed milk in PBS for 60 min at room temperature and incubated with primary antibody at 1:1000 in PBS-T buffer overnight at 4 °C. Membranes were washed several times in PBS-T, incubated with fluorescent dye-conjugated secondary anti-rabbit Ab  in blocking buffer (PBS-T) at 1:10,000 and visualized using Li-Cor system. The custom-made anti-scC11 Ab was raised in rabbit against full-length S. cerevisiae C11 and affinity-purified against the same full-length protein by GenScript USA, Piscataway.In total, 50 ml of 0.5 ODS. cerevisiae Pol III EC (PDB 5FJ8) with the CTD-Linker of yeast TFIIS modeled in, as reported previously34, was a kind gift of N Hoffman and C. Muller .PyMOL molecular graphics software v2.0.1 was used for structure representations. The Each and every experiment depicting results in any form and including all that are considered representative have been repeated independently with similar results a minimum of two times. We note that a minimum of two independent repeats with similar results was atypical for many of the most relevant samples/constructs tested as part of a series of samples/constructs. More common was that most of the individual samples/constructs were tested four or more times with similar results. Moreover, multiple experiments performed as biological replicates with similar results were then repeated as biological replicates using a different batch of purified proteins, again producing similar results.Supplementary InformationPeer Review File


Sex (p = 0.30) and breed (p = 0.65) had no significant effects. A large variation in the squares visited per hour was determined for the individual horses. Most frequently visited were the individual resources, such as the feed stalls or lying halls. An open tarp skin lying hall was preferred over a metal hall. The shelters were only partly popular. An overview of the usage frequency of the paddock could be created by using heatmaps. Overall, specific conclusions about the use of individual areas and potential for improvement could be pointed out.Horses are increasingly being kept in group stables where they are given the opportunity to move freely and to contact conspecifics. This study examined how the horses of a large group move in an active stable and which areas or resources they use over a period of 7.5 months with the help of linear mixed models. Therefore, a grid with the size of 3 × 3 m per square was applied. The horses used, on average, 53.2 ± 19 different squares per hour. It was shown that observation day  and the covariate age (p < 0.001) had significant effects on the different squares visited per hour. No significant effects were found for sex (p = 0.30) and breed (p = 0.65) as only geldings and no stallions were part of the group and the distribution of the breeds was unfavorable. The random effect animal showed that the horse-individual estimates from −19.2 to 17.6 different squares visited per hour were quite large. Furthermore, it could be shown that the horses used resources such as feed stalls with a frequency of up to 0.14% more than other paddock areas without resources. Open lying halls with tarp skin were also preferred over the metal hall. The shelters were only partly popular. Use could be visualized with the help of heat maps. This study gives a good overview of the use of individual areas and resources and possible improvements.The aim of this study was to analyze the utilization of different stable areas of a total of 52 group-housed horses as well as their preferred stable parts and the use of resources. The study was situated in a “HIT Active Stable In Germany, keeping horses in groups is becoming an increasingly common practice. This type of husbandry enables these highly social animals to better live out their species-specific behaviors, such as all-day movement and the possibility of social interaction with conspecifics. The positive effect of group housing and social interactions is adequately described in the literature ,3,4,5,6.®  ) and the “German Order for the Protection of Animals used for Experimental Purposes and other Scientific Purposes” (German designation: Tierschutz-Versuchstierverordnung [TierSchVersV]) were applied. The owners of the individual horses had previously agreed to the participation of their animals.p = 0.65) (p = 0.30). When considering age, it was found that with each increased year of life, the squares per hour visited decreased by 0.6 (p < 0.001). Similar significances, but with lower Least Square Means (LSM), were also found for the dataset without pasture.Including data with pasture, each horse was located in 53.2 ± 19 squares/h on average. No significant effect was found for the breed ( = 0.65) . Warmblop < 0.001)  . During p < 0.001) and location (p < 0.001) in both variants with and without consideration of the pasture. Due to the very high number of squares, the percentage values are very low. The LSMs of the individual days were between 0.06 and 0.08%. The usage frequency of the different resources of the farm is shown in The evaluation of the second model revealed significant differences in the LSMs of day  and breed (p = 0.65) had no significant effect on different visited squares/h. This confirmed previous results in Hildebrandt et al. [The fixed factors sex . At a younger age, horses have an increased urge to play and explore, which leads to more movement within the barn. This corresponds with Krueger et al. [The factor observation day had a significant effect on the squares visited per hour. Certainly, the possibility of visiting the pasture has a high influence. This can be seen especially by considering the summer data. Activity phases are longer due to greater daylight periods. While grazing, horses typically move physiologically forward bit by bit. The occasional escape from insects could also have influenced the number of visited squares. Considering only the paddock without pasture, the observation day also had a significant effect. Furthermore, the inclusion of new horses and the establishment of a new rank order also had an effect, which had already been proven in Hildebrandt et al. . Individr et al. , who repWhen considering the random effect animal in In general, the frequencies of the visited squares in Horses are adapted to temperate grasslands. In the natural environment they spend more than 14 h of a day foraging . The impThe results showed that the horses use some areas higher frequently than others, especially the resources. The individual frequencies were only so low because of the high number in total. For example, the frequency of 0.14% of FS_7 resulted from the average of the assigned squares of the feed stall. Nevertheless, it can be seen that at least small differences along the individual areas could be identified. Thus, for example, potential for improvement could be determined, since the metal lying hall was used less frequently than the one with tarp skin. The metal hall had only small entrances and the horses could not observe their environment. The fact that all areas were used shows that the stable itself was generally well designed. Furthermore, it must be pointed out that these results are only valid for this specific active stable and that, for example, differences in the available space or a modification of the number of horses could be investigated in further studies. Likewise, in general, other stables which are structured or designed differently could be used. Special focus could also be made on group size, breeds sex. The results of the occupancy of the areas showed that the correct positioning of the individual resources is of great importance. This should be taken into account when planning the stable. Furthermore, the effect of the individual horses in the group should not be underestimated and stable areas should be individually adapted to the individual group.The present study demonstrated the frequency of visits of the different squares and its influences. In general, it was quite variable as to how many different squares/h an animal visited. The individual horse, beside the day and the age, represented the biggest influence factor. Different events can influence movement in the stable. The utilization of farm areas was analyzed with the help of the second model and heatmaps. It can be speculated that by prioritizing the resources, the horses are motivated to move to reach necessary resources. Thus, it is important to separate the resources as far as possible from each other spatially to create an additional movement stimulation. In general, all paddock areas were used and, with a few individual exceptions, all horses basically used all areas.


Epitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt–290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing. With the advent of multi-modality measurements for personalized medicine, clinical samples will be routed to many more molecular tests posing the pressure to improve every sample preparation step to derive insights2. The ideal extraction method extracts all intended analytes from a sample and enables automation. The conventional extraction systems such as spin columns and magnetic beads are commonly used for nucleic acids isolation from biological samples; however, these methods are labor intensive and difficult to automate3.Extraction of nucleic acids for molecular testing is a critical step in generating high quality sequencing libraries or input to PCR-based tests5. We designed and fabricated an easily automatable nucleic acids extraction system based on electrophoresis principles6. Epitachophoresis (ETP) takes advantage of a discontinuous electrolyte system consisting of leading and trailing electrolytes to focus nucleic acids into a narrow band8. In the ETP system, an agarose gel stabilizes the electrolyte solutions while allowing for nucleic acid size selection applications. When applicable, the gel can be replaced by a non-sieving matrix9 to allow for concentration of large DNA fragments.Non-affinity based separation methods such as electrophoresis, take advantage of differences in electro-migration to separate nucleic acids6. Here we employed a challenging clinical sample, Formalin fixed paraffin embedded (FFPE)10 tissue, and a widely used biological model (cell line) to showcase the strength of ETP in extracting different types and sizes of nucleic acids from biological samples.ETP utilizes a unique circular architecture where the sample is loaded into the outer ring and pipetted out as a liquid extract from the central well Fig. . We prev11  FFPE samples  using the ETP platform, and DNA FFPE column extraction kit (Promega) was used as the control  ETP condition were used to extract DNA from lung carcinoma cell line NCI-H1975). A DNA precipitation kit (Promega Wizard HMW DNA) was used as a reference. Pulsed-field gel electrophoresis was employed to estimate the size of the extracted fragments  applications. The sequencing analysis for miRNA extracted from lung carcinoma cell line (NCI-H1975) is presented in Fig. les Fig. b. For theen Fig. c compareeen Fig. b. ETP is2 = 0.95).We also focus on the total RNA profiles extracted from the lung carcinoma cell line (NCI-H1975) in addition to the targeted miRNA extractions. Pairwise comparisons of RNA from simultaneous RNA/DNA ETP  and gDNA including large molecular weight. DNA and RNA quantity and quality was sufficient for Next-Generation Sequencing analysis.l-histidine monohydrochloride monohydrate (99%), l-histidine (99%), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid , tris(hydroxymethyl) aminomethane , anionic dye Brilliant Blue FCF (Sigma-Aldrich). Low-electroendosmosis—multi purpose and molecular biology grade agarose (Fisher). DNA and RNA ladders (New England BioLabs). ERCC RNA Spike-In Mix (Thermo Fisher Scientific). SYBR Gold (Invitrogen).The following chemicals were used: Biological samples: Colorectal Cancer FFPE tissue blocks were purchased from Indivumed Germany  and curls were cut in-house with a microtome instrument . NCI-H975 Lung carcinoma cell line was purchased from ATCC  catalog number of CRL-5908 and Lot number: 70023980.List of kits used for sample preparation and extraction comparison: All following kits were purchased from Promega: ReliaPrep FFPE gDNA Miniprep System, ReliaPrep FFPE Total RNA Miniprep System, Wizard HMW DNA Extraction Kit, ReliaPrep miRNA Cell and Tissue Miniprep System, ReliaPrep RNA Tissue Miniprep System, ReliaPrep gDNA Tissue Miniprep.List of analytical kits used in the presented study: Qubit assay kits (Thermo Fisher Scientific)—1x dsDNA HS Assay Kit, RNA HS Assay Kits, microRNA Assay Kit. Bioanalyzer kits (Agilent)—High Sensitivity DNA Kit and High Sensitivity RNA kit. Tapestation kits (Agilent)—High Sensitivity DNA ScreenTape, Genomic DNA ScreenTape, High Sensitivity RNA ScreenTape.List of kits used for sequencing libraries: Amicon Ultra-0.5 mL filters (3 and 10 KDa) were purchased from Sigma Millipore. Sequencing libraries for FFPE DNA extraction were prepared using the AVENIO Tumor Tissue Expanded kit Analysis workflow . miRNA libraries were prepared using NebNext small RNA preparation for Illumina . Sequencing libraries for total RNA were prepared using the KAPA RNA HyperPrep Kit with RiboErase .Fabrication of ETP device: We used Autocad (Autodesk) 3D modeling software for designing the platform Fig. a. The fi2 power source was used (Trotec). Qubit 4 fluorometer (Thermo Fisher Scientific) was used for quantitative amounts of DNA and RNA from extracts. Size of extracted DNA and RNA was checked on Bioanalyzer 2100 (Agilent), Tapestation 4200 (Agilent) and Pulse Field Electrophoresis Pulse Pippin (Sage Science). Extracts were processed on Roche 480 lightCycler (Roche), Illumina NextSeq 500 (Illumina), Veriti Dx (Thermofisher).Equipment: PowerPac™ HC High-Current Power Supply was purchased from BioRad and uEP300-12 Power Supply was from Labsmith . Benchtop pH Meter Orion Star A211 with ROSS pH Buffer calibration kit were obtained from Thermo Scientific. Laser cutter—Speedy 360 with a 120 W COl-histidine monohydrochloride monohydrate 11 g of l-histidine in 500 ml water). Trailing electrolyte (TE) solution—20 mM TAPS-TRIS, pH 8.30 (0.605 g of TAPS and 1.625 g of TRIS in 500 ml of water). The agarose gel—in 20 mM LE . All buffers were prepared in deionized and nuclease-free water (Fisher). 0.5% (or 0.7%) agarose gel—500 mg (or 700 mg) of agarose was mixed with 100 ml of 20 mM Histidine-HCl LE buffer in a glass Erlenmeyer flask and heated on the hot plate till boiling while stirring. The mixture was kept at boiling for 1 min. After cooling the mixture to approximately 60 C, the solution was transferred to the round gel mold. Gel should be prepared at least 1 h before extraction and can be kept at 4 C for overnight. 0.7% agarose gel was used for FFPE experiments, 0.5–0.7% agarose gel was used for cell line experiments.Electrolytes and gel were prepared by following procedure: Leading electrolyte (LE) solution—100 mM Histidine-HCl, pH 6.25 .The sample/TE mixture was injected into the gap between the gel and the electrode.Constant power 2 W was applied for 40 min.Detection and extract collection: The Brilliant blue was used to indirectly track the nucleic acid band : Using Promega ReliaPrep FFPE gDNA Miniprep System, FFPE curls (2 × 10 µm curls per extraction) were deparaffinized, lysed and Proteinase K treated as described by the manufacturer. Lysates were then de-crosslinked at 80 C for 1 h before cold shock on ice for 2 min and stored at 4 C overnight. Samples were then thawed at room temperature for at least 10 min prior to extraction via ETP. This extraction type had two biological replicas for each FFPE block .Control extractions used for FFPE study: Promega ReliaPrep FFPE gDNA Miniprep system was used according to the manufacturer to extract DNA from FFPE curls (2 × 10 µm curls per extraction) with minor modification of RNase treatment step being omitted. This extraction type had two biological replicas for each FFPE block .Nucleic Acids yield measurement: In order to have true measurement of dsDNA and RNA without background interference, small portions of each extract underwent RNase A (Promega: A797C from FFPE gDNA kit) or DNase I  treatments. RNase treated portion was used to measure the true dsDNA amount (Qubit dsDNA HS Assay Kits) and DNase treated samples were used to measure the accurate RNA amount (Qubit RNA HS Assay Kit).FFPE DNA Quality measurement: DNA Quality was assessed using the AVENIO Tumor DNA Isolation and QC Kit (Roche Diagnostics). The quality of each FFPE DNA sample was assessed by quantitative PCR (qPCR) using 2 PCR amplicons (66 bp and 191 bp). The normalized quality ratio (Q-ratio) was calculated using QC PCR DNA standard of known quality. We used the 3 technical replicates of the Cp values per amplicon to calculate the Q score for each sample including the QC PCR DNA standard by using the following equation: Q score = 2[average(Cp66) − average(Cp191)]. We obtained a Q-ratio for each sample through the following equation: Q-ratio = sample Q score/QC PCR DNA Standard Q score.FFPE DNA Library preparation & sequencing: Sequencing libraries for FFPE DNA extraction were prepared using the AVENIO Tumor Tissue Expanded kit Analysis workflow . Input was calculated (100/(Q-ratio) + 10) ng of input DNA where Q-ratio is the normalized quality score from qPCR assay. Libraries were sequenced using Illumina NextSeq 500 High Output v3 . The sequencing data were analyzed using an internal analysis pipeline equivalent to the commercially available AVENIO Oncology Analysis Software version 2.0.0. Reads were subsampled to 20 million paired end reads. Two extraction replicas and two libraries per replica (n = 4) from CRC FFPE block for each extraction method.Sample pretreatment (cell lysing and Proteinase K treatment) for ETP samples: 100 µl Lysis buffer and 10 µl Proteinase K from Promega ReliaPrep FFPE Total RNA Miniprep System kit was used to pretreat the ~ 325,000 cells per extraction. The mixture was digested at 56 C for 15 min. Lysates were then cold shocked on ice for 2 min before overnight incubation at 4 C. All samples were thawed at room temperature for at least 10 min prior to extraction.Extracts post-treatment (DNase treatment + RNA Column Clean-up): Prior to downstream analysis, DNA portion of the extraction was digested by 20 min treatment with NEB DNase I (RNase Free) at 37 C. Total RNA was retrieved from the treated sample using NEB Monarch RNA Clean-up kit (10 µg) with 3X ethanol used alongside the binding buffer to ensure retention of the small RNA in the extracts (> 6 nt). This protocol was suggested by the manufacturer in keeping the small RNA during clean-up. This condition had three extraction replicas.miRNA extraction control: The Promega ReliaPrep miRNA cell and Tissue miniprep System was used to pretreat and extract total RNA from chosen cell lines as recommended by the manufacturer. Extracts were then further concentrated by NEB Monarch RNA Clean-up (10 µg) kit prior to small RNA library preparations. Please note that conditions used for the RNA Column clean-up was tailored to keeping any RNA above 6 nt size (3X used for ethanol).Nucleic Acids yield measurement: Qubit RNA HS assay kit (Thermo Fisher Scientific) was used to measure total RNA from each extraction while Qubit microRNA assay kit (Thermo Fisher Scientific) was employed to estimate the amount of small RNA (< 1000 nt) extracted per sample.RNA size range measurement following extractions: RNA size was profile was checked by Tapestation capillary gel electrophoresis  and by Bioanalyzer 2100 chip gel electrophoresis .miRNA cell line Sequencing Analysis and data analysis: Total RNA was extracted from the (cell line). 500 ng was used as an input into the NebNext small RNA preparation for Illumina . Library preparation was performed according to manufacturing instructions. RNA extract was normalized 500 ng prior to generating the small RNA libraries. Ampure XP beads was used as the size selection method. Two rounds of bead clean-up was preformed. The purified libraries were pooled and then sequenced with a NextSeq 500 instrument using the Illumina NextSeq High Output kit v3 (300 cycles) 2x 75 (Illumina).https://github.com/nf-core/smrnaseq. The raw sequencing data and the normalized CPM (counts per million) values for each of the samples is available at: https://www.ncbi.nlm.nih.gov/geo/. Venn diagram input data: Only miRNA transcripts with abundance  in all the ETP replicas or in all Column replicas are considered to be extracted by the respective method. Scatter plot: The average of detected miRNA across 6 replicas was graphed. Zero counts were assigned to miRNAs not found in all replicates even if they were found in some of the libraries  for ETP: 100 µl Lysis buffer and 10 µl Proteinase K from Promega ReliaPrep FFPE Total RNA Miniprep System kit was used to pretreat the ~ 557,000 cells per extraction. Treatment was done at 56 C for 15 min. Lysates were then cold shocked on ice for 2 min prior to being incubated overnight at 4 C. Following overnight incubation, samples were first incubated at room temperature for at least 10 min prior to the next steps. ERCC was added into the cell lysate—1 µl of 1:100 dilution of ERCC RNA Spike-In Mix (Thermo Fisher Scientific). Lysates were fragmented into smaller pieces (500 bp) using Covaris E220 Focused UltraSonicator . For the fragmentation step, 130 µl of lysate solution was placed into covaris microtubes and the following sonication program was used: duty cycle 10%, intensity 3, cycles per burst 200, time 80 s.Control conditions: DNA Column Clean-up Control—Promega Wizard SV Genomic DNA Purification System was used according to the suggested protocol by the manufacturer to extract gDNA from ~ 557,000 cells per extraction. ERCC were added into the cell lysate prior to extraction of nucleic acids by the columns. This condition had two technical replicates.RNA Column Clean-up Control: Promega ReliaPrep RNA cell Miniprep System was used according to the suggested protocol by the manufacturer to extract RNA from ~ 557,000 cells per extraction. ERCC were added into the cell lysate prior to extraction of nucleic acids by the columns. This condition had three technical replicates.Nucleic Acids yield measurement: Qubit RNA HS assay kit and Qubit dsDNA HS assay kit were used to measure total RNA and dsDNA respectively from each extraction which was used to calculate the input for each sequencing Library preparations.DNA & RNA size profile measurement: Size of profile extracted DNA and RNA was determined using Tapestation 4200 (Agilent) by Genomic DNA ScreenTape Analysis and High Sensitivity RNA ScreenTape (Agilent) respectively. For RNA, samples were first treated with DNase I  and underwent RNA Column clean up before size profile analysis and the RNA sequencing library preparations to avoid any background signal from DNA.Cell line RNA Library preparation & sequencing: Sequencing libraries for RNA extraction were prepared using the KAPA RNA HyperPrep Kit with RiboErase (Roche Diagnostics). Libraries were sequenced using Illumina NextSeq 500 High Output v3 . The sequencing data were analyzed using an internal analysis pipeline equivalent to the commercially AVENIO Oncology Analysis Software (version 2.0.0). Reads were subsampled to 20 million paired end reads.Cell line RNA Sequencing Analysis: We used HISAT2 to align the sequencing reads to the genome and then use Kallisto to quantify the abundance of RNA transcripts. Gene abundances were calculated by summing over the abundance of the corresponding transcripts for the gene.Cell line DNA Library preparation & sequencing: Sequencing libraries for cell line DNA extraction were prepared using the AVENIO Tumor Tissue Expanded T Analysis workflow (Roche Diagnostics). Input in Library preparation with 100 ng. Libraries were sequenced using Illumina NextSeq 500 High Output v3 . The sequencing data were analyzed using an internal analysis pipeline equivalent to the commercially AVENIO Oncology Analysis Software (version 2.0.0). Reads were subsampled to 20 million paired end reads. Two extraction replicas and two libraries per replica (n = 4) from CRC FFPE block for each extraction method.Sample pretreatment (cell lysing and Proteinase K treatment): Promega wizard HMW DNA extraction lysis buffer and Proteinase K were used as described by manufacturing to lyse ~ 290,000 cells per extraction. Wide bore pipette tips were used to reduce the DNA shearing. This condition had three extraction replicas. Following lysing and proteinase K treatment, lysate was loaded onto ETP and long DNA was extracted.Gel ETP conditions were same as described in “Gel-free ETP condition and set-up: A miniaturized version of the EPT device was used. The polycarbonate top dish  was fabricated by injection molding . A 6-well plate served as the bottom dish . The anode (3 mm height) was laser-cut from MinGraph Flexible Graphite Sheet with Adhesive. It was gently taped around the perimeter of the outside top dish. Stainless steel ring  served as the top cathode electrode. Separation semipermeable membrane (10 × 30 mm) was cut from 0.45 µm Durapore Membrane Filter . The separation membrane was then carefully rolled around and attached to insert by using Double side adhesive tape . Inserts were CNC machined from polycarbonate . The insert equipped with a separation membrane and o-ring 1.9 × 5.7 mm was inserted to the top dish central hole (9 mm ID). Dialysis membrane  was attached by double side adhesive (M3) acrylic at the bottom of the top dish with insert.ETP separation condition: Separation—8.3 ml of 100 mM LE in the bottom dish and 200 µl of 100 mM LE inside the central well. Sample: 0.9 ml of 20 mM TE buffer, Brilliant blue (10 µl 0.1 mg/ml in water) and 320 µl of lysate. Constant power 0.5 W was applied for 15 min.Control condition/commercial kit for long DNA extraction: Wizard HMW DNA Extraction kit was used to extract long DNA from chosen cell line  with three extraction replicas for this condition. Protocol suggested by the manufacturer was followed with RNase treatment omitted. Wide orifice pipette tips (Rainin) were used to reduce the DNA shearing.Gel-pulse field analysis: The Pulse field gel electrophoresis was run using the Pulse Pippin instrument (Sage Science). We used a 0.75% agarose gel  in a 0.5X KBB buffer. Lambda PFG Ladder was used as a standard (New England Biolabs). The power supply was set for 5–430 Kbp Waveform protocol . After electrophoresis, the gel was stained for 30 min in SYBR safe gel stain solution in 0.5X solution (Invitrogen). The pictures of the gel were imaged using UV light (Protein Simple).Supplementary Information.