02309nas a2200217 4500008004100000022001400041245011600055210006900171260007100240300001400311490000700325520147400332100002501806700002201831700002001853700001901873700003001892700002001922700001801942856013101960 2009 eng d a0743-746300aDendritic effect of ligand-coated nanoparticles: enhanced apoptotic activity of silica-berberine nanoconjugates0 aDendritic effect of ligandcoated nanoparticles enhanced apoptoti a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcFEB a2339-23470 v253 a

We describe the synthesis and biological characterization of a novel prototype, namely, silica nanoconjugates bearing a covalently linked berberine, a plant alkaloid known to have antiproliferative activity. The effect of synthesized nanoconjugates on cell proliferation, the cell cycle profile, and apoptosis in the human cervical carcinoma cell line (HeLa), human hepatocellular liver carcinoma cell line (HepG2), and human embryonic kidney (HEK) 293T cell line has been studied and compared with the results obtained for free berberine. Our results show that all the nanoconjugates display higher antiproliferative activity than free berberine. The ability of these nanoconjugates to inhibit cellular proliferation is mediated by the cell cycle arrest at the G1 phase. Moreover, silica nanoconugates caused selective apoptotic arrest with a higher efficiency than free berberine followed by apoptotic cell death as shown by quantitative fluorescence-activated cell sorting analyses. Efficiency of the nanoconjugates increases upon an increase in the linker chain length, demonstrating the distinct role of the spacer chain that conjugates nanoparticles and ligands. The actual reason to show enhanced efficiency by the nanoconjugates has not been elucidated in the present study; however, we hypothesize that an increase in local concentration due to the confinement of a ligand on the nanosurface (''dendritic'' effect) might have led to the observed effect.

1 aHalimani, Mahantappa1 aChandran, Prathap1 aKashyap, Sudhir1 aJadhav, V., M.1 aPrasad, Bhagavatula L. V.1 aHotha, Srinivas1 aMaiti, Souvik uhttps://library.ncl.res.in/content/dendritic-effect-ligand-coated-nanoparticles-enhanced-apoptotic-activity-silica-berberine-002740nas a2200217 4500008004100000022001400041245015100055210006900206260007100275300001400346490000800360520189600368100001802264700001602282700001402298700001602312700002202328700002602350700001802376856012802394 2009 eng d a1520-610600aEffects for the incorporation of five-atom thioacetamido nucleic acid (TANA) backbone on hybridization thermodynamics and kinetics of DNA duplexes0 aEffects for the incorporation of fiveatom thioacetamido nucleic  a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcMAR a2944-29510 v1133 a

Thermodynamic studies of nucleic acids serve not only to widen our understanding on the nature and strength of forces that stabilize nucleic acids in a myriad of structural states they assume but also to facilitate the development of databases that could be used to predict the stability and selectivity of probe/primer-sets required in a broad range of nucleic acid-based diagnostic and therapeutic protocols. In the current study, we investigated the effect of a novel, backbone-modified ``thioacetamido linkage'' (TANA) on thermodynamics of hybridization, binding kinetics, and conformation of a DNA duplex. The modification comprises of an extended five-atom amide (N3'-CO-CH(2)-S-CH(2)) linker, as opposed to four-atom phosphodiester linker backbone present in DNA. One to three TANA-substitutions have been introduced in the linker backbone of two thymidine residues of one of the strand of the DNA duplex. Using spectroscopic and calorimetric techniques, we observed that TANA destabilizes the DNA helix by lowering the favorable enthalpy parameter of duplex formation. TANA center dot DNA duplexes were found to adopt a conformation toward an A-type duplex as shown by circular dichroism spectroscopy studies. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, Delta C(p), accompanying the duplex formation. The average Delta C(p) change per duplex was found to be 832.5 cal mol(-1) K(-1), giving an average base-pair change of 59.5 cal (mol of base pairs)(-1) K(-1). Hybridization kinetic measurements using surface plasmon resonance indicated a decrease in binding affinity parameter (K(A)) that originates from higher dissociation rate constants (k(d)). Furthermore, optical melting studies showed that increasing the number of modifications results in a modest change in the number of counterions taken up during duplex formation.

1 aKaur, Harleen1 aArora, Amit1 aGogoi, K.1 aSolanke, P.1 aGunjal, Anita, D.1 aKumar, Vaijayanti, A.1 aMaiti, Souvik uhttps://library.ncl.res.in/content/effects-incorporation-five-atom-thioacetamido-nucleic-acid-tana-backbone-hybridization-002326nas a2200181 4500008004100000022001400041245014600055210006900201260007100270300001400341490000700355520156100362100001801923700003301941700001801974700002301992856012902015 2011 eng d a0006-296000aSingle nucleotide polymorphism in transcobalamin II (15V) induces structural changes in the protein as revealed by molecular modeling studies0 aSingle nucleotide polymorphism in transcobalamin II 15V induces  a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcMAR a1396-14020 v503 a

Cobalamin is an essential micronutrient in mammals. Deficiencies of this micronutrient have been implicated as risk factors for various complex diseases. Cobalamin is transported to the cells by the transport protein transcobalamin II (TCII), and hence genetic variations (like single nucleotide polymorphisms) in TCII could be perceived to affect the binding of cobalamin to TCII, thereby modulating the intracellular concentrations of cobalamin. To understand whether three nonsynonymous mutations in TCII (I5V, P241R, and R381Q) alter the structure of the protein which could potentially affect cobalamin binding, we performed molecular dynamics simulation in silico. Superimposition of active sites of the four simulated models (wild type and three variants) with the human TCII crystal structure revealed that the distance between the N epsilon nitrogen atom of His-173 and the cobalt ion of cobalamin deviated considerably in the I5V model as compared to wild type and other variants. His-173 directly coordinates with the cobalt ion of cobalamin. Further, from our dynamic cross-correlation and principal component analysis it appears that in the I5V model the beta-domain moves apart from the alpha-domain creating a wide gap between the two domains. This might facilitate the initial binding of cobalamin in the I5V model as cobalamin enters the binding site through the gap between the two domains. These observations were not found in the other variants. We thus speculate that binding of cobalamin will be more facile in the I5V variant.

1 aSilla, Yumnam1 aChandamouli, Balasubramanian1 aMaiti, Souvik1 aSengupta, Shantanu uhttps://library.ncl.res.in/content/single-nucleotide-polymorphism-transcobalamin-ii-15v-induces-structural-changes-protein-001689nas a2200277 4500008004100000022001400041245010500055210006900160260007700229300001200306490000700318520072700325653001201052653002501064653001301089653001501102653001401117100002401131700002701155700002301182700001601205700001801221700002301239700001801262856013101280 2012 eng d a1439-422700aAntagonism of microRNA function in zebrafish embryos by using locked nucleic acid enzymes (LNAzymes)0 aAntagonism of microRNA function in zebrafish embryos by using lo aPO BOX 10 11 61, D-69451 WEINHEIM, GERMANYbWILEY-V C H VERLAG GMBHcMAR a584-5890 v133 a

MicroRNAs (miRNAs) have crucial functions in many cellular processes, such as differentiation, proliferation and apoptosis; aberrant expression of miRNAs has been linked to human diseases, including cancer. Tools that allow specific and efficient knockdown of miRNAs would be of immense importance for exploring miRNA function. Zebrafish serves as an excellent vertebrate model system to understand the functions of miRNAs involved in a variety of biological processes. We designed and employed a strategy based on locked nucleic acid enzymes (LNAzymes) for in vivo knockdown of miRNA in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos.

10aenzymes10alocked nucleic acids10amicroRNA10amorpholino10azebrafish1 aSuryawanshi, Hemant1 aLalwani, Mukesh, Kumar1 aRamasamy, Soundhar1 aRana, Rajiv1 aScaria, Vinod1 aSivasubbu, Sridhar1 aMaiti, Souvik uhttps://library.ncl.res.in/content/antagonism-microrna-function-zebrafish-embryos-using-locked-nucleic-acid-enzymes-lnazymes-002140nas a2200217 4500008004100000022001400041245011900055210006900174260008700243300001200330490000700342520129800349100001801647700002301665700001701688700002401705700002201729700002301751700001801774856013001792 2012 eng d a2159-333700aImproved inhibition of telomerase by short twisted intercalating nucleic acids under molecular crowding conditions0 aImproved inhibition of telomerase by short twisted intercalating a140 HUGUENOT STREET, 3RD FL, NEW ROCHELLE, NY 10801 USAbMARY ANN LIEBERT INCcDEC a399-4040 v223 a

Human telomeric DNA has the ability to fold into a 4-stranded G-quadruplex structure. Several G-quadruplex ligands are known to stabilize the structure and thereby inhibit telomerase activity. Such ligands have demonstrated efficient telomerase inhibition in dilute conditions, but under molecular crowding conditions mimicking physiological milieu, stabilization of the telomeric G-quadruplex is often lost. We attempted to demonstrate the enhanced G-quadruplex stabilizing ability under molecular conditions by using twisted intercalating nucleic acids (TINA)-modified oligonucleotides. We have shown using circular dichroism and ultraviolet spectroscopic methods that these TINA-modified short oligonucleotides function as G-quadruplex, inducing agents and participate in the formation of stabilized 3:1 G-quadruplex with the human telomeric oligonucleotide. Using enzyme-linked immunosorbent assay-based telomerase repeat amplification assay (TRAP) assay as well as nondenaturing polyacrylamide gel electrophoresis-based TRAP, we demonstrate remarkable enhancement in their anti-telomerase activity even under molecular crowding conditions. This is the first time in which a G-quadruplex stabilizing agent has demonstrated enhanced activity even under molecular crowding conditions.

1 aAgarwal, Tani1 aPradhan, Devranjan1 aGeci, Imrich1 aEl-Madani, Amro, M.1 aPetersen, Michael1 aPedersen, Erik, B.1 aMaiti, Souvik uhttps://library.ncl.res.in/content/improved-inhibition-telomerase-short-twisted-intercalating-nucleic-acids-under-molecular-001970nas a2200217 4500008004100000022001400041245006600055210006500121260007100186300001400257490000800271520123400279100002101513700001901534700001801553700002501571700002201596700001901618700001801637856009701655 2012 eng d a1520-610600aKinetic studies of amino acid-based surfactant binding to DNA0 aKinetic studies of amino acidbased surfactant binding to DNA a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcMAY a5831-58370 v1163 a

In this work, the binding kinetics of amino acid-based surfactants, presenting different linkers and head groups, with calf thymus (CT)-DNA was studied using stopped-flow fluorescence spectroscopy. The kinetic studies were carried out as a function of Na+ concentration and surfactant-to-DNA charge ratio. The surfactant binding on DNA took place in two consecutive steps, for which the corresponding first and second relative rate constants (k(1) and k(2)) were determined. The fast step was attributed to the surfactant binding to DNA and micelle formation in its vicinity, the slower step to DNA condensation and possible rearrangement of the surfactant aggregates. In general, both relative rate constants increase with surfactant concentration and decrease with the ionic strength of the medium. The architecture of the surfactant was found to have a significant impact on the kinetics of the DNA surfactant complexation. Surfactants with amide linkers showed larger relative rate constants than those with ester linkers. The variation of the relative rate constants with the head groups of the surfactants, alanine and proline, was found to be less obvious, being partially dependent on the surfactant concentration.

1 aSanthiya, Deenan1 aDias, Rita, S.1 aDutta, Sounak1 aDas, Prasanta, Kumar1 aMiguel, Maria, G.1 aLindman, Bjorn1 aMaiti, Souvik uhttps://library.ncl.res.in/content/kinetic-studies-amino-acid-based-surfactant-binding-dna-000987nas a2200265 4500008004100000022001400041245013600055210006900191260007600260300001400336490000700350653001400357653001900371653002000390653001500410653000800425100001800433700003100451700002400482700002100506700002500527700002300552700001800575856012800593 2012 eng d a1433-785100aTuberculosis drug streptomycin as a potential cancer therapeutic: inhibition of miR-21 function by directly targeting its precursor0 aTuberculosis drug streptomycin as a potential cancer therapeutic aCOMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USAbWILEY-BLACKWELLcDEC a1019-10230 v5110aApoptosis10adrug discovery10aGene expression10ainhibitors10aRNA1 aBose, Debojit1 aJayaraj, Gopal, Gunanathan1 aSuryawanshi, Hemant1 aAgarwala, Prachi1 aPore, Subrata, Kumar1 aBanerjee, Rajkumar1 aMaiti, Souvik uhttps://library.ncl.res.in/content/tuberculosis-drug-streptomycin-potential-cancer-therapeutic-inhibition-mir-21-function-002646nas a2200217 4500008004100000022001400041245010800055210006900163260010300232300001400335490000700349520178900356653001002145653003602155653002402191653002802215653001902243100001902262700001802281856012902299 2013 eng d a0300-908400aEffect of different arginine methylations on the thermodynamics of tat peptide binding to HIV-1 TAR RNA0 aEffect of different arginine methylations on the thermodynamics  a23 RUE LINOIS, 75724 PARIS, FRANCEbELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIERcJUL a1422-14310 v953 a

RNA-binding proteins are an important class of mediators that regulate cell function and differentiation. Methylation of arginine, a post-translational modification (PTM) found in these proteins, can modulate their function. Arginine can be monomethylated or dimethylated, depending on the type of methyl transferases involved. This paper describes a comparative study of the thermodynamics of unmodified and modified Tat peptide interaction with TAR RNA, where the peptide is methylated at epsilon (epsilon) and eta (eta) nitrogen atoms of guanidinium group of arginine side chain at position 52 or 53. The results indicate that monomethylation of arginine at epsilon (epsilon) nitrogen atom enhances binding affinity, owing to a more favourable enthalpy component which overrides the less favourable entropy change. In contrast, monomethylation of arginine residue at eta nitrogen results in reduced binding affinity originating exclusively from a less favourable enthalpy change leaving entropic component unaffected. However, in case of simultaneous methylation at epsilon and eta positions, the binding parameters remain almost unaffected, when compared to the unmodified peptide. In case of symmetric dimethylation at eta position the observed enthalpy change of the binding was found to be smaller than the values obtained for the unmodified peptide. Asymmetric dimethylation at eta position showed the most reduced binding affinities owing to less favourable enthalpy changes. These results provide insights that enable elucidation of the biological outcome of arginine methylation as PTMs that regulate protein function, and will contribute to our understanding of how these PTMs are established in vitro and in vivo. (C) 2013 Elsevier Masson SAS. All rights reserved.

10aHIV-110aPost-translational modification10aProtein methylation10aRNA-protein interaction10aThermodynamics1 aKumar, Santosh1 aMaiti, Souvik uhttps://library.ncl.res.in/content/effect-different-arginine-methylations-thermodynamics-tat-peptide-binding-hiv-1-tar-rna-001862nas a2200169 4500008004100000022001400041245007400055210006900129260007100198300001400269490000800283520123900291100002501530700002101555700001801576856009801594 2013 eng d a1520-610600aEffect of loops and G-quartets on the stability of RNA G-quadruplexes0 aEffect of loops and Gquartets on the stability of RNA Gquadruple a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcJUN a6896-69050 v1173 a

The loop length, loop composition, salt concentration, and number of G-quartets are major determinants of G-quadruplex stability. We examined the effect of each of these factors on the thermal stability and folding topology of a library of RNA quadruplexes. The thermal stability of G2 and G3 RNA quadruplexes was investigated upon varying the loop length (from 1-1-1 to 15-15-15) and salt concentration (from sequence 1 to 100 mM KCl), while the effect of loop composition was explored using 18 naturally occurring potential RNA quadruplexes predicted in untranslated regions (UTRs). We found loop length and quadruplex stability to be inversely related for G2 RNA quadruplexes and G3 RNA quadruplexes with shorter loops. However, melting temperature saturates for G3 RNA quadruplexes with longer loops. RNA G-quadruplexes with longer loops (G3 15-15-15) displayed T-m values significantly higher than the physiological temperature. This study thus highlights the need to modify the consensus motif presently used by quadruplex prediction tools. An increase in the loop size from 7 bases to IS bases in the consensus motif will add to its predictive value for the discovery of potential RNA quadruplexes across transcriptomes.

1 aPandey, Satyaprakash1 aAgarwala, Prachi1 aMaiti, Souvik uhttps://library.ncl.res.in/content/effect-loops-and-g-quartets-stability-rna-g-quadruplexes-002428nas a2200157 4500008004100000022001400041245011700055210006900172260009100241300001600332490000600348520175000354100001902104700001802123856012902141 2013 eng d a1932-620300aEffect of N-acetylation and N-methylation of lysine residue of tat peptide on its interaction with HIV-1 TAR RNA0 aEffect of Nacetylation and Nmethylation of lysine residue of tat a1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USAbPUBLIC LIBRARY SCIENCEcOCT aUNSP e775950 v83 a

Post-translational modification (PTM) of RNA binding proteins (RBPs) play a very important role in determining their binding to cognate RNAs and therefore regulate the downstream effects. Lysine can undergo various PTMs and thereby contribute to the regulation of different cellular processes. It can be reversibly acetylated and methylated using a pool of respective enzymes, to act as a switch for controlling the binding efficiency of RBPs. Here we have delineated the thermodynamic and kinetic effects of N-acetylation and N-monomethylation of lysine on interaction between HIV-1 TAR RNA and its cognate binder Tat peptide (a model system). Our results indicate that acetylation of lysine 50 (K50), leads to eight-fold reduction in binding affinity, originating exclusively from entropy changes whereas, lysine 51 (K51) acetylation resulted only in three fold decrease with large enthalpy-entropy compensation. The measurement of kinetic parameters indicated major change (4.5 fold) in dissociation rate in case of K50 acetylation however, K51 acetylation showed similar effect on both association and dissociation rates. In contrast, lysine methylation did not affect the binding affinity of Tat peptide to TAR RNA at K50, nonetheless three fold enhancement in binding affinity was observed at K51 position. In spite of large enthalpy-entropy compensation, lysine methylation seems to have more pronounced position specific effect on the kinetic parameters. In case of K50 methylation, simultaneous increase was observed in the rate of association and dissociation leaving binding affinity unaffected. The increased binding affinity for methylated Tat at K51 stems from faster association rate with slightly slower dissociation rate.

1 aKumar, Santosh1 aMaiti, Souvik uhttps://library.ncl.res.in/content/effect-n-acetylation-and-n-methylation-lysine-residue-tat-peptide-its-interaction-hiv-1-002321nas a2200181 4500008004100000022001400041245010600055210006900161260007100230300001400301490000700315520160500322100002101927700002501948700001701973700001801990856013102008 2013 eng d a0006-296000aG-quadruplex augments translation in the 5 ` untranslated region of transforming growth factor beta 20 aGquadruplex augments translation in the 5 untranslated region of a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcMAR a1528-15380 v523 a

Transforming growth factor beta 2 (TGF beta 2) is a versatile cytokine with a prominent role in cell migration, invasion, cellular development, and immunomodulation. TGF beta 2 promotes the malignancy of tumors by inducing epithelial-mesenchymal transition, angiogenesis, and immunosuppression. As it is well-documented that nucleic acid secondary structure can regulate gene expression, we assessed whether any secondary motif regulates its expression at the post-transcriptional level. Bioinformatics analysis predicts an existence of a 23-nucleotide putative G-quadruplex sequence (PG4) in the 5' untranslated region (UTR) of TGF beta 2 mRNA. The ability of this stretch of sequence to form a highly stable, intramolecular parallel quadruplex was demonstrated using ultraviolet and circular dichroism spectroscopy. Footprinting studies further validated its existence in the presence of a neighboring nucleotide sequence. Following structural characterization, we evaluated the biological relevance of this secondary motif using a dual luciferase assay. Although PG4 inhibits the expression of the reporter gene, its presence in the context of the entire 5' UTR sequence interestingly enhances gene expression. Mutation or removal of the G-quadruplex sequence from the 5' UTR of the gene diminished the level of expression of this gene at the translational level. Thus, here we highlight an activating role of the G-quadruplex in modulating gene expression of TGF beta 2 at the translational level and its potential to be used as a target for the development of therapeutics against cancer.

1 aAgarwala, Prachi1 aPandey, Satyaprakash1 aMapa, Koyeli1 aMaiti, Souvik uhttps://library.ncl.res.in/content/g-quadruplex-augments-translation-5-untranslated-region-transforming-growth-factor-beta-2-001528nas a2200229 4500008004100000022001400041245005000055210004900105260007700154300001400231490000700245520082100252653001901073653001401092653002201106653001301128653001301141100002101154700002501175700001801200856008001218 2013 eng d a1439-422700aG-Quadruplexes as tools for synthetic biology0 aGQuadruplexes as tools for synthetic biology aBOSCHSTRASSE 12, D-69469 WEINHEIM, GERMANYbWILEY-V C H VERLAG GMBHcNOV a2077-20810 v143 a

With the potential to engineer biological systems, synthetic biology is an emerging field that combines various disciplines of sciences. It encompasses combinations of DNA, RNA and protein modules for constructing desired systems and the rewiring of existing signalling networks. Despite recent advances, this field still lags behind in the artificial reconstruction of cellular processes, and thus demands new modules and switches to create genetic circuits. The widely characterised noncanonical nucleic acid secondary structures, G-quadruplexes are promising candidates to be used as biological modules in synthetic biology. Structural plasticity and functional versatility are significant G-quadruplex traits for its integration into a biological system and for diverse applications in synthetic circuits.

10aG-quadruplexes10aribozymes10asynthetic biology10atopology10azipcodes1 aAgarwala, Prachi1 aPandey, Satyaprakash1 aMaiti, Souvik uhttps://library.ncl.res.in/content/g-quadruplexes-tools-synthetic-biology-001905nas a2200181 4500008004100000022001400041245008400055210006900139260007100208300001200279490000600291520122600297100001801523700003101541700001901572700001801591856011401609 2013 eng d a1554-892900aMolecular-beacon-based screen for small molecule inhibitors of miRNA maturation0 aMolecularbeaconbased screen for small molecule inhibitors of miR a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcMAY a930-9380 v83 a

miRNAs are small non-coding RNAs that regulate about 60% of mammalian genes by modulating their transcript levels. Network scale studies of miRNA-mediated regulatory circuits demonstrate the central importance of this class of small RNA in the maintenance of biological robustness. More recently, several reports have described the deregulation of numerous miRNA to be causally associated with many diseases, including cancer. These studies have highlighted the potential for development of therapeutic modalities against miRNA. Previous screening protocols, for small molecules targeting miRNA function, are either costly or technically too complex to be applied in a high-throughput manner in standard chemical laboratories. We describe a simple in vitro screening method using a DNA-based molecular beacon that overcomes the limitations associated with earlier screens. We used this method to identify inhibitors of miR-27a function from a library of 14 aminoglycosides as a pilot study. Inhibitory molecules identified were further scrutinized to identify the validity of screen. With this proof of concept we illustrate the utility of a scalable molecular-beacon-based screening strategy for miRNA inhibitors.

1 aBose, Debojit1 aJayaraj, Gopal, Gunanathan1 aKumar, Santosh1 aMaiti, Souvik uhttps://library.ncl.res.in/content/molecular-beacon-based-screen-small-molecule-inhibitors-mirna-maturation-002079nas a2200169 4500008004100000022001400041245008100055210006900136260011000205300001000315490000600325520141000331100001601741700001901757700001801776856011501794 2013 eng d a1742-206X00aThermodynamics and solvation dynamics of BIV TAR RNA-Tat peptide interaction0 aThermodynamics and solvation dynamics of BIV TAR RNATat peptide  aTHOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLANDbROYAL SOC CHEMISTRYcOCT a88-980 v93 a

The interaction of the trans-activation responsive (TAR) region of bovine immunodeficiency virus (BIV) RNA with the Tat peptide is known to play important role in viral replication. Despite being thoroughly studied through a structural point of view, the nature of binding between BIV TAR RNA and the BIV Tat peptide requires information related to its thermodynamics and the nature of hydration around the TAR-Tat complex. In this context, we carried out the thermodynamic study of binding of the Tat peptide to the BIV TAR RNA hairpin through different calorimetric and spectroscopic measurements. Fluorescence titration of 2-aminopurine tagged BIV TAR RNA with the Tat peptide gives their binding affinity. The isothermal titration calorimetric experiment reveals the enthalpy of binding between BIV TAR RNA and the Tat peptide to be largely exothermic with the value of -11.7 (SEM 0.2) kcal mol(-1). Solvation dynamics measurements of BIV TAR RNA having 2-AP located at the bulge region have been carried out in the absence and presence of the BIV Tat peptide using the time correlated single photon counting technique. The solvent cage around the Tat binding site of RNA appears to be more rigid in the presence of the Tat peptide as compared to the free RNA. The displacement of solvent and ions on RNA due to peptide binding influences the entropic contributions to the total binding energy.

1 aGoel, Teena1 aKumar, Santosh1 aMaiti, Souvik uhttps://library.ncl.res.in/content/thermodynamics-and-solvation-dynamics-biv-tar-rna-tat-peptide-interaction-001335nas a2200193 4500008004100000022001400041245010900055210006900164260011000233300001400343490000700357520055400364100001700918700001800935700002400953700001700977700001800994856012901012 2014 eng d a1359-734500aAnti-cancer therapeutic potential of quinazoline based small molecules via global upregulation of miRNAs0 aAnticancer therapeutic potential of quinazoline based small mole aTHOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLANDbROYAL SOC CHEMISTRYcMAR a4639-46420 v503 a

Three quinazoline based small molecules showed global upregulation of miRNA expression with a selective enrichment of tumor suppressor miRNAs. The target genes of the upregulated miRNAs were predicted to be enriched for apoptotic pathways. Apoptotic induction following treatment with quinazoline compounds was confirmed by in cellulo studies. Thus, these small molecules having the core structural moiety (2,4-diphenyl-quinazoline) can be used as scaffolds to design activators of miRNA expression paving the way for novel anti-cancer drugs.

1 aNahar, Smita1 aBose, Debojit1 aPanja, Sumit, Kumar1 aSaha, Satyen1 aMaiti, Souvik uhttps://library.ncl.res.in/content/anti-cancer-therapeutic-potential-quinazoline-based-small-molecules-global-upregulation-002229nas a2200193 4500008004100000022001400041245011400055210006900169260007100238300001400309490000700323520147700330100001801807700001601825700001901841700003101860700001801891856012601909 2014 eng d a0006-296000aIn the sense of transcription regulation by G-quadruplexes: asymmetric effects in sense and antisense strands0 aIn the sense of transcription regulation by Gquadruplexes asymme a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcJUN a3711-37180 v533 a

G-Quadruplexes occupy important regulatory regions in the genome. DNA G-quadruplexes in the promoter regions and RNA quadruplexes in the UTRs (untranslated regions) have been individually studied and variously implicated at different regulatory levels of gene expression. However, the formation of G-quadruplexes in the sense and antisense strands and their corresponding roles in gene regulation have not been studied in much detail. In the present study, we have elucidated the effect of strand asymmetry in this context. Using biophysical methods, we have demonstrated the formation of stable G-quadruplex structure in vitro using CD and UV melting. Additionally, ITC was employed to demonstrate that a previously reported selective G-quadruplex ligand was able to bind and stabilize the G-quadruplex in the present sequence. Further, we have shown using reporter constructs that although the DNA G-quadruplex in either strand can reduce translation efficiency, transcriptional regulation differs when G-quadruplex is present in the sense or antisense strand. We demonstrate that the G-quadruplex motif in the antisense strand substantially inhibits transcription, while when in the sense strand, it does not affect transcription, although it does ultimately reduce translation. Further, it is also shown that the G-quadruplex stabilizing ligand can enhance this asymmetric transcription regulation as a result of the increased stabilization of the G-quadruplex.

1 aAgarwal, Tani1 aRoy, Saumya1 aKumar, Santosh1 aChakraborty, Tushar, Kanti1 aMaiti, Souvik uhttps://library.ncl.res.in/content/sense-transcription-regulation-g-quadruplexes-asymmetric-effects-sense-and-antisense-002196nas a2200217 4500008004100000022001400041245009200055210006900147260007100216300001400287490000700301520139700308100001801705700002701723700001901750700001601769700003101785700002301816700001801839856012101857 2014 eng d a0006-296000aMorphological effects of G-quadruplex stabilization using a small molecule in zebrafish0 aMorphological effects of Gquadruplex stabilization using a small a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcFEB a1117-11240 v533 a

Zebrafish (Danio rerio) embryos are transparent and advantageous for studying early developmental changes due to ex utero development, making them an appropriate model for studying gene expression changes as a result of molecular targeting. Zebrafish embryos were injected with a previously reported G-quadruplex selective ligand, and the phenotypic changes were recorded. We report marked discrepancies in the development of intersegmental vessels. In silico analysis determined that the putative G-quadruplex motif occur in the upstream promoter region of the Cdh5 (N-cadherin) gene. A real-time polymerase chain reaction-based investigation indicated that in zebrafish, CDH-2 (ZN-cad) was significantly downregulated in the ligand-treated embryos. Biophysical characterization of the interaction of the ligand with the G-quadruplex motif found in this promoter yielded strong binding and stabilization of the G-quadruplex with this ligand. Hence, we report for the first time the phenotypic impact of G-quadruplex targeting with a ligand in a vertebrate organism. This study has unveiled not only G-quadruplex targeting in non-human animal species but also the potential that G-quadruplexes can provide a ready tool for understanding the phenotypic effects of targeting certain important genes involved in differentiation and developmental processes in a living eukaryotic organism.

1 aAgarwal, Tani1 aLalwani, Mukesh, Kumar1 aKumar, Santosh1 aRoy, Saumya1 aChakraborty, Tushar, Kanti1 aSivasubbu, Sridhar1 aMaiti, Souvik uhttps://library.ncl.res.in/content/morphological-effects-g-quadruplex-stabilization-using-small-molecule-zebrafish-002537nas a2200241 4500008004100000022001400041245005100055210005000106260007300156300001400229490000900243520183400252653001002086653001202096653000902108653001002117653001702127653000802144100002102152700002502173700001802198856007902216 2014 eng d a0304-416500aRole of G-quadruplex located at 5 end of mRNAs0 aRole of Gquadruplex located at 5 end of mRNAs aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcDEC a3503-35100 v18403 a

Background: Secondary structures in 5' UTR of mRNAs play a critical role in regulating protein synthesis. Though studies have indicated the role of secondary structure G-quadruplex in translational regulation, position-specific effect of G-quadruplex in naturally occurring mRNAs is still not understood. As a pre-initiation complex recognises 5' cap of the mRNA and scans along the untranslated region (UTR) before initiating translation, the presence of G-quadruplex in 5' region may have a significant contribution in regulating translation. Here, we investigate the role of G-quadruplex located at the 5' end of an mRNA. Methods: Biophysical characterisation of putative G-quadruplexes was performed using UV and CD spectroscopy. Functional implication of G-quadruplex in the context of their location was assessed in cellulo using gRT-PCR and dual luciferase assay system. Results: PG4 sequences in 5' UTR of AICT interacting protein (AKTIP), cathepsin B (CTSB) and forkhead box E3 (FOXE3) mRNAs form G-quadruplex whereas it is unable to form G-quadruplex in apolipoprotein A-I binding protein (APOA1BP). Our results demonstrated diverse roles of G-quadruplex located at 5' end of mRNAs. Though G-quadruplex in AKTIP and CTSB mRNA act as inhibitory modules, it activates translation in FOXE3 mRNA. Conclusions: Our works suggests that G-quadruplex present at the 5' terminal of an mRNA behaves differently in a different gene context. It can activate or inhibit gene expression. General significance: This study demonstrated that it is difficult to predict the role of G-quadruplex on the basis of its position in 5' UTR. The neighbouring nucleotide sequence, the intracellular milieu and the interacting partners might render diverse functions to this secondary structure. (C) 2014 Elsevier B.V. All rights reserved.

10aAKTIP10aAPOA1BP10aCTSB10aFOXE310aG-quadruplex10aUTR1 aAgarwala, Prachi1 aPandey, Satyaprakash1 aMaiti, Souvik uhttps://library.ncl.res.in/content/role-g-quadruplex-located-5-end-mrnas-002312nas a2200169 4500008004100000022001400041245018500055210006900240260007100309300001400380490000700394520156300401100001901964700001701983700001802000856012402018 2014 eng d a0006-296000aUnderstanding the effect of locked nucleic acid and 2 `-O-methyl modification on the hybridization thermodynamics of a miRNA-mRNA pair in the presence and absence of AfPiwi protein0 aUnderstanding the effect of locked nucleic acid and 2 Omethyl mo a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcMAR a1607-16150 v533 a

miRNAs are some of the key epigenetic regulators of gene expression. They act through hybridization with their target mRNA and modulate the level of respective proteins via different mechanisms. Various cancer conditions are known to be associated with up- and downregulation of the oncogenic and tumor suppressor miRNAs, respectively. The levels of aberrantly expressed oncogenic miRNAs can be downregulated in different ways. Similarly, restoration of tumor suppressor miRNAs to their normal levels can be achieved using miRNA mimics. However, the use of miRNA mimics is limited by their reduced biostability and function. We have studied the hybridization thermodynamics of the miRNA 26a (11-mer, including the seed sequence) guide strand with the mRNA (11-mer) target strand in the absence and presence of AfPiwi protein. We have also inserted locked nucleic acids (LNAs) and 2'-O-methyl-modified nucleotides into the guide strand, in a walk-through manner, to assess their effect on the binding efficiency between guide and target RNA. Insertion of LNA and 2'-O-methyl-modified nucleotides into the guide strand helped to strengthen the binding affinity irrespective of the position of insertion. However, in the presence of AfPiwi protein, these modifications reduced the binding affinity to different extents depending on the position of insertion. Insertion of a modification leads to an increase in the enthalpic contribution with an increased unfavorable entropic contribution, which negatively compensates for the higher favorable enthalpy.

1 aKumar, Santosh1 aMapa, Koyeli1 aMaiti, Souvik uhttps://library.ncl.res.in/content/understanding-effect-locked-nucleic-acid-and-2-o-methyl-modification-hybridization-001217nas a2200193 4500008004100000022001400041245013800055210006900193260011000262300001400372490000700386520040200393100001700795700002000812700001700832700001800849700002600867856013000893 2015 eng d a1359-734500aBeta, gamma-Bis-substituted PNA with configurational and conformational switch: preferred binding to cDNA/RNA and cell-uptake studies0 aBeta gammaBissubstituted PNA with configurational and conformati aTHOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLANDbROYAL SOC CHEMISTRYcMAR a7693-76960 v513 a

(S,S)- and (R,R)-beta,gamma-Bis-substituted PNAs were synthesized from the C-2 symmetric vicinal diamine system embedded in 1,4 dihydroxybutane and 1,4-dimethoxybutane scaffolds. (R,R)-beta,gamma-Bis-methoxymethyl-PNA derived from D-tartaric acid was found to be in the right configuration and conformation to be an excellent mimic of PNA, endowed with superior ability to enter into cells.

1 aBose, Tanaya1 aBanerjee, Anjan1 aNahar, Smita1 aMaiti, Souvik1 aKumar, Vaijayanti, A. uhttps://library.ncl.res.in/content/beta-gamma-bis-substituted-pna-configurational-and-conformational-switch-preferred-binding01959nas a2200193 4500008004100000022001400041245014100055210006900196260008800265300001200353490000700365520116900372100001701541700001801558700001501576700003101591700001801622856012501640 2015 eng d a2159-333700aCyclic cationic peptides containing sugar amino acids selectively distinguishes and inhibits maturation of pre-mirnas of the same family0 aCyclic cationic peptides containing sugar amino acids selectivel a140 HUGUENOT STREET, 3RD FL, NEW ROCHELLE, NY 10801 USAbMARY ANN LIEBERT, INCcDEC a323-3290 v253 a

The discovery of microRNAs (miRNAs) has added a new dimension to the gene regulatory networks, making aberrantly expressed miRNAs as therapeutically important targets. Small molecules that can selectively target and modulate miRNA levels can thus serve as lead structures. Cationic cyclic peptides containing sugar amino acids represent a new class of small molecules that can target miRNA selectively. Upon treatment of these small molecules in breast cancer cell line, we profiled 96 therapeutically important miRNAs associated with cancer and observed that these peptides can selectively target paralogous miRNAs of the same seed family. This selective inhibition is of prime significance in cases when miRNAs of the same family have tissue-specific expression and perform different functions. During these conditions, targeting an entire miRNA family could lead to undesired adverse effects. The selective targeting is attributable to the difference in the three-dimensional structures of precursor miRNAs. Hence, the core structure of these peptides can be used as a scaffold for designing more potent inhibitors of miRNA maturation and hence function.

1 aNahar, Smita1 aBose, Debojit1 aPal, Sudip1 aChakraborty, Tushar, Kanti1 aMaiti, Souvik uhttps://library.ncl.res.in/content/cyclic-cationic-peptides-containing-sugar-amino-acids-selectively-distinguishes-and-001999nas a2200181 4500008004100000022001400041245008200055210006900137260007100206300001400277490000800291520132600299100002101625700001901646700002501665700001801690856010901708 2015 eng d a1520-610600aHuman telomeric RNA G-quadruplex response to point mutation in the G-quartets0 aHuman telomeric RNA Gquadruplex response to point mutation in th a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcAPR a4617-46270 v1193 a

Many putative G-quadruplex forming sequences have been predicted to exist in the human genome and transcriptome. As these sequences are subject to point mutations or SNPs (single nucleotide polymorphisms) during the course of evolution, we attempt to understand impact of these mutations in context of RNA G-quadruplex formation using human telomeric RNA (TERRA) as a model sequence. Our studies suggest that G-quadruplex stability is sensitive to substitution of the guanines comprising G-quartets. While central G-quartet plays a crucial role in maintaining the DNA G-quadruplex stability as evident in literature, there is equal importance of three G-quartets in the stability of RNA quadruplex structure. The work here highlights the alterations in the G-quartet are detrimental to the integrity of overall RNA G-quadruplex structure. Furthermore, TmPyP4 molecules are shown to exhibit similar binding behavior toward telomeric RNA G-quadruplex harboring base substitutions employing CD titrations and isothermal titration calorimetry; well indicating that mutation does not influence TmPyP4 recognition ability as it affects the stability of RNA G-quadruplex. Thus, our study implicates that mutation in G-quartets causes destabilization of RNA G-quadruplex without affecting its trans factor binding ability.

1 aAgarwala, Prachi1 aKumar, Santosh1 aPandey, Satyaprakash1 aMaiti, Souvik uhttps://library.ncl.res.in/content/human-telomeric-rna-g-quadruplex-response-point-mutation-g-quartets-002675nas a2200169 4500008004100000022001400041245007100055210006900126260011000195300001200305490000700317520201100324100003102335700001702366700001802383856010402401 2015 eng d a1359-734500aNonconventional chemical inhibitors of microRNA: therapeutic scope0 aNonconventional chemical inhibitors of microRNA therapeutic scop aTHOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLANDbROYAL SOC CHEMISTRYcOCT a820-8310 v513 a

MicroRNAs (miRNAs) are a class of genomically encoded small RNA molecules (similar to 22nts in length), which regulate gene expression post transcriptionally. The term microRNA or miRNA was coined in 2001, and research in the past decade has shed light on their widespread occurrence, evolutionary conservation and tissue specific functions. It is estimated that they modulate the gene expression of approximately 60% of the mammalian genes by regulating the levels of target mRNAs to which they can bind on the basis of sequence complementarities. miRNAs are produced in a well coordinated series of steps from being transcribed in the nucleus to exerting their function in the cytoplasm. miRNAs are now implicated in diverse biological phenomena ranging from development to stress response which makes miRNAs one of the central regulatory molecules which modulate information flow along the central dogma of gene expression. More importantly, like any regulatory molecule, deregulation of miRNAs is causally associated with several diseases (mainly cancer) and is directly involved in a variety of pathophysiologies owing to their aberrant expression. Thus, modulation of miRNA levels is of prime therapeutic importance. Conventional methods of miRNA knockdown using chemically modified antisense-oligonucleotides have been explored extensively but face the challenges of modes of delivery, biostability and biodistribution. This calls for the development of more alternative and non-conventional methods to target miRNA. Small molecules targeting RNA chemical and structural space provide one such timely opportunity. In this article we first provide a brief overview of miRNA biogenesis and its disease associations. We then summarize the major developments in conventional oligonucleotide based approaches to miRNA knockdown and its status. We then focus on the more non-conventional methods like oligonucleotide enzymes and small molecules and provide an outlook on the future of such methods.

1 aJayaraj, Gopal, Gunanathan1 aNahar, Smita1 aMaiti, Souvik uhttps://library.ncl.res.in/content/nonconventional-chemical-inhibitors-microrna-therapeutic-scope-001803nas a2200193 4500008004100000022001400041245007300055210006900128260011000197300001400307490000600321520109200327100001701419700001801436700001501454700001801469700001801487856010401505 2015 eng d a2041-652000aPotent inhibition of miR-27a by neomycin-bisbenzimidazole conjugates0 aPotent inhibition of miR27a by neomycinbisbenzimidazole conjugat aTHOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLANDbROYAL SOC CHEMISTRYcJUL a5837-58460 v63 a

miRNAs are important components of regulatory networks that control gene expression and have implications in various diseases including cancer. Targeting oncogenic miRNAs with small molecules is currently being explored to develop cancer therapeutics. Here, we report the development of dual binding neomycin-bisbenzimidazole conjugates that target oncogenic miR-27a with high affinity (K-a = 1.2 to 7.4 x 10(8) M-1). These conjugates bring significant reduction (similar to 65% at 5 mu M) in mature miRNA levels and penetrate easily in the cells where they localise both in the cytoplasm and the nucleus. Cell cycle analysis showed significant increase in the G0/G1 phase (similar to 15%) and decrease in the S phase (similar to 7%) upon treatment with neomycin-bisbenzimidazole conjugates, suggesting inhibition of cell proliferation. Using the conjugation approach, we show that moderately binding ligands can be covalently combined into high affinity binders. This study also highlights the role of linker optimization in designing high affinity ligands for miR-27a targeting.

1 aNahar, Smita1 aRanjan, Nihar1 aRay, Arjun1 aArya, Dev, P.1 aMaiti, Souvik uhttps://library.ncl.res.in/content/potent-inhibition-mir-27a-neomycin-bisbenzimidazole-conjugates-002126nas a2200193 4500008004100000022001400041245009800055210006900153260007100222300001400293490000700307520137200314100002501686700002101711700003101732700002301763700001801786856012801804 2015 eng d a0006-296000aRNA stem-loop to G-quadruplex equilibrium controls mature MicroRNA production inside the cell0 aRNA stemloop to Gquadruplex equilibrium controls mature MicroRNA a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcDEC a7067-70780 v543 a

The biological role of the existence of overlapping structures in RNA is possible yet remains very unexplored. G-Rich tracts of RNA form G-quadruplexes, while GC-rich sequences prefer stem-loop structures. The equilibrium between alternate structures within RNA may occur and influence its functionality. We tested the equilibrium between G-quadruplex and stem-loop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem-loop structures in pre-miRNA to produce mature miRNAs. Deviation from stem-loop leads to deregulated mature miRNA levels, providing readout of the existence of an alternate structure per se G-quadruplex-mediated structural interference in miRNA maturation. In vitro analysis using beacon and Dicer cleavage assays indicated that mature miRNA levels depend on relative amounts of K+: and Mg2+ ions, suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex to stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence of the formation of noncanonical G-quaciruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.

1 aPandey, Satyaprakash1 aAgarwala, Prachi1 aJayaraj, Gopal, Gunanathan1 aGargallo, Raimundo1 aMaiti, Souvik uhttps://library.ncl.res.in/content/rna-stem-loop-g-quadruplex-equilibrium-controls-mature-microrna-production-inside-cell-001733nas a2200205 4500008004100000022001400041245006900055210006800124260007200192300001400264490000700278520102700285100001801312700001701330700002301347700001501370700002401385700001801409856010001427 2015 eng d a0305-104800aSelective inhibition of miR-21 by phage display screened peptide0 aSelective inhibition of miR21 by phage display screened peptide aGREAT CLARENDON ST, OXFORD OX2 6DP, ENGLANDbOXFORD UNIV PRESScAPR a4342-43520 v433 a

miRNAs are nodal regulators of gene expression and deregulation of miRNAs is causally associated with different diseases, including cancer. Modulation of miRNA expression is thus of therapeutic importance. Small molecules are currently being explored for their potential to downregulate miRNAs. Peptides have shown to have better potency and selectivity toward their targets but their potential in targeting and modulating miRNAs remain unexplored. Herein, using phage display we found a very selective peptide against pre-miR-21. Interestingly, the peptide has the potential to downregulate miR-21, by binding to pre-miR-21 and hindering Dicer processing. It is selective towards miR-21 inside the cell. By antagonising miR-21 function, the peptide is able to increase the expression of its target proteins and thereby increase apoptosis and suppress cell proliferation, invasion and migration. This peptide can further be explored for its anti-cancer activity in vivo and may be even extended to clinical studies.

1 aBose, Debojit1 aNahar, Smita1 aRai, Manish, Kumar1 aRay, Arjun1 aChakraborty, Kausik1 aMaiti, Souvik uhttps://library.ncl.res.in/content/selective-inhibition-mir-21-phage-display-screened-peptide-001466nas a2200169 4500008004100000022001400041245002900055210002800084260011000112300001400222490000700236520092600243100002101169700002501190700001801215856006301233 2015 eng d a1477-052000aTale of RNA G-quadruplex0 aTale of RNA Gquadruplex aTHOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLANDbROYAL SOC CHEMISTRYcAPR a5570-55850 v133 a

G-quadruplexes are non-canonical secondary structures found in guanine rich regions of DNA and RNA. Reports have indicated the wide occurrence of RNA G-quadruplexes across the transcriptome in various regions of mRNAs and non-coding RNAs. RNA G-quadruplexes have been implicated in playing an important role in translational regulation, mRNA processing events and maintenance of chromosomal end integrity. In this review, we summarize the structural and functional aspects of RNA G-quadruplexes with emphasis on recent progress to understand the protein/trans factors binding these motifs. With the revelation of the importance of these secondary structures as regulatory modules in biology, we have also evaluated the various advancements towards targeting these structures and the challenges associated with them. Apart from this, numerous potential applications of this secondary motif have also been discussed.

1 aAgarwala, Prachi1 aPandey, Satyaprakash1 aMaiti, Souvik uhttps://library.ncl.res.in/content/tale-rna-g-quadruplex-001981nas a2200169 4500008004100000022001400041245013500055210006900190260007100259300001400330490000800344520127300352100001801625700002401643700001801667856012601685 2016 eng d a1520-610600aInfluence of ionic liquids on thermodynamics of small molecule DNA interaction: the binding of ethidium bromide to calf thymus DNA0 aInfluence of ionic liquids on thermodynamics of small molecule D a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcMAR a2691-27000 v1203 a

Ionic liquids (ILs) are salts with poor ionic coordination, resultantly remaining in liquid state below 100 degrees C and some may retain liquid state even at room temperature. ILs are known to provide a conducive environment for many biological enzymatic reactions, but their interaction with biomacromolecules are poorly understood. In the present study, we investigate the effect of various ionic liquids on DNA small molecule interaction using calf thymus DNA (ctDNA) ethidium bromide (EB) as a model system. The effect of various ionic liquids on these interactions is studied by an array of techniques such as circular dichroism (CD), UV melting, fluorescence exclusion and isothermal titration calorimetry. Interestingly, we observed that presence of IL increased the stability of ctDNA without altering its structure. The binding affinities Kbs for EB binding to ctDNA in the presence of 300 mM ILs are about half order of magnitude smaller than the Kbs in absence of ILs and correspond to a less favorable free energy. We noted that, when adjusted to corresponding buffer condition, the unfavorable shift in AG of ctDNA-EB interaction is attributed to decreased entropy in the case of ILs, whereas the same effect by NaCI was due to increased enthalpy.

1 aMishra, Arpit1 aEkka, Mary, Krishna1 aMaiti, Souvik uhttps://library.ncl.res.in/content/influence-ionic-liquids-thermodynamics-small-molecule-dna-interaction-binding-ethidium01541nas a2200241 4500008004100000022001400041245013200055210006900187260000800256300001200264490000700276520072300283653001501006653001101021653001301032653001801045653002101063100001701084700002201101700002601123700001801149856013201167 2016 eng d a2159-333700aInhibition of miR-21 by 3 `/5 `-serinyl-capped 2 `-O-Methyl RNA Interspersed with 2 `-O-(2-Amino-3-Methoxypropyl) uridine units0 aInhibition of miR21 by 3 5 serinylcapped 2 OMethyl RNA Intersper cOCT a327-3340 v263 a

miRNAs are highly conserved class of small ncRNAs whose involvement in human pathophysiologies is extensively investigated. MiR-21 is a well established oncogenic miRNA whose deregulation plays a significant role in onset and progression of cancer. The need of novel approaches to downregulate miR-21 is rapidly expanding. Potent inhibition of miR-21 is achieved by chemically modified 2-O-methyl RNA oligonucleotide. The serinol capping at 3 and 5ends and the interspersed 2-O-(R-2-amino-3-methoxypropyl) uridine units enhance the nuclease resistance and efficacy of 2-O-methyl RNA for the inhibition of miR-21. This represents a simple and novel modification for developing oligonucleotide-based therapeutics.

10aantagomirs10acancer10amicroRNA10anoncoding RNA10aoligonucleotides1 aNahar, Smita1 aKotikam, Venubabu1 aKumar, Vaijayanti, A.1 aMaiti, Souvik uhttps://library.ncl.res.in/content/inhibition-mir-21-3-5-serinyl-capped-2-o-methyl-rna-interspersed-2-o-2-amino-3-methoxypropyl02388nas a2200205 4500008004100000245014100041210006900182260000800251300001400259490000700273520164000280100001701920700001801937700002301955700002001978700002001998700001902018700001802037856012702055 2016 eng d00aSystematic evaluation of biophysical and functional characteristics of selenomethylene-locked nucleic acid-mediated inhibition of miR-210 aSystematic evaluation of biophysical and functional characterist cDEC a7023-70320 v553 amiRNAs constitute an important layer of gene regulation mediated by sequence-specific targeting of mRNAs. Aberrant expression of miRNAs contributes to a host of pathological states. Promoting cancer, miR-21 is upregulated in variety of cancers and promotes tumor progresion by suppressing a network of tumor suppressor genes. Here we describe a novel class of bicyclic RNA analogues, selenomethylene-locked nucleic acid (SeLNA), that display high affinity, improved metabolic stability, and increased potency for miR-21 inhibition. The thermal stability (T-m) for duplexes was increased significantly with incorporation of SeLNA monomers as compared to that of the unmodified DNA RNA hybrid. A comprehensive thermodynamic profile obtained by isothermal titration calorimetry revealed a favorable increase in the enthalpy of hybridization for SeLNA containing DNA and target RNA heteroduplexes. SeLNA modifications displayed remarkable binding affinity for miR-21 target RNA with a K-a of <= 1.05 x 10(8) M-1. We also observed enhanced serum stability for SeLNA RNA duplexes with a half-life of <= 36 h. These in vitro results were well correlated with the antisense activity in cancer cells imparting up to similar to 91% inhibition of miR-21. The functional impact of SeLNA modifications on miR-21 inhibition was further gauged by investigating the migration and invasion characterisitics of cancer cells, which were drastically reduced to similar to 49 and similar to 55%, respectively, with SeLNA having four such modifications. Our findings demonstrate SeLNA as a promising candidate for therapeutics for disease associated miRNAs.1 aNahar, Smita1 aSingh, Amrita1 aMorihiro, Kunihiko1 aMoai, Yoshihiro1 aKodama, Tetsuya1 aObika, Satoshi1 aMaiti, Souvik uhttps://library.ncl.res.in/content/systematic-evaluation-biophysical-and-functional-characteristics-selenomethylene-locked01757nas a2200229 4500008004100000022001400041245010000055210006900155260000800224300001400232490000700246520099300253100001701246700001801263700002001281700001601301700001801317700002301335700002701358700001801385856012401403 2018 eng d a1359-734500aG-quadruplex motif at the 3' end of sgRNAs improves CRISPR-Cas9 based genome editing efficiency0 aGquadruplex motif at the 3 end of sgRNAs improves CRISPRCas9 bas cMAR a2377-23800 v543 aOriginating as a component of prokaryotic adaptive immunity, the type II CRISPR/Cas9 system has been repurposed for targeted genome editing in various organisms. Although Cas9 can bind and cleave DNA efficiently under in vitro conditions, its activity inside a cell can vary dramatically between targets owing to differences between genomic loci and availability of enough Cas9/sgRNA (single guide RNA) complex molecules for cleavage. Most methods to improve CRISPR/Cas9 activity have so far relied on Cas9 protein engineering or base modifications in the sgRNA sequence. Here we demonstrate that a structure based rational design of sgRNAs can enhance the efficiency of Cas9 cleavage in vivo. By appending a naturally forming RNA G-quadruplex motif to the 3′ end of sgRNAs we can improve its stability and target cleavage efficiency in zebrafish embryos without inducing off-target activity, thereby underscoring its value in the design of better and optimized genome editing triggers.1 aNahar, Smita1 aSehgal, Paras1 aAzhar, Mohammad1 aRai, Manish1 aSingh, Amrita1 aSivasubbu, Sridhar1 aChakraborty, Debojyoti1 aMaiti, Souvik uhttps://library.ncl.res.in/content/g-quadruplex-motif-3-end-sgrnas-improves-crispr-cas9-based-genome-editing-efficiency01918nas a2200265 4500008004100000022001400041245011100055210006900166260000900235300001200244490000700256520103600263653002101299653002001320653000801340653002601348653002001374100002301394700002201417700001901439700002001458700001801478700002401496856013201520 2018 eng d a1860-717900aSuperior HIV-1 TAR binders with conformationally constrained R52 arginine mimics in the Tat(48-57) peptide0 aSuperior HIV1 TAR binders with conformationally constrained R52  cFEB  a220-2260 v133 a

We report a 100-fold increase in binding affinity of the Tat(48-57) peptide to HIV-1 transcriptional activator-responsive element (TAR) RNA by replacing Arg52, an essential and critical residue for Tat's specific binding, with (2S,4S)-4-guanidinoproline. The resulting Tat1M peptide is a far superior binder than Tat1M, a peptide containing another conformationally constrained arginine mimic, (2S,4S)-4-amino-N-(3-guanidinopropyl)proline, or even the control Tat peptide (CtrlTat) itself. Our observations are supported by circular dichroism (CD), isothermal titration calorimetry (ITC), gel electrophoresis and UV spectroscopy studies. Molecular dynamics simulations suggest increased interactions between the more compact Tat1M and TAR RNA, relative to CtrlTat. The CD signature of the RNA itself remains largely unchanged upon binding of the peptides. The Tat mimetics further have better cell uptake properties than the control Tat peptide, thus increasing their potential application as specific TAR-binding molecules.

10aantiviral agents10aarginine mimics10aRNA10aTat peptide analogues10aTat-TAR binding1 aBhosle, Govind, S.1 aKharche, Shalmali1 aKumar, Santosh1 aSengupta, Durba1 aMaiti, Souvik1 aFernandes, Moneesha uhttps://library.ncl.res.in/content/superior-hiv-1-tar-binders-conformationally-constrained-r52-arginine-mimics-tat48-57-peptide02341nas a2200397 4500008004100000022001400041245012700055210006900182260000800251300001600259490000800275520110900283653001601392653001701408653001901425653001001444653002301454100002201477700001801499700002001517700002701537700001601564700001701580700001801597700002001615700001801635700002101653700001901674700001801693700001501711700001601726700002801742700001801770700002701788856012801815 2019 eng d a0027-842400aFrancisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing0 aFrancisella novicida Cas9 interrogates genomic DNA with very hig cOCT a20959-209680 v1163 a

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

10aCRISPR Cas910agene therapy10aGenome editing10aiPSCs10asickle cell anemia1 aAcharya, Sundaram1 aMishra, Arpit1 aPaul, Deepanjan1 aAnsari, Asgar, Hussain1 aAzhar, Mohd1 aKumar, Manoj1 aRauthan, Riya1 aSharma, Namrata1 aAich, Meghali1 aSinha, Dipanjali1 aSharma, Saumya1 aJain, Shivani1 aRay, Arjun1 aJain, Suman1 aRamalingam, Sivaprakash1 aMaiti, Souvik1 aChakraborty, Debojyoti uhttps://library.ncl.res.in/content/francisella-novicida-cas9-interrogates-genomic-dna-very-high-specificity-and-can-be-used02557nas a2200229 4500008004100000022001400041245011000055210006900165260000800234300001600242490000800258520175500266100002702021700001602048700001802064700002102082700002102103700001802124700002702142700002902169856012902198 2020 eng d a0002-786300aTerminal uridylyl transferase mediated site-directed access to clickable chromatin employing CRISPR-dCas90 aTerminal uridylyl transferase mediated sitedirected access to cl cAUG a13954-139650 v1423 a

Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemoenzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogues at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on the CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase, and the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.

1 aGeorge, Jerrin, Thomas1 aAzhar, Mohd1 aAich, Meghali1 aSinha, Dipanjali1 aAmbi, Uddhav, B.1 aMaiti, Souvik1 aChakraborty, Debojyoti1 aSrivatsan, Seergazhi, G. uhttps://library.ncl.res.in/content/terminal-uridylyl-transferase-mediated-site-directed-access-clickable-chromatin-employing02716nas a2200313 4500008004100000022001400041245011400055210006900169260000900238300001100247490000700258520169700265100001701962700001801979700002201997700002002019700002202039700001602061700002002077700002202097700002002119700002002139700003302159700001502192700001902207700001802226700002702244856013102271 2021 eng d a2050-084X00aFnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip0 aFnCas9based CRISPR diagnostic for rapid and accurate detection o cJUN  ae671300 v103 aThe COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.1 aKumar, Manoj1 aGulati, Sneha1 aAnsari, Asgar, H.1 aPhutela, Rhythm1 aAcharya, Sundaram1 aAzhar, Mohd1 aMurthy, Jayaram1 aKathpalia, Poorti1 aKanakan, Akshay1 aMaurya, Ranjeet1 aVasudevan, Janani, Srinivasa1 aAparna, S.1 aPandey, Rajesh1 aMaiti, Souvik1 aChakraborty, Debojyoti uhttps://library.ncl.res.in/content/fncas9-based-crispr-diagnostic-rapid-and-accurate-detection-major-sars-cov-2-variants-paper02847nas a2200529 4500008004100000022001400041245009900055210006900154260000900223300001100232490000800243520130300251653001301554653001101567653001101578653000801589653001401597653001801611100001601629700002001645700001701665700002701682700001801709700001801727700002001745700002101765700001901786700001901805700002201824700001801846700002001864700002201884700001801906700001801924700001701942700002201959700002201981700001602003700002602019700002102045700002802066700001702094700002702111700001802138700003002156856013102186 2021 eng d a0956-566300aRapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis0 aRapid and accurate nucleobase detection using FnCas9 and its app cJUL  a1132070 v1833 a

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without transcleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

10aCRISPRDx10aFELUDA10aFnCas910aLFA10aSARS-CoV210aSNV detection1 aAzhar, Mohd1 aPhutela, Rhythm1 aKumar, Manoj1 aAnsari, Asgar, Hussain1 aRauthan, Riya1 aGulati, Sneha1 aSharma, Namrata1 aSinha, Dipanjali1 aSharma, Saumya1 aSingh, Sunaina1 aAcharya, Sundaram1 aSarkar, Sajal1 aPaul, Deepanjan1 aKathpalia, Poorti1 aAich, Meghali1 aSehgal, Paras1 aRanjan, Gyan1 aBhoyar, Rahul, C.1 aSinghal, Khushboo1 aLad, Harsha1 aPatra, Pradeep, Kumar1 aMakharia, Govind1 aChandak, Giriraj, Ratan1 aPesala, Bala1 aChakraborty, Debojyoti1 aMaiti, Souvik1 aEpide, Indian, CoV2 Genom uhttps://library.ncl.res.in/content/rapid-and-accurate-nucleobase-detection-using-fncas9-and-its-application-covid-19-diagnosis00550nas a2200157 4500008004100000022001400041245007200055210006900127260000800196300001400204490000800218100002700226700002000253700001800273856010100291 2021 eng d a0140-673600aRapid identification and tracking of SARS-CoV-2 variants of concern0 aRapid identification and tracking of SARSCoV2 variants of concer cAPR a1346-13470 v3971 aChakraborty, Debojyoti1 aAgrawal, Anurag1 aMaiti, Souvik uhttps://library.ncl.res.in/content/rapid-identification-and-tracking-sars-cov-2-variants-concern01709nas a2200241 4500008004100000022001400041245008900055210006900144260000800213300001200221490000700233520090000240100001801140700002701158700002701185700003101212700001901243700001801262700002401280700002701304700001801331856011801349 2022 eng d a0305-104800aAlternative splicing modulation mediated by G-quadruplex structures in MALAT1 lncRNA0 aAlternative splicing modulation mediated by Gquadruplex structur cJAN a378-3960 v503 aMALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNAG-quadruplexes (rG4s) present in the 3' region of MALAT1 which regulates this function. Using rG4 domain-specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation, and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, result in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3' region of MALAT1 orchestrating AS.1 aGhosh, Arpita1 aPandey, Satya, Prakash1 aAnsari, Asgar, Hussain1 aSundar, Jennifer, Seematti1 aSingh, Praveen1 aKhan, Yasmeen1 aEkka, Mary, Krishna1 aChakraborty, Debojyoti1 aMaiti, Souvik uhttps://library.ncl.res.in/content/alternative-splicing-modulation-mediated-g-quadruplex-structures-malat1-lncrna02519nas a2200385 4500008004100000245010900041210006900150260000900219300001200228490000700240520127600247653002001523653002301543653003101566653003001597653002101627100002501648700001801673700002601691700002101717700001701738700001901755700001801774700002001792700002801812700001801840700001801858700001801876700002301894700001801917700002601935700001801961700002401979856013002003 2023 eng d00aG4-binding drugs, chlorpromazine and prochlorperazine, repurposed against COVID-19 infection in hamsters0 aG4binding drugs chlorpromazine and prochlorperazine repurposed a cMAR  a11331230 v103 a

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >5 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA-approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ or PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in Vero cell cultures were clear in case of both CPZ and PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against viruses like SARS-CoV-2, which spread fast and accumulate mutations quickly.

10aConserved motif10aFDA-approved drugs10aG-quadruplex binding drugs10ahamster model of COVID-1910aRNA G-quadruplex1 aRoy, Shuvra, Shekhar1 aSharma, Shalu1 aRizvi, Zaigham, Abbas1 aSinha, Dipanjali1 aGupta, Divya1 aRophina, Mercy1 aSehgal, Paras1 aSadhu, Srikanth1 aTripathy, Manas, Ranjan1 aSamal, Sweety1 aMaiti, Souvik1 aScaria, Vinod1 aSivasubbu, Sridhar1 aAwasthi, Amit1 aHarshan, Krishnan, H.1 aJain, Sanjeev1 aChowdhury, Shantanu uhttps://library.ncl.res.in/content/g4-binding-drugs-chlorpromazine-and-prochlorperazine-repurposed-against-covid-19-infection02096nas a2200265 4500008004100000022001400041245011000055210006900165260000800234300001400242490000700256520118500263100001801448700002701466700002801493700001601521700002701537700003101564700001901595700001801614700002401632700002701656700001801683856012901701 2023 eng d a0305-104800aIdentification of G-quadruplex structures in MALAT1 lncRNA that interact with nucleolin and nucleophosmin0 aIdentification of Gquadruplex structures in MALAT1 lncRNA that i cSEP a9415-94310 v513 a

Nuclear-retained long non-coding RNAs (lncRNAs) including MALAT1 have emerged as critical regulators of many molecular processes including transcription, alternative splicing and chromatin organization. Here, we report the presence of three conserved and thermodynamically stable RNA G-quadruplexes (rG4s) located in the 3 & PRIME; region of MALAT1. Using rG4 domain-specific RNA pull-down followed by mass spectrometry and RNA immunoprecipitation, we demonstrated that the MALAT1 rG4 structures are specifically bound by two nucleolar proteins, Nucleolin (NCL) and Nucleophosmin (NPM). Using imaging, we found that the MALAT1 rG4s facilitate the localization of both NCL and NPM to nuclear speckles, and specific G-to-A mutations that disrupt the rG4 structures compromised the localization of both NCL and NPM in speckles. In vitro biophysical studies established that a truncated version of NCL (& UDelta;NCL) binds tightly to all three rG4s. Overall, our study revealed new rG4s within MALAT1, established that they are specifically recognized by NCL and NPM, and showed that disrupting the rG4s abolished localization of these proteins to nuclear speckles

1 aGhosh, Arpita1 aPandey, Satya, Prakash1 aJoshi, Dheeraj, Chandra1 aRana, Priya1 aAnsari, Asgar, Hussain1 aSundar, Jennifer, Seematti1 aSingh, Praveen1 aKhan, Yasmeen1 aEkka, Mary, Krishna1 aChakraborty, Debojyoti1 aMaiti, Souvik uhttps://library.ncl.res.in/content/identification-g-quadruplex-structures-malat1-lncrna-interact-nucleolin-and-nucleophosmin02100nas a2200229 4500008004100000022001400041245016900055210006900224260000800293300001200301490000700313520127500320100001801595700001901613700001501632700002301647700001701670700001601687700001701703700001801720856013201738 2024 eng d a0006-296000aDevelopment of an in silico platform (TRIPinRNA) for the identification of novel RNA intramolecular triple helices and their validation using biophysical techniques0 aDevelopment of an in silico platform TRIPinRNA for the identific cDEC a250-2650 v643 a

There are surprisingly few RNA intramolecular triple helices known in the human transcriptome. The structure has been most well-studied as a stability-element at the 3 ` end of lncRNAs such as MALAT1 and NEAT1, but the intrigue remains whether it is indeed as rare as it is understood to be or just waiting for a closer look from a new vantage point. TRIPinRNA, our Python-based in silico platform, allows for a comprehensive sequence-pattern search for potential triplex formation in the human transcriptome-noncoding as well as coding. Using this tool, we report the putative occurrence of homopyrimidine type (canonical) triple helices as well as heteropurine-pyrimidine strand type (noncanonical) triple helices in the human transcriptome and validate the formation of both types of triplexes using biophysical approaches. We find that the occurrence of triplex structures has a strong correlation with local GC content, which might be influencing their formation. By employing a search that encompasses both canonical and noncanonical triplex structures across the human transcriptome, this study enriches the understanding of RNA biology. Lastly, TRIPinRNA can be utilized in finding triplex structures for any organism with an annotated transcriptome.

1 aRakheja, Isha1 aBharti, Vishal1 aSahana, S.1 aDas, Prosad, Kumar1 aRanjan, Gyan1 aKumar, Ajit1 aJain, Niyati1 aMaiti, Souvik uhttps://library.ncl.res.in/content/development-silico-platform-tripinrna-identification-novel-rna-intramolecular-triple-helices01773nas a2200181 4500008004100000022001400041245016000055210006900215260000800284300001400292490000800306520108200314100001801396700001901414700001801433700001501451856012501466 2025 eng d a1520-610600aMolecular modeling of non-canonical intramolecular RNA triple helix structures predicted from TRIPinRNA and their in vitro biophysical structure validation0 aMolecular modeling of noncanonical intramolecular RNA triple hel cAPR a4298-43080 v1293 a

RNA triple helices have traditionally been characterized by pyrimidine-type UA-U or CG-C triplets, with other base triplets considered to be destabilizing. However, the presence of non-canonical triplets in riboswitches and self-splicing introns suggests that triplexes containing longer stretches of such triplets may exist in the human genome too. Using molecular modeling, we investigated a chimeric triple helix derived from the FLRT2-AS1 lncRNA, confirming its stability over a 500 ns simulation. Biophysical analyses further support the formation of this triplex in vitro. Although these non-canonical structures exhibit less thermal stability compared to traditional UA-U triplets found in lncRNAs like metastasis associated lung adenocarcinoma transcript 1 and NEAT1, they may serve distinct biological functions, suggesting a dynamic and more temporal role in cellular processes. The triplex selected for this study is found in a human long non-coding RNA gene, paving the way for investigating the intriguing roles of these triple helices in cell biology.

1 aRakheja, Isha1 aPanda, Gayatri1 aMaiti, Souvik1 aRay, Arjun uhttps://library.ncl.res.in/content/molecular-modeling-non-canonical-intramolecular-rna-triple-helix-structures-predicted