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Publications (10 of 13) Show all publications
Nawale, G. N., Bahadorikhalili, S., Sengupta, P., Kadekar, S., Chatterjee, S. & Varghese, O. P. (2019). 4 '-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading. Chemical Communications, 55(62), 9112-9115
Open this publication in new window or tab >>4 '-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading
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2019 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 55, no 62, p. 9112-9115Article in journal (Refereed) Published
Abstract [en]

We designed novel 4 '-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in approximate to 4.9-fold more efficient knockdown than unmodified siRNA.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-392130 (URN)10.1039/c9cc04141a (DOI)000477960000008 ()31298670 (PubMedID)
Available from: 2019-09-03 Created: 2019-09-03 Last updated: 2019-09-03Bibliographically approved
Paidikondala, M., Rangasami, V. K., Nawale, G. N., Casalini, T., Perale, G., Kadekar, S., . . . Varghese, O. P. (2019). An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method. Angewandte Chemie International Edition, 58(9), 2815-2819
Open this publication in new window or tab >>An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method
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2019 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 58, no 9, p. 2815-2819Article in journal (Refereed) Published
Abstract [en]

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular-matrix-derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size- and concentration-dependent gene silencing in a CD44-positive human osteosarcoma cell line (MG-63) and in human mesenchymal stromal cells (hMSCs). This native HA-based siRNA transfection represents the first report on an anionic, non-viral delivery method that resulted in approximately 60% gene knockdown in both cell types tested, which correlated with a reduction in translation levels.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2019
Keywords
nanoparticles, extracellular matrix, hyaluronic acid, RNAi, transfection
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-379591 (URN)10.1002/anie.201900099 (DOI)000459807200045 ()30644615 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SBE13-0028
Available from: 2019-03-18 Created: 2019-03-18 Last updated: 2019-03-18Bibliographically approved
Paidikondala, M., Nawale, G. N. & Varghese, O. P. (2019). Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel. Biomacromolecules, 20(3), 1317-1324
Open this publication in new window or tab >>Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel
2019 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 20, no 3, p. 1317-1324Article in journal (Refereed) Published
Abstract [en]

Small interfering RNAs (siRNAs) are powerful toolsfor post-transcriptional gene silencing, which offers enormousopportunities for tissue engineering applications. However, poorserum stability, inefficient intracellular delivery, and inevitabletoxicity of transfection reagents are the key barriers for their clinicaltranslation. Thus, innovative strategies that allow safe and efficientintracellular delivery of the nucleic acid drugs at the desired site isurgently needed for a smooth clinical translation of therapeuticallyappealing siRNA-based technology. In this regard, we havedeveloped an innovative siRNA transfection protocol that employsa short incubation time of just 5 min. This allows easy transfection insuspension followed by transplantation of the cells in a hyaluronicacid (HA) hydrogel system. We also report here the unique ability ofsiRNA to bind HA that was quantified by siRNA release andrheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA insuspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to beessential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with orwithout removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removalof excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improvedknockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) asdetermined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the proteinlevels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allowsminimal manipulation of cells that are to be transplanted and reduce toxicity.

National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-379682 (URN)10.1021/acs.biomac.8b01712 (DOI)000461270500019 ()30642167 (PubMedID)
Funder
Swedish Foundation for Strategic Research , 2009-1035Swedish Foundation for Strategic Research , SBE13-0028EU, FP7, Seventh Framework Programme, FP7/2007-2013/607868
Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-04-12Bibliographically approved
Han, Y., Qiu, Z., Nawale, G. N., Varghese, O. P., Hilborn, J., Tian, B. & Leifer, K. (2019). MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification. Biosensors & bioelectronics, 127, 188-193
Open this publication in new window or tab >>MicroRNA detection based on duplex-specific nuclease-assisted target recycling and gold nanoparticle/graphene oxide nanocomposite-mediated electrocatalytic amplification
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2019 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 127, p. 188-193Article in journal (Refereed) Published
Abstract [en]

DNA technology based bio-responsive nanomaterials have been widely studied as promising tools for biomedical applications. Gold nanoparticles (AuNPs) and graphene oxide (GO) sheets are representative zero- and two-dimensional nanomaterials that have long been combined with DNA technology for point-of-care diagnostics. Herein, a cascade amplification system based on duplex-specific nuclease (DSN)-assisted target recycling and electrocatalytic water-splitting is demonstrated for the detection of microRNA. Target microRNAs can form DNA: RNA heteroduplexes with DNA probes on the surface of AuNPs, which can be hydrolyzed by DSN. MicroRNAs are preserved during the reaction and released into the suspension for the digestion of multiple DNA probes. After the DSN-based reaction, AuNPs are collected and mixed with GO to form AuNP/GO nanocomposite on an electrode for the following electrocatalytic amplification. The utilization of AuNP/GO nanocomposite offers large surface area, exceptional affinity to water molecules, and facilitated mass diffusion for the water-splitting reaction. For let-7b detection, the proposed biosensor achieved a limit detection of 1.5 fM in 80 min with a linear detection range of approximately four orders of magnitude. Moreover, it has the capability of discriminating non-target microRNAs containing even single-nucleotide mismatches, thus holding considerable potential for clinical diagnostics.

Keywords
Gold nanoparticles, Graphene oxide, MicroRNA detection, Electrocatalytic amplification, Duplex-specific nuclease
National Category
Analytical Chemistry Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-377203 (URN)10.1016/j.bios.2018.12.027 (DOI)000457508800026 ()30611105 (PubMedID)
Funder
Swedish Research Council, 2016-05259Knut and Alice Wallenberg FoundationEU, Horizon 2020, 713683
Available from: 2019-02-25 Created: 2019-02-25 Last updated: 2019-04-24Bibliographically approved
Kadekar, S., Nawale, G. N., Karlsson, K., Ålander, C., Podiyan, O. & Varghese, O. P. (2019). Synthetic design of asymmetric miRNA with engineered 3′-overhang to improve strand selection. Molecular Therapy - Nucleic Acids, 16, 597-604
Open this publication in new window or tab >>Synthetic design of asymmetric miRNA with engineered 3′-overhang to improve strand selection
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2019 (English)In: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 16, p. 597-604Article in journal (Refereed) Published
Abstract [en]

We have developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3′-end by adding extra nucleotides. Addition of seven nucleotides at the 3′-ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two-ends, which resulted in selective RISC recruitment as demonstrated by the stem-loop quantitative PCR experiment. Such selective recruitment was also corroborated at the protein level by Western blot analysis. In order to investigate the functional effect due to selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that the recruitment of miR strand is responsible for inducing apoptosis as well as to inhibit invasiveness of cancer cells. Recruitment of miR* strand, on the other hand, showed opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNA to enhance their therapeutic potential.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
RNA interference, miRNA, miR34a, strand selection, anticancer therapy
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-382299 (URN)10.1016/j.omtn.2019.04.012 (DOI)000470250900053 ()31085353 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SBE13-0028National initiative on Stem Cells for Regenerative Therapy, 2009-1035
Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-06-26Bibliographically approved
Wang, S., Nawale, G. N., Kadekar, S., Oommen, O. P., Jena, N. K., Chakraborty, S., . . . Varghese, O. P. (2018). Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH. Scientific Reports, 8, Article ID 2193.
Open this publication in new window or tab >>Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 2193Article in journal (Refereed) Published
Abstract [en]

We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-347090 (URN)10.1038/s41598-018-20735-0 (DOI)000423787500168 ()29391582 (PubMedID)
Funder
Swedish Foundation for Strategic Research
Available from: 2018-03-26 Created: 2018-03-26 Last updated: 2018-03-26Bibliographically approved
Mirajkar, A. L., Mittapelli, L. L., Nawale, G. N. & Gorea, K. R. (2018). Synthetic green fluorescent protein (GFP) chromophore analog for rapid, selective and sensitive detection of cyanide in water and in living cells. Sensors and actuators. B, Chemical, 265, 257-263
Open this publication in new window or tab >>Synthetic green fluorescent protein (GFP) chromophore analog for rapid, selective and sensitive detection of cyanide in water and in living cells
2018 (English)In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 265, p. 257-263Article in journal (Refereed) Published
Abstract [en]

Here, we report Green Fluorescent Protein (GFP) chromophore analog as a turn-on fluorescent chemodosimeter (THBI) for selective detection of cyanide in water, on solid state and in living cells. The detection limit was found to be 0.17 mu M (4.5 ppb). The time dependent study revealed that there is a rapid enhancement in fluorescence intensity (in less than 5s) and was constant over the period of 1 h. Cell imaging data exhibited that THBI was successfully crossed cell membrane and visualized fluorescence response in live HCT cells. 

Place, publisher, year, edition, pages
ELSEVIER SCIENCE SA, 2018
Keywords
GFP, HBI, THBI, Cyanide chemodosimeter, Fluorogenic chemosensor
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356375 (URN)10.1016/j.snb.2018.03.068 (DOI)000430232500031 ()
Available from: 2018-08-15 Created: 2018-08-15 Last updated: 2018-08-15Bibliographically approved
Bermejo-Velasco, D., Nawale, G. N., Oommen, O. P., Hilborn, J. & Varghese, O. P. (2018). Thiazolidine chemistry revisited: a fast, efficient and stable click-type reaction at physiological pH. Chemical Communications, 54(88), 12507-12510
Open this publication in new window or tab >>Thiazolidine chemistry revisited: a fast, efficient and stable click-type reaction at physiological pH
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2018 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 54, no 88, p. 12507-12510Article in journal (Refereed) Published
Abstract [en]

We describe the fast reaction kinetics between 1,2-aminothiols and aldehydes. Under physiological conditions such a click-type reaction afforded a thiazolidine product that remains stable and did not require any catalyst. This type of bioorthogonal reaction offers enormous potential for the coupling of biomolecules in an efficient and biocompatible manner.

National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-364896 (URN)10.1039/c8cc05405c (DOI)000448947000019 ()30345438 (PubMedID)
Available from: 2018-11-06 Created: 2018-11-06 Last updated: 2019-06-26Bibliographically approved
Nawale, G. N. (2012). Incorporation of 4'-C-aminomethyl-2'-O-methylthymidine into DNA by thermophilic DNA polymerases. Chemical Communications, 48(77), 9619-9621
Open this publication in new window or tab >>Incorporation of 4'-C-aminomethyl-2'-O-methylthymidine into DNA by thermophilic DNA polymerases
2012 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 48, no 77, p. 9619-9621Article in journal (Refereed) Published
Abstract [en]

The dual modified nucleotide 4'-C-aminomethyl-2'-O-methylthymidine 5'-triphosphate was synthesized and enzymatically incorporated into DNA by the thermophilic DNA polymerases Pfu and Therminator III. The dual ribose modification imparted increased exonuclease resistance to DNA compared to the well-known 2'-O-methyl modification.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-364934 (URN)10.1039/c2cc35222b (DOI)22908130 (PubMedID)
Available from: 2018-11-06 Created: 2018-11-06 Last updated: 2019-03-29Bibliographically approved
Nawale, G. N. (2012). Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs.. Journal of Organic Chemistry, 77(7), 3233-3245
Open this publication in new window or tab >>Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs.
2012 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 77, no 7, p. 3233-3245Article in journal (Refereed) Published
Abstract [en]

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (∼1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.

National Category
Organic Chemistry
Research subject
Chemistry with specialization in Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-364935 (URN)10.1021/jo202666m (DOI)22372696 (PubMedID)
Available from: 2018-11-06 Created: 2018-11-06 Last updated: 2019-03-29Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-7256-0758

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