Systematic Evaluation of Biophysical and Functional Characteristics of

Subscriber access provided by University of Otago Library

Article

Systematic evaluation of biophysical and functional characteristics of Selenomethylene Locked Nucleic Acid mediated inhibition of miR-21 Smita Nahar, Amrita Singh, Kunihiko Morihiro, Yoshihiro Moai, Tetsuya Kodama, Satoshi Obika, and Souvik Maiti Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00895 • Publication Date (Web): 15 Nov 2016 Downloaded from http://pubs.acs.org on November 17, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Systematic evaluation of biophysical and functional characteristics of Selenomethylene Locked Nucleic Acid mediated inhibition of miR-21 Smita Nahar,a,b Amrita Singh a,b, Kunihiko Morihirod,e, Yoshihiro Moai f, Tetsuya Kodama f Satoshi Obikad,e and Souvik Maitia,b,c* a

Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi-110001,India.

*b

CSIR- Institute of Genomics and Integrative Biology, Mathura Road, Delhi-110025, India. Fax: +91-11- 2766-7471; Tel: +91-11- 2766-6156; E-mail: [email protected]

c

Organic Chemistry Division, CSIR-National Chemical Laboratory, Pashan Road, Pune 411008 India.

d

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. e

National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8-SaitoAsagi, Ibaraki, Osaka 567-0085, Japan.

ACS Paragon Plus Environment

1

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

f

Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.

ABBREVIATIONS PS, Phosphorothioate; AMO, Anti miRNA Oligonucleotide; SeLNA, Selenomethylene Locked Nucleic Acid;


2

Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ABSTRACT

miRNAs constitute an important layer of gene regulation mediated by sequence-specific targeting of mRNAs. Aberrant expression of miRNAs contributes to host of pathological states. Cancer promoting, miR-21 is upregulated in variety of cancers and promotes tumor progresion by suppressing network of tumor suppressor genes. Here we describe a novel class of bicyclic RNA analog, Selenomethylene Locked nucleic acid (SeLNA) which display high affinity, improved metabolic stability and increased potency for miR-21 inhibition. The thermal stability (Tm) for duplexes was increased significantly with incorporation of SeLNA monomers as compared to unmodified DNA-RNA hybrid. A comprehensive thermodynamic profile obtained by ITC experiments revealed favourable increase in enthalpy of hybridization for SeLNA containing DNA and target RNA heteroduplexes. SeLNA modifications displayed remarkable binding affinity towards miR-21 target RNA with Ka upto 1.05 x 108 M-1. We also observed enhanced serum stability for SeLNA-RNA duplexes with half-life upto 36 hrs. These in vitro results were well correlated with the antisense activity in cancer cells imparting upto ~91 % of inhibition of miR-21. The functional impact of SeLNA modifications on miR-21 inhibition was further gauged by investigating the migration and invasion characterisitics in cancer cells, which got drastically reduced to ~49% and ~ 55% respectively with SeLNA having four such modifications. Our findings demonstrate SeLNA as a promising candidate for therapeutics for disease-associated miRNAs.

KEYWORDS: MiRNA, miR-21, SeLNA, cancer, Selenomethylene Locked Nucleic acid, invasion, migration, characterization, functional


3

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

Precisely controlled gene expression is mediated by a fine balance between transcription, RNA processing and translation. It has been increasingly clear that miRNAs are key regulatory components in this process. miRNAs (19-24 ntds) are endogenously expressed small non-coding RNAs that regulate gene expression mainly at the post transcriptional level through association with the multiprotein silencing complex usually referred as the RISC complex.1 Mostly, miRNAs originate by RNA pol II mediated transcription from hairpin precursors embedded in longer primary transcripts called pri-miRNA. The stem loops in pri-miRNA are processed by the microprocessor component that has Drosha as the catalytic subunit and DGCR8 as the RNA binding protein. This primary processing by the DGCR8-Drosha complex is carried out in the nucleus which releases ~ 70 ntd long pre-miRNA. The pre-miRNAs are exported out of the nucleus by exportin 5 and processed further by Dicer near the terminal loop into short ~22 ntd mature miRNA duplexes. These duplexes are subsequently loaded onto the Ago proteins resulting in assembly of the RISC complex. Herein, the duplexes get unwound and the guide strand is selected on the basis of thermodynamic stability.2 This mature miRNA then guides the RISC complex to 3′UTR of its target mRNA for its degradation or translational inhibition.3, 4 A 2-8 nucleotide seed region in miRNA starting from 5′ end is required for targeting mRNA via Watson-Crick hybridisation.1 This seed region is the prime stretch exploited for the use of antisense oligonucleotides to bind miRNA and relieve its inhibition on their target mRNAs. These tiny mediators orchestrate sequence dependent regulation of thousands of targets5, 6, thus playing diverse roles in crucial fundamental biological processes.7, 8 Accordingly, dysregulation of miRNA expression have profound effects on pathology of wide range of diseases 9 10, majorly in cancer.11, 12 Thus, it is not surprising that miRNAs have been appreciated as the new class of therapeutic targets and have added new dimension to oligonucleotide based drugs.13 AntimiRs


4

Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

represent antisense oligonucleotides that hybridizes with miRNAs by Watson-Crick pairing and block their function either by RNase H mediated degradation or by sequestration of the intended miRNA. Conceptually simple, the design and synthesis of antimiRs is relatively rapid and inexpensive method for inhibiting miRNA function than creating gene knockouts. However, the unmodified antimiRs containing natural phosphodiester bonds are susceptible to nuclease attack and are degraded rapidly reducing their functional half life significantly in the cells. Moreover, their poor cellular uptake and binding affinity limit their applications in cells severely. This has led to an upsurge in discovery of novel chemical modifications aimed to improve their PK-PD properties. Successful clinical trials illustrate the rapid progress of development of antimiR based tools for miRNA inhibition.14 A variety of chemical modifications such as phosphorothioates, peptide nucleic acids (PNA) 15, 16

, morpholino phosphoroamidate17, 18, 2′-O-methyl

and Locked nucleic acids (LNA)

23, 24

19, 20

, 2′-O-methoxyethyl nucleic acids

21, 22

have been extensively explored for their antisense

applications owing to their high target hybridization affinities and increased nuclease resistance. 25, 26

Locked Nucleic Acid (LNA)27, also known as 2′-O,4′-C-methylene bridged nucleic acid

(2′,4′-BNA)

28

is a bicyclic ribonucleoside analog that has been extensively studied in the past

decades. In this modification, the 2′-O-oxygen is bridged to the 4′-position via a methylene linker to confine the nucleotides in local 3′- endo structural conformation commonly found in the A-form RNA complex. Locking it in enhances base stacking and H-bonding interactions thereby increasing stability of the oligonucleotide.29 Wide spectrums of reports have described the efficacy and specificity of LNA- based silencing method.24,

30, 31

In general, modifications

containing 2′ heteroatom resulted in more stable duplexes as compared to other sugar modifications. 32


5

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

A variety of LNA analogs have been studied which uses LNA-monomer as a template with replacement of 2′-oxygen by other substituents. Prominent examples include amino-LNA, thioLNA33 which has similar 2′-, 4′- bicyclic planar structure but 2′-oxygen is substituted by NH2 and S group respectively. The positioning of these chemical moieties in the minor groove influences the duplex stability by electrostatic interactions. In need to develop novel modifications with improved hybridization properties towards target miRNA, our group reported Selenomethylene Locked Nucleic Acid (SeLNA) in which the Selenium atom replaces the 2′oxygen of the ribose sugar.34,35 We observed that SeLNA conferred high affinity to target RNA as compared to its reversible oxidizable state (SeOLNA) and LNA because of large entropy gain. Moreover, the excellent hybridization of SeLNA modification was comparable to six-membered bridging structure of 2’, 4’-BNANC. This prompted us to test the potency of SeLNA in inhibiting oncogenic miR-21. MiR-21 is a highly oncogenic miRNA that regulates cancer cell proliferation, migration, invasion and apoptosis by suppressing a number of tumor suppressor genes.36

37

We

systematically explored the biophysical and biological features in SeLNA mediated targeting of miR-21 by a battery of in vitro and in cellulo assays.

Experimental Syntheses of SeLNA-modified oligonucleotides SeLNA phosphoramidite was synthesized according to our reported procedure.34 Solid-phase oligonucleotide syntheses were performed on an nS-8 Oligonucleotides Synthesizer (GeneDesign Inc., Ibaraki, Japan). Oligonucleotides were synthesized with trityl-on mode on a 500 A CPG solid support column in 1.0 μmol scale using 5-ethylthio-1H-tetrazole (0.25 M in MeCN) as an activator. SeLNA phosphoramidite was incorporated into oligonucleotides with a


6

Page 7 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

coupling efficiency comparable to the commercially available phosphoramidites by increasing the coupling time from 32 sec to 10 min. Cleavage from the solid support and deprotection were accomplished with concentrated ammonium hydroxide solution at 55 °C for 12 h. The crude oligonucleotides were purified on a Sep Pack column (Waters, MA, USA) followed by RPHPLC on an XBridgeTM OST C18 column, 2.5 µm, 10 × 50 mm (Waters) using MeCN in 0.1 M triethylammonium acetate buffer (pH 7.0). The purified oligonucleotides were quantified by UV absorbance at 260 nm and their structures were confirmed by MALDI-TOF mass spectrometry fig S1-S6). CD- spectroscopy The conformations of single stranded unmodified DNA and of all the duplexes were evaluated by CD spectroscopy. The duplexes were mixed at equimolar ratio at a final concentration of 3 µM. The CD spectra were obtained on Jasco circular dichroism spectrometer from 350 nm to 200 nm using a 10 mm quartz cuvette with a wavelength step of 1 nm. The reported spectra are the average of three scans measured at 25°C.

Temperature dependent UV - spectroscopy The UV melting experiments were performed in Cary 100 (Varian) spectrophotometer equipped with a thermoelectrically controlled cell holder. The concentrations of each 1:1 heteroduplexes were 1 µM prepared in 10 mM sodium cacodylate pH (7.0) or 10 mM sodium cacodylate containing 100 mM NaCl. Optical melting curves of Absorbance vs temperature profiles were measured for each 1:1 duplex at 260 nm at a temperature range of 25 °C to 85°C. The temperature was scanned at a rate of 0.5°C/min. All data points were collected at 1°C. The


7

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

plot of mole fraction of the unfolded heteroduplex vs temperature was plotted and Tm was determined using previously established methods43.

Isothermal Titration Calorimetry (ITC) Isothermal titration experiments for the SeLNA modified duplex formation were carried out on a VP-ITC system (Microcal Inc., U.S.A.) at 30 °C. All the samples were prepared in 10mM sodium cacodylate pH (7.0). The SeLNA incorporated DNA strand was loaded into injection syringe with concentration as 45 µM (titrant) and was titrated to complementary RNA strand with concentration 1.5 µM into the sample cell. Titrations were performed with 7 µl injections, with 300 sec interval during subsequent injections. The titrations were continued till 25 injections, past saturation such that the heat of dilution is determined. The raw data was analysed in Origin (Microcal software) to generate integrated heat effect per injection. The resultant plot was fitted by non-linear least square minimization method to yield a sigmoidal curve and thermodynamic parameters like binding constant (Ka), stoichiometry (n), enthalpy change (∆H) were obtained by curve fitting. The Gibbs free energy change, ∆G, and entropy (∆S) were calculated from the equation, ∆G = -RTlnKa = ∆H - T∆S,

where R is gas constant and T is temperature (Kelvin). Serum Stability 7µM duplex of miR-21 with each oligo i.e. SeLNA0 ,SeLNA1, SeLNA2, SeLNA3, SeLNA4 was prepared by heating the oligo RNA mixture at 95°C for 5 minutes and annealing at 55°C for 30 minutes. Prepared duplex of each oligo (single stranded) was incubated with 10% FBS (Fetal


8

Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Bovine Serum) at 37°C for 48 hours. Similar 7µM reaction mixture was also made for each oligo (single stranded without RNA) in10% FBS. 10µl sample was collected at each time point and reaction was stopped with 3 times 1.5X TBE buffer and subsequently freeze in liquid nitrogen. Collected aliquots were analyzed for their stability on a 15% Native PAGE stained with SYBR Green(Life Technologies) and scanned using Typhoon FLA 7000. Density plot was made using open source software ImageJ.

Cell Culture Human breast cancer cell line, MCF-7, obtained from European Collection of Authenticated Cell Cultures (ECACC) was routinely passaged in Dulbecco's Modified Eagle Medium (DMEM medium, High Glucose, GIBCO) supplemented with 10% fetal bovine serum (FBS) without antibiotic and antimycotic at 37°C in humidifed air containing 5% CO2 air atmosphere.

Cell viability assay MCF-7 cells were seeded in 96 well-plates (8000 cells/well), grown to 80% confluency and then transfected with SeLNA oligos at different concentrations ranging from 20-200 nM. After 24 h of growth, cells were treated with 1 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (Sigma-Aldrich) and incubated for 3hrs at 37 °C in O2 incubator. After incubation, the medium was removed and formazan crystals were dissolved in 200µl of DMSO solution. Sample absorbance was measured at 570 nm with a reference wavelength of 630 nm. Data was normalized with untreated healthy control set.


9

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

Luciferase Assay MCF-7 cells were grown in 24 well plate at a density of 2 × 104 cells/ well. Post 24 h, the cells were co-transfected with luciferase target construct all the oligonucleotides i.e. Scramble, SeLNA0, SeLNA1, SeLNA2, SeLNA3, SeLNA4 at a final concentration of 50 nM using Lipofectamine 2000 transfection reagent (Life technologies) and incubated for 24 h. The sequence for Scramble used was ATTAACAGCCGTAGT with four SeLNA-T modifications. The cells were incubated at 37 °C for 4 h followed by the replenishment of transfection media with DMEM growth media (500 µL). Post treatment, cells were lysed in 100 µl of 1X Passive Lysis Buffer (Promega) and centrifuged at 14,000 rpm for 15 min. The supernatants were assayed for renilla and firefly luciferase signal using the dual-luciferase reporter assay kit (Promega), according to manufacturer’s protocol. Renilla luciferase values were normalized using firefly luciferase values.

RT-PCR MCF-7 cells were transfected with all the oligos i.e. Scramble, SeLNA0, SeLNA1, SeLNA2, SeLNA3, SeLNA4 at a final concentration of 50 nM for 24 h using Lipofectamine2000 transfection reagent (Life technologies). RNA was isolated using Trizol method and cDNA was prepared using QuantiMir kit. RT-PCR (Roche RT-PCR system) with three technical replicates was done using KAPA SYBR green master mix, specific forward primers for miR-21 and a universal reverse primer for all RNAs. U6 RNA was used as an internal normalization control. All the oligos were in the end normalized to the scrambled control oligo. The analysis was done by 2-∆∆Ct method.44


10

Page 11 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Western Blot MCF-7 cells (3 x 105 cells/well) were seeded in 6-well plates, grown to ~70% confluency 24 h before transfection. They were transfected with 50 nM each of Scramble, SeLNA0, SeLNA1, SeLNA2, SeLNA3 and SeLNA4 for 24 h using Lipofectamine 2000 (Lifetechnologies), according to manufacturer’s protocol. After 24 h, whole cells were lysed with Cell Lytic solution (Sigma-Aldrich). The supernatant were collected after centrifugation at 12000g for 15 min at 4°C. The protein contents were measured with BCA reagent kit (Pierce Chemical Co., Rockland, IL). 40µg of lysate protein was separated on 12%

SDS PAGE, transferred and blotted into

PVDF membrane (GE healthcare). After blockage of non-specific sites with 5% BSA, the membrane was incubated overnight with primary antibody, PDCD4 (1:1000, Abcam) and GAPDH (1:2000, CST). The blots were washed with 0.1%TBST solution thrice for 15 min each. This was followed by incubation with AP conjugated secondary antibody (1:10000) for 2h at RT. After the repeated washings, the blot was developed by BCIP-NBT substrate solution (SigmaAldrich), followed by densitometric analysis by IMAGE J software, NIH.

Migration and Invasion Assay Migration invasion assay was performed using Cytoselect 24 well cell migration and invasion assay (8µm colorimetric format) from Cell Biolabs. MCF-7 cells were transfected with all the oligos i.e. Scramble, SeLNA0, SeLNA1, SeLNA2, SeLNA3, SeLNA4 at a final concentration of 50 nM for 48 h using Lipofectamine2000 transfection reagent (Life technologies). Cells were trypsinzed and suspended in serum free media. In a fresh 24-well plate 500µl of serum containing media was added to each lower well and 300µl of cell suspension was added to


11

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

migration and invasion chambers/inserts. Cells were allowed to migrate/invade for 24 hours. Media was carefully aspirated from each insert and interiors gently swabbed with cotton swabs to remove non migratory cells. Cells on the insert were stained in 400µl cell stain solution for 20 minutes and after gentle washing and subsequent drying of inserts cells were observed under light microscope at 10X magnification. At least 5 individual fields per insert were captured. Inserts were then transferred to an empty well and 200ul of extraction solution was added. After 30 minutes of orbital shaking, 100µl from each sample was transferred to 96 well microtiter plate and absorbance measured at 560nm in Tecan plate reader.

RESULTS AND DISCUSSION In order to explore the scope of SeLNA, a series of SeLNA modifications were introduced in a 15 mer DNA strand in a walk in manner (Table 1). The oligonucleotides chosen for the study were complementary to miR-21. Five oligonucleotides with SeLNA modification incorporated in thymidine (T residue) ranging from zero to four modifications were studied (Table S1). In order to evaluate the conformation of SeLNA modified duplexes (1:1), we employed Circular Dichroism (CD) spectroscopy. As shown in fig 1, the single stranded unmodified DNA displayed a disordered conformation which upon binding to target RNA, transits to A-form conformation. We observed a gradual blue shift from 268 nm of unmodified duplex as SeLNA monomers were increased. A stark 8nm blue shift in SeLNA4 spectra was seen as compared to SeLNA0. Apart from a positive peak at 260 nm, we observed a negative peak at 207 nm for all duplexes typical of A-type conformation indicating strong conformational steering by SeLNA modifications.38 The ellipticity of negative peak increased with increase in SeLNA modifications, and was highest for duplex having four SeLNA monomers (SeLNA4). Thus,


12

Page 13 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

overall the duplex geometry progressed towards A-type conformation as a function of SeLNA monomers. We evaluated the hybridization properties of unmodified DNA, SeLNA-DNA strand with its complementary RNA via absorbance vs temperature profiles. The UV-melting curves for duplexes at 260 nm and the Tm dependence on concentration is shown in Fig 2. Each melting curve shows characteristic sigmoidal behaviour for the unfolding of nucleic acid duplex. The analysis of the shapes of melting curves yielded transition temperatures (Tm), which are midpoint temperatures of helix-coil transition. We observed that all duplexes showed significantly higher Tm values as compared to unmodified duplex indicating that each modification led to an increment in Tm and stabilization of the duplex (Table 2). UV melting spectroscopy was also carried as a function of salt concentration from 10-110 mM. Increase in salt concentrations led to shift of the curves to higher temperature. At a higher salt concentration of 110 mM, further prominent increase in Tm was observed for all duplexes as compared to unmodified duplex (Table 2). Hybridization of the oxidizable state of SeLNA was also studied by thermal denaturation UV experiments (Fig S7). The temperature dependent unfolding of SeOLNA and target RNA heteroduplex showed higher Tm values as compared to unmodified duplex (Fig S8, Table S2). However, the stability of SeOLNA duplexes was lower than SeLNA modified duplexes. The Tm increment per modification was higher for each SeLNA incorporated base as compared to SeOLNA. For four modifications, the Tm difference was of 10°C in 100 mM Na+ concentration (Table S2), reinforcing the enhanced thermal stability and high hybridization affinity of SeLNA modifications. These encouraged us to biophysically and functionally characterize the effects of SeLNA modifications in the present study.


13

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

Sodium ions bind to DNA and contribute to the stability of the duplex by shielding the electrostatic repulsive forces imparted by the negatively charged phosphate groups. The presence of counterions associated with nucleic acids is a significant factor that influences stability. When the duplex melts, there is a release of Na+ ions due to reduction of charge density along the chain during conversion from the helical to single stranded coil conformation. To estimate the effect of SeLNA modifications on counterion uptake associated with duplex formation, we calculated the net counterion uptake for each of the modification over a range of Na+ concentrations (10-110 mM). The dependence of Tm on salt concentration is shown in fig S8. The slopes of each curves is calculated using a linear regression analysis (Table S3). The net uptake of Na+ is obtained from the following equation:

∆nNa+ = 1.11 (∆H/RTm2) ∂ Tm/∂ (ln[Na+)]

where 1.11 is a proportionality constant for converting ionic activity into concentrations and δTm/δ(ln [Na+]) represents the slope of a plot of Tm versus the logarithm of sodium ions (ln [Na+]) at different concentrations (10−110 mM Na+). The slopes are proportional to the difference in the number of bound counterions in the single and double stranded states. There is an increase in the counterions with increase in modification which can be ascribed to the SeLNA induced change of helix conformation to A-type geometry. Compared to the unmodified heteroduplexes, the higher charge density of SeLNA modified duplexes leads to higher uptake of counterions upon duplex formation. However, the observed change in net Na+ uptake was within experimental error and thus, insignificant. To obtain insight into thermodynamic parameters of heteroduplex formation, we performed ITC experiments between unmodified DNA (SeLNA0) and SeLNA modified DNA (SeLNA1,


14

Page 15 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

SeLNA2, SeLNA3, SeLNA4) with its target RNA. Representative isotherm for SeLNA0 and SeLNA4 is shown in fig 3. Thermodynamic parameters describing the association of SeLNAmodified DNA with RNA is presented in Table 3. As shown in table 3, the stoichiometries for all the duplexes were close to unity showing 1:1 binding. SeLNA4 was shown to have two orders of increased affinity for the 15mer complementary RNA strand displaying Ka of 1.05 x 108 M-1 as compared to unmodified strand (SeLNA0) with Ka of 3.2 x 106 M-1. With incorporation of SeLNA modification and its interaction with complementary RNA strand resulted in exothermic reaction displayed by negative ∆H. The increase in each of the SeLNA modification was accompanied by favourable enthalpy of hybridisation (FigS9, S10, and S11) which is attributed to the low polarity of selenium atoms favouring hydrophobic interactions. The overall Gibbs free energy (∆G) was favourable due to highly negative favourable enthalpic and unfavourable entropic compensation represented in Table 3. One of the prime factors influencing the antimiR efficacy is their metabolic stability. Unmodified oligonucleotides are rapidly decomposed in biological systems, thereby restricting their application profoundly. Since metabolic stability is conferred by the inherent ability of oligonucleotide for nuclease resistance as well as stabilizing the duplex structure crucial for its activity. We investigated the effect of incorporation of SeLNA modification on single stranded (ss) and double standed (ds) forms upon incubation in 10% fetal bovine serum. As shown in fig4A and B, the serum stability of both ss and ds forms follows: SeLNA4> SeLN3> SeLNA2> SeLNA1> SeLNA0. The stability of the double-stranded complexes with SeLNA-DNA with target RNA was increased with every additional substitution (FigS12). SeLNA 4 showed most prominent serum stability in ss form (t1/2 ~ 24 h) as well as in ds forms (t1/2 ~ 36 h) as compared to all the other counterparts.


15

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

To dispel concerns about effect mediated solely by SeLNA modifications, we employed a scramble sequence with highest number of SeLNA modifications (four) in each of our cellular studies. MCF-7 was our model cell line as it expresses high endogenous levels of miR-21.39 To investigate the cytotoxicity effects imparted by SeLNA modifications, we transfected all the SeLNA modified oligos at a concentration range from 20 nM to 200 nM in MCF-7 cell line (Fig S13). The introduction of SeLNA modification did not cause significant toxicity in MCF-7 cells upto 24 h as compared to untreated cells. ~80 % of the cells were viable even at a high dosage of 200 nM of transfected oligos implying that these modifications are non-toxic to the cells. Next, we wanted to assess the impact of different SeLNA modification on miR-21 knockdown. To accomplish this, we used a dual luciferase plasmid harbouring target sites complementary to miR-21, downstream of Renilla luciferase gene. In MCF-7 cells where miR-21 expression is high, the luminescence due to suppression of target is low. However, after transfection of SeLNA antimiRs, we observe a higher luminescence suggesting the knockdown of miR-21. Thus, the functional potency of each of the SeLNA antimiRs was studied as shown in Fig 5. The various SeLNA incorporated antimiRs were transfected at a concentration range of 10 nM-100 nM in MCF-7 cells. We observed increment in luminescence signal of targets with increase in SeLNA modification. At 10 nM, the SeLNA0, SeLNA1 and SeLNA2 oligos did not impart much effect on the luminiscence signal, suggesting that it requires higher concentration to have its functional activity. At 20 nM-100 nM, significant upregulation of target was seen with antimiR SeLNA3 (upto 4 fold) and SeLNA4 (upto 5 fold) as compared to Scramble control. Thus, we see a concentration and modification dependent increase in luminiscence signal of targets as compared to scramble oligonucleotide, implying the inhibition of miR-21.


16

Page 17 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Even more convincing evidence for SeLNA as highly efficient set of oligonucleotides were observed after measurement of the relative levels of miR-21 inhibition upon their transfection in MCF-7 cells by qPCR after 24 h. We observed ~16.3% reduction in miR-21 levels with unmodified antimiR (SeLNA0) as compared to scramble oligo. The inhibition was concomitant with increasing number of SeLNA modifications, with ~23% reduction in SeLNA1, ~72% with SeLNA2, ~87% with SeLNA3 and ~91% with SeLNA4 modifications. MiR-21 post-transcriptionally downregulates its important target programmed cell death 4 (PDCD4) and promotes tumour progression and proliferation.40,

41

Since miR-21 and PDCD4

levels are negatively correlated, we tested the efficacy of SeLNA mediated downregulation of miR-21 on PDCD4 protein level expression. Here also, we observed a significant increase in PDCD4 levels with SeLNA3 (~2.2 fold) and SeLNA4 (2.5 fold) modifications (Fig S14). The excellent correlation of biophysical and in vitro data with in cellular studies encouraged us to evaluate the utility of these chemically modified oligonucleotides by monitoring the migratory and invasiveness of cancer cells. MiR-21 is overexpressed in diverse type of tumors and contributes to proliferation, invasion and metastasis.42 We envisaged the endpoint biological effect of SeLNA modifications on invasive and migratory properties of cancerous cell. We found SeLNA2 suppressed migration by ~38% (Fig 7) and invasion by ~26% (Fig 8). Furthermore, we found concomitant decrease in reduction in migration and invasion on increasing SeLNA modifications. SeLNA3 and SeLNA4 suppressed migration by ~42% and ~ 45% respectively. The invasiveness was reduced to ~40.3% with SeLNA3 and ~55.3% with SeLNA4.

Conclusions


17

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

miRNAs function as master regulators by modulating expression of thousands of mRNAs and controlling several pathways simultaneously. In this perspective, disease associated miRNAs have emerged as new therapeutic targets and have gained attraction as compared to conventional individual gene knockout strategies owing to their combinatorial effect relevant in complex diseases especially cancer. This has been highlighted by rapid progress from discovery to viable therapies in miRNA therapeutics. Promising results for ON-based drugs in clinical studies has led to revival of interests in chemical biology community to discover novel chemical modifications to improve target binding affinity and nuclease resistance. Here, we describe a novel and potentially remarkable SeLNA modification with improved therapeutic properties. By monitoring the CD properties for the SeLNA modified duplexes as compared to unmodified DNA strand, it was observed that SeLNA-DNA duplexes have tendency to adopt conformation resembling A-form geometry. We investigated the energetics of SeLNA modified DNA binding to its complementary RNA strand. Presence of SeLNA modification significantly increased the thermal stability as revealed by increase in Tm with incorporation of each modification in a step-wise manner. The presence of SeLNA modifications enhanced the thermal stability significantly implying formation of stable heteroduplexes. The binding affinities drastically increased for its target strand for SeLNA4 in the order of 108 M-1. The presence of SeLNA modification contributes to favourable enthalpy of hybridisation. Moreover, SeLNA modifications substantially stabilized the oligos to nucleases prone degradation and increased its half life in serum for upto 24 h and 36 h for heteroduplexes. The thermodynamics of hybridization, improved affinity to complement RNA and improved serum stability correlated well with the antisense efficacy observed in cellular assays. Efficient silencing of miR-21 was achieved till ~91% by SeLNA4 modified oligo as compared to only ~


18

Page 19 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

16 % inhibition achieved by unmodified oligo. Exploiting the inverse correlation of miR-21 and its functional target protein PDCD4, we found concomitant increase in tumor suppressor, PDCD4 target levels by western blot upon miR-21 knockdown by SeLNA modifications. The increase in PDCD4 levels were highest for SeLNA4 modified oligos, thereby emphasizing the correlation of increasing no. of SeLNA modifications with increasing miR-21 silencing. Overall, the most encouraging outcome was seen with SeLNA having four modifications with associated increase in potency for silencing miR-21 and reducing migration and invasion of cancer cells. Thus, novel SeLNA modification can be very well rendered as a potential therapeutics for inhibition of disease associated miRNAs. Not only it can result in functional inhibition of target miRNA but can also serve as valuable tool for identification and validation of miRNA targets and loss of function studies.

ASSOCIATED CONTENT Supporting Information Supplementary figures (FigS1-S14) and Tables (table S1-S3) are provided in the supplementary information. AUTHOR INFORMATION Author Contributions All authors have given approval to the final version of the manuscript. FUNDING INFORMATION This work was supported by the Council of Scientific and Industrial Research (CSIR) (Project Title: Genome Dynamics in Cellular Organization, Differentiation and Enantiostasis Project code BSC0123), India.


19

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 37

REFERENCES [1] Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116, 281-297. [2] Tomari, Y., Matranga, C., Haley, B., Martinez, N., and Zamore, P. D. (2004) A protein sensor for siRNA asymmetry, Science 306, 1377-1380. [3] Kim, V. N. (2005) MicroRNA biogenesis: coordinated cropping and dicing, Nat Rev Mol Cell Biol 6, 376-385. [4] Ha, M., and Kim, V. N. (2014) Regulation of microRNA biogenesis, Nat Rev Mol Cell Biol 15, 509-524. [5] Bushati, N., and Cohen, S. M. (2007) microRNA functions, Annu Rev Cell Dev Biol 23, 175205. [6] Lewis, B. P., Burge, C. B., and Bartel, D. P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets, Cell 120, 1520. [7] Ambros, V. (2004) The functions of animal microRNAs, Nature 431, 350-355. [8] Mattick, J. S., and Makunin, I. V. (2005) Small regulatory RNAs in mammals, Hum Mol Genet 14 Spec No 1, R121-132. [9] Soifer, H. S., Rossi, J. J., and Saetrom, P. (2007) MicroRNAs in disease and potential therapeutic applications, Mol Ther 15, 2070-2079. [10] Li, Y., and Kowdley, K. V. (2012) MicroRNAs in common human diseases, Genomics Proteomics Bioinformatics 10, 246-253. [11] Lu, J., Getz, G., Miska, E. A., Alvarez-Saavedra, E., Lamb, J., Peck, D., Sweet-Cordero, A., Ebert, B. L., Mak, R. H., Ferrando, A. A., Downing, J. R., Jacks, T., Horvitz, H. R., and Golub, T. R. (2005) MicroRNA expression profiles classify human cancers, Nature 435, 834-838. [12] Ventura, A., and Jacks, T. (2009) MicroRNAs and cancer: short RNAs go a long way, Cell 136, 586-591. [13] Jiang, K. (2013) Biotech comes to its 'antisenses' after hard-won drug approval, Nat Med 19, 252. [14] Janssen, H. L., Reesink, H. W., Lawitz, E. J., Zeuzem, S., Rodriguez-Torres, M., Patel, K., van der Meer, A. J., Patick, A. K., Chen, A., Zhou, Y., Persson, R., King, B. D., Kauppinen, S., Levin, A. A., and Hodges, M. R. (2013) Treatment of HCV infection by targeting microRNA, N Engl J Med 368, 1685-1694. [15] Hyrup, B., and Nielsen, P. E. (1996) Peptide nucleic acids (PNA): synthesis, properties and potential applications, Bioorg Med Chem 4, 5-23. [16] Fabani, M. M., Abreu-Goodger, C., Williams, D., Lyons, P. A., Torres, A. G., Smith, K. G., Enright, A. J., Gait, M. J., and Vigorito, E. (2010) Efficient inhibition of miR-155 function in vivo by peptide nucleic acids, Nucleic Acids Res 38, 4466-4475.


20

Page 21 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[17] Summerton, J., and Weller, D. (1997) Morpholino antisense oligomers: design, preparation, and properties, Antisense Nucleic Acid Drug Dev 7, 187-195. [18] Kloosterman, W. P., Lagendijk, A. K., Ketting, R. F., Moulton, J. D., and Plasterk, R. H. (2007) Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development, PLoS Biol 5, e203. [19] Meister, G., Landthaler, M., Dorsett, Y., and Tuschl, T. (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing, RNA 10, 544-550. [20] Hutvagner, G., Simard, M. J., Mello, C. C., and Zamore, P. D. (2004) Sequence-specific inhibition of small RNA function, PLoS Biol 2, E98. [21] Esau, C., Davis, S., Murray, S. F., Yu, X. X., Pandey, S. K., Pear, M., Watts, L., Booten, S. L., Graham, M., McKay, R., Subramaniam, A., Propp, S., Lollo, B. A., Freier, S., Bennett, C. F., Bhanot, S., and Monia, B. P. (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting, Cell Metab 3, 87-98. [22] Davis, S., Propp, S., Freier, S. M., Jones, L. E., Serra, M. J., Kinberger, G., Bhat, B., Swayze, E. E., Bennett, C. F., and Esau, C. (2009) Potent inhibition of microRNA in vivo without degradation, Nucleic Acids Res 37, 70-77. [23] Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hokfelt, T., Broberger, C., Porreca, F., Lai, J., Ren, K., Ossipov, M., Koshkin, A., Jakobsen, N., Skouv, J., Oerum, H., Jacobsen, M. H., and Wengel, J. (2000) Potent and nontoxic antisense oligonucleotides containing locked nucleic acids, Proc Natl Acad Sci U S A 97, 56335638. [24] Obad, S., dos Santos, C. O., Petri, A., Heidenblad, M., Broom, O., Ruse, C., Fu, C., Lindow, M., Stenvang, J., Straarup, E. M., Hansen, H. F., Koch, T., Pappin, D., Hannon, G. J., and Kauppinen, S. (2011) Silencing of microRNA families by seed-targeting tiny LNAs, Nat Genet 43, 371-378. [25] Esau, C. C. (2008) Inhibition of microRNA with antisense oligonucleotides, Methods 44, 55-60. [26] Stenvang, J., Petri, A., Lindow, M., Obad, S., and Kauppinen, S. (2012) Inhibition of microRNA function by antimiR oligonucleotides, Silence 3, 1. [27] K. Singh, S., A. Koshkin, A., Wengel, J., and Nielsen, P. (1998) LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition, Chemical Communications, 455-456. [28] Obika, S., Nanbu, D., Hari, Y., Morio, K.-i., In, Y., Ishida, T., and Imanishi, T. (1997) Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering, Tetrahedron Letters 38, 8735-8738. [29] Owczarzy, R., You, Y., Groth, C. L., and Tataurov, A. V. (2011) Stability and mismatch discrimination of locked nucleic acid-DNA duplexes, Biochemistry 50, 9352-9367. [30] Elmen, J., Lindow, M., Schutz, S., Lawrence, M., Petri, A., Obad, S., Lindholm, M., Hedtjarn, M., Hansen, H. F., Berger, U., Gullans, S., Kearney, P., Sarnow, P., Straarup,


21

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

E. M., and Kauppinen, S. (2008) LNA-mediated microRNA silencing in non-human primates, Nature 452, 896-899. [31] Elmen, J., Lindow, M., Silahtaroglu, A., Bak, M., Christensen, M., Lind-Thomsen, A., Hedtjarn, M., Hansen, J. B., Hansen, H. F., Straarup, E. M., McCullagh, K., Kearney, P., and Kauppinen, S. (2008) Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver, Nucleic Acids Res 36, 1153-1162. [32] Freier, S. M., and Altmann, K. H. (1997) The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes, Nucleic Acids Res 25, 4429-4443. [33] Kumar, R., Singh, S. K., Koshkin, A. A., Rajwanshi, V. K., Meldgaard, M., and Wengel, J. (1998) The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2'thio-LNA, Bioorg Med Chem Lett 8, 2219-2222. [34] Morihiro, K., Kodama, T., Kentefu, Moai, Y., Veedu, R. N., and Obika, S. (2013) Selenomethylene locked nucleic acid enables reversible hybridization in response to redox changes, Angew Chem Int Ed Engl 52, 5074-5078. [35] Wheeler, M., Chardon, A., Goubet, A., Morihiro, K., Tsan, S. Y., Edwards, S. L., Kodama, T., Obika, S., and Veedu, R. N. (2012) Synthesis of selenomethylene-locked nucleic acid (SeLNA)-modified oligonucleotides by polymerases, Chem Commun (Camb) 48, 1102011022. [36] Medina, P. P., Nolde, M., and Slack, F. J. (2010) OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma, Nature 467, 86-90. [37] Pfeffer, S. R., Yang, C. H., and Pfeffer, L. M. (2015) The Role of miR-21 in Cancer, Drug Dev Res 76, 270-277. [38] Kypr, J., Kejnovska, I., Renciuk, D., and Vorlickova, M. (2009) Circular dichroism and conformational polymorphism of DNA, Nucleic Acids Res 37, 1713-1725. [39] Yan, L. X., Wu, Q. N., Zhang, Y., Li, Y. Y., Liao, D. Z., Hou, J. H., Fu, J., Zeng, M. S., Yun, J. P., Wu, Q. L., Zeng, Y. X., and Shao, J. Y. (2011) Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth, Breast Cancer Res 13, R2. [40] Asangani, I. A., Rasheed, S. A., Nikolova, D. A., Leupold, J. H., Colburn, N. H., Post, S., and Allgayer, H. (2008) MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer, Oncogene 27, 2128-2136. [41] Frankel, L. B., Christoffersen, N. R., Jacobsen, A., Lindow, M., Krogh, A., and Lund, A. H. (2008) Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells, J Biol Chem 283, 1026-1033. [42] Zhu, S., Wu, H., Wu, F., Nie, D., Sheng, S., and Mo, Y. Y. (2008) MicroRNA-21 targets tumor suppressor genes in invasion and metastasis, Cell Res 18, 350-359.


22

Page 23 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[43] Marky, L. A., and Breslauer, K. J. (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves, Biopolymers 26, 1601-1620. [44] Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25, 402-408.


23

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

SCHEMES Scheme 1. Structure of SeLNA monomer.


24

Page 25 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

TABLES Table 1. Oligonucleotides used with SeLNA modifications (in boldface)

Nomenclature

Sequence (15 mer)

SeLNA0

5′ CAGTCTGATAAGCTA 3′

SeLNA1


SeLNA2


SeLNA3


SeLNA4


Table 2. Melting Temperatures (Tm) obtained by UV thermal melting experiments at two different salt concentrations increment 10 Tm increment Tm in 100 Tm per mM Na+ per modification modification

Duplex

Tm in mM Na+

SeLNA0

31.9 ± 0.2

SeLNA1

37.1 ± 0.3

5.2

52.9 ± 0.2

6.8

SeLNA2

43.8 ± 0.4

6.7

59.9 ± 0.4

7.0

SeLNA3

48.7 ± 0.2

4.9

65.8 ± 0.5

5.9

SeLNA4

51.7 ± 0.5

3.0

68.0 ± 0.5

2.2

46.1 ± 0.3

a

All the parameters were obtained from UV experiments conducted in 10 mM sodium cacodylate pH (7.0) or 10 mM sodium cacodylate containing 100 mM NaCl. Concentrations of heteroduplexes used were 1 µM. The values of Tm are within ± 0.5°C error.


25

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 37

Table 3. Thermodynamic Parameters obtained by ITC for the formation of Duplexes Dulpex

n

Ka ( x 107 M-1)

∆H( kcal/mol)

∆S (cal/mol/deg)

∆G (30°C) (kcal/mol)

SeLNA0

1.0

0.32

-20.9

-39

-8.8

SeLNA1

1.1

0.72

-49.0

-130

-9.2

SeLNA2

0.8

1.78

-80.5

-232

-10.2

SeLNA3

0.9

9.6

-91.9

-267

-10.9

SeLNA4

1.0

10.5

-129

-389

-11.1

a

All the parameters were obtained from the ITC experiments conducted in 10 mM sodium cacodylate pH (7.0) at 30°C. Concentration of RNA in the sample cell was 1.5 µM and each DNA strand with SeLNA modification was used in syringe with a concentration of 45 µM. Ka values are within 10%error and ∆H, ∆S, ∆G values are within 5% error obtained from triplicate experiments


26

Page 27 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

FIGURE LEGENDS Figure 1.

Circular dichroism spectra of dulpexes consisting of varying modifications-

unmodified (SeLNA0), SeLNA1, SeLNA2, SeLNA3 and SeLNA4 in 10 mM sodium cacodylate (pH 7.0) at 25°C. Spectra of single stranded DNA were also measured as compared to all duplexes. Figure 2. UV melting curves of unmodified (■), single (○), double (∆), triple(∇) and quadruple (◊) heteroduplexes of SeLNA in 10 mM sodium cacodylate buffer sodium cacodylate buffer with 100 mM NaCl. Figure 3. ITC titration plots for heteroduplex formation in 10mM sodium cacodylate buffer (pH 7.0) at 30°C for A) SeLNA0 (□) and B) SeLNA4 (◊) with target RNA. Calorimetric titrations were performed in which 45 µM of SeLNA-DNA strand was sequentially injected into the sample cell containing 1.5 µM of complementary RNA. Upper panel shows baseline corrected experimental data and the lower

panel shows the molar heat of binding for SeLNA0 (□) and

SeLNA4 (◊) against target 15-mer RNA. Figure 4. Serum stability of different SeLNA modified oligonucleotides in a) single stranded form and b) in SeLNA-RNA duplexes. The samples (7 µM each) were incubated at 37 °C in 10% FBS and withdrawn at the indicated time points. All samples were separated in 15% polyacrylamide gels and stained by SYBR gold. ds’ depicts double-stranded siRNA and ‘ss’ single-stranded. Figure5. Relative luminescence (RLU) of miR -21 target biosensor construct after treatment with different SeLNA modified antisense oligonucleotides. Highest RLU indicates maximum miR-21 inhibition which was observed after treatment of MCF-7 cells with SeLNA4. RLU is


27

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 37

calculated by normalizing with firefly luciferase. Error bars represent ± SD calculated from three independent biological experiments,* p

Systematic Evaluation of Biophysical and Functional Characteristics of

Recommend Documents