The story so far...

1.1.1. miRNA in Disease

 

miRNAs in Cancer and apoptosis:

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. We found that the over expression of miR-23a,27a,24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway.  Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Moreover, we also showed that over expression of miR-23a-27a-24-2 sensitized HEK293T cells to TNF-a cytotoxicity.

 

We have also initiated studies on miRNA profiling in cancer samples collected in collaboration with clinical collaborators.  A cluster of miRNAs was found to be differentially expressed in Glioma and certain specific miRNA were implicated in hepatocellular carcinoma.  Further, we have initiated studies on development of antagomiRs to modulate the exprerssion of well known cancer related miRNAs like miR-21, miR-34 and miR-27a.

 

Anti-viral role of miRNAs:

 

Computational predictions revealed earlier that the HIV-1 genome includes regions that may be potentially targeted by human miRNAs. The functionality of predicted miR-29a target site in the HIV-1 nef gene was established through experimental studies. Locked nucleic acid modified anti-miRNAs targeted against hsa-miR-29a and 29b specifically reversed the inhibitory effect mediated by cellular miRNAs on the target site. Ectopic expression of the miRNA results in repression of the target Nef protein and reduction of virus levels. Our results show that the cellular miRNA hsa-miR29a downregulates the expression of Nef protein and interferes with HIV-1 replication.

We are also interested in studying the expression level of this miRNA in cohorts of AIDS patients and normal subjects.  Therefore, we collected samples from HIV patients showing various rates of disease progression and assessed the miRNA level.  Preliminary results indicate that the miRNA is expressed highly in Long term progressors, while rapid progressors have lower levels of the miRNA.

The discovery of viral encoded microRNAs, especially from a family of oncogenic viruses, has attracted immense attention towards the possibility of microRNAs as critical modulators of viral oncogenesis. The host-virus crosstalk mediated by microRNAs, messenger RNAs and proteins, is complex and involves different cellular regulatory layers.  

 

1.1.2. Correlating transcriptome, miRnome and proteome

 

miRNA proteomics: The interaction of the miRNAs with the target transcript can accommodate for wobble base pairing or even non-complementarity. This makes prediction of targets, a highly inaccurate task. Currently, the most commonly used method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and the evolutionary conservation of the targeted regions in the mRNAs.  Such algorithms predict 1000s of targets for any miRNA, necessitating tedious experimentation to identify handful of true functional targets. Recently developed high throughput experimental methods such as HITS-CLIP that rely on the co-immunoprecipitation of miRNA and target mRNA also do not ensure functional relevance of the targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional targets of a miRNA in the cell. We used SILAC based proteomics on HEK293T cells to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. miR-34a is a tumor suppressor miRNA with a known role in regulation of cell proliferation. The differences in protein expression we found in the miR-34a over-expressing cells were consistent with its role in cell cycle regulation. Intriguingly, the over-expression of miR-29a, a pro-apoptotic miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of an unexpected set of proteins.  We compared the miR-34a and miR-29a targets we identified through proteomics to the targets identified by computational prediction as well as by experimental methods such as HITS-CLIP and 3’UTR reporter assays and found a high degree of congruence between them. We conclude that miRNA-proteomics can be an efficient approach for identifying direct targets of miRNA that are functionally relevant. We propose miRNA-proteomics as a high-throughput alternative for miRNA target identification that is both sensitive and unbiased. This approach is valuable for rapid assessment of the impact of a miRNA on the proteome and thus discovering its biological role in the cell.

 

 

1.1.3. miRNA & genomic context:

The efficiency of binding of miRNAs to target transcripts depends on the sequence as well as intramolecular structure of the transcript. Single Nucleotide Polymorphisms (SNPs) can contribute to alterations in the structure of regions flanking them, thereby influencing the accessibility for miRNA binding. Collated information on SNPs that affect 3’UTR structure and miRNA function was ported in a MySQL database accessible through the URL: http://miracle.igib.res.in/ dbSMR. This tool was later used in prediction of a model for incomplete penetrance and variable expressivity (see below).

 

We have comprehensively curated genetic variations in miRNA loci in the human genome and established a computational pipeline to assess potential functional consequences of these variants along with methods for systematic curation and reporting of variations in these loci. The data is made available on the Leiden Open (source) Variation Database (LOVD) platform at http://genome.igib.res.in/mirlovd to provide ease of aggregation and analysis and is open for community curation efforts.

 

1.1.4. miRNA in Development

Incomplete penetrance and variable expressivity are non-Mendelian phenomena resulting in the lack of correlation between genotype and phenotype. These phenomena are seen most often in dominantly inherited diseases, implying that they are sensitive to concentration of the gene product. In this framework and the advances in understanding the role of microRNA (miRNA) in fine-tuning gene expression at translational level, we proposed miRNA-mediated regulation as a mechanism for incomplete penetrance and variable expressivity. We finally showed that in target genes important in alagille syndrome and cancer, the presence of miRNA binding sites at 3( UTR, co-expression of target gene–miRNA pairs for genes showing incomplete penetrance, and variable expressivity derived from available data lend support to our hypothesis. Single nucleotide polymorphisms in the miRNA target site facilitate the implied differential targeting of the transcripts from homologous alleles. We demonstrated this using target UTR clones designed to assess the differential targeting by miRNAs.

 

We have also initiated a zebrafish screen for miRNAs predicted to play a role in development of blood cell lineage and vasculature.  miRNA mimics were injected into embryos at single cell stage and subsequently the phenotypic effects observed.  One of the striking phenotypes involve a fish with a severe hemorrage in the head region.  Functional characterization of these miRNAs is underway.  Sensor assays for screening of targets was also carried out.  Finally, key targets of miR-143 was identified and validated in zebrafish.

 

 

 

1.1.5. Development of modified Nucleic Acids to enhance miRNA –target hybridization

 

Design and Synthesis of positively charged  conformationally constrained  PNA analogs: 

TANA modified sequences originally developed for detection of miRNAs were synthesized at NCL and further tested at IGIB.

 

Code No.

Sequence

( c,u,g  = 2’-OMe )

O D / ml

Conc.

µmol/ml

Mass (Maldi Tof)

Cal.                       Obs.

P-2’-ome-1

5’AcAACCAGCTAAGA3’

13.0

75.58

4279.8              4282.55

P-2’-ome-2

5’AcAACcAGCTAAGA3’

7.6

44.18

4309.8              4310.30

P-2’-ome-3

5’AcAACcAGCuAAGA3’

20.4

118.60

4325.8              4323.42

P-2’-ome-4

5’AcAACcAGCuAAgA3’

7.6

44.18

4355.0              4353.24

TANAcc-1

5’ACA Acc AGC TAA GA 3’

5.4

31.395

4256.80     4254.03

Detection of the miR34a from zebrafish embryos using modified backbone oligonucleotides showed that TANA had a sensitivity comparable to LNA and O-methyl modifications.

 

1.1.6. Sugar based small molecule to recognize RNA secondary structure in Untranslated regions of RNA

It is known that the cationic antibiotics of the aminoglycoside family bind to several RNA motifs by specifically recognizing their 3D structure.  In particular, they have been proven to exert an inhibitory effect on the binding of the Tat-protein to the TAR-RNA. The structure of the TAR-RNA in complex with neomycin B has been investigated by NMR.  It has been shown that the neomycin B binds the TAR-RNA in the minor groove of the lower stem, which results in an allosteric inhibition of Tat binding to TAR caused by a reduction of the major groove volume in the upper stem. Aminoglycosides are valuable small molecules to study the structural basis of RNA recognition and inhibition of function by cationic organic molecules. However, their use as RNA targeting drugs is impeded by several drawbacks, such as low specificity, high toxicity and development of resistance. These considerations underline the continuous need for new compounds that share an aminoglycosidic scaffold but can be easily derivatized to obtain drug leads with a better pharmacological profile.

The synthesis of this unique neooligomer exploit the concepts of Click chemistry wherein a suitable alkyne and an azide will be reacting in the presence of Cu(I) to give a conjugate of both the reacting partners. 

Cartoon representation of the proposed library of cyclic neooligoaminodeoxysaccharide

 

This class of aminoglycoside analogues is unique in the fact that they are easily synthesized via 1,3-dipolar cycloaddition and provide novel methods of conjugation and functional group compatibility.

Small molecules that can modulate the function of miRNA are required in order to understand their function as well as therapeutic agents in variety of biological processes where miRNAs are involved.  We took a two-pronged approach to develop the small molecule identification programme.  The first is to modify natural product scaffolds and the second one is to make random but de novo designer amino glycosides which can be checked for miRNA binding studies.  In the first instance, we have selected Berberine, a Chinese plant based natural product with variety of biological functions.  Berberine has been found in many medicinally important plants including Coptis chinensis (Coptis or goldenthread), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), and Coscinium fenestratum.  

Our synthetic endeavour started with the preparation of C-9 modified berberine analogues using click reaction by an SN2 displacement.  All these compounds are sent to Dr. G Sureshkumar at the Indian Institute of Chemical Biology, Kolkata for biophysical studies and Dr. Souvik Maiti for biology work. (see Below)

It has been documented in the literature that aminoglycosides are potential RNA binding compounds and during the process of incubation of the project, dimeric to tetrameric polyhydroxylated amine-containing compounds were reported to inhibit some RNA molecules.  The main hypothesis given for the binding was the electrostatic interaction between the phosphate back bone and amine of the sugar molecule.  They have carried out with various inositol derivatives.  In this study, a library of dimeric to tetrameric carbohydrate molecules were planned to synthesize with multiple amine groups being displayed on their peripherial sites so that they can have interactions with targeted miRNA.   We exploited acid mediated glycosylations, Sonogashira reaction for dimerization and esterification for installing the amino acid moiety.  Some of the members of the library are shown in Figure 1.  It is pertinent to mention that interesting observations while performing this investigation resulted in the identification of a novel glycosyl donor (Chem Commn 2009).  Synthesized Dimeric sugar derivatives will be sent to Dr. Souvik Maiti of IGIB for further biology work.

The work at IICB has been directed in three different aspects

1.                   Understanding and characterizing the binding of small molecules to diverse RNA structures.

2.                   Towards synthesis of thermodynamically stable RNA oligomers.

3.                   Effects of cellular microenvironment on miRNA activity in mammalian cells

 

1. Understanding and characterizing the structural and energetic aspects of binding of small molecules to various RNA structures

Rational design of RNA based small molecule therapeutics requires detailed knowledge of the structural aspects of RNA itself on one hand and the molecular nature of the interaction profile to various conformations that RNA can adopt. The development of RNA binding molecules are still at the primitive stage due to the complex diversity of the various RNA structures and also the lack of information on the mode mechanism, specificity and energetics. In this context studies were performed studies to enhance the fundamental knowledge in this area by studying the interaction of some naturally occurring alkaloids that were previously characterized to be DNA binders with natural ans synthetic RNA structures.

(a) Binding of small molecule alkaloids to tRNA

The binding of two natural isoquinoline alkaloids berberine and palmatine and a synthetic coralyne to tRNAPhe was investigated using various biophysical techniques and also molecular modeling. The alkaloid binds to the RNA exhibiting positive cooperativity. A partial intercalative binding was envisaged from absorption, fluorescence and circular dichroism studies. Differential scanning calorimetry showed three melting transitions revealing heterogeneity in the RNA fine structure that were all affected on binding of the alkaloids. Coralyne also exhibited positive cooperativity in its binding to tRNA. Evidences for a true intercalative binding of the alkaloid were evaluated from fluorescence quenching studies with significant perturbation of the RNA conformation. The affinity of coralyne to tRNA was in the order of 106 M-1, while that of berberine and palmatine was two orders of magnitude lower. The binding of coralyne to tRNA was predominantly enthalpy driven. It was also observed that coralyne interaction was accompanied by moderate heat capacity change and showed enthalpy entropy compensation that confirmed the involvement of multiple weak non-covalent interactions in the binding process. Molecular docking studies further confirmed the proposal of partial intercalative binding of berberine and palmatine and true intercalation posture of coralyne on tRNA (see figure).

(b) Binding of alkaloids to double stranded RNA structures

The binding aspects of the alkaloids berberine, palmatine and coralyne to double stranded (ds) RNA polymers poly(A).poly(U), poly(I).poly(C) and poly(C).poly(G) was investigated to elucidate the specificity of these alkaloids to ds RNA. Cooperative binding with affinity in the order 104 M-1 was observed. Binding was characterized to be partial intercalation from various techniques for berberine and palmatine, but true intercalation for coralyne with significant conformational change in the RNA polymers on alkaloid binding. Energetic of the binding revealed that the binding was driven by negative enthalpy and positive entropy changes with significant hydrophobic contribution in the binding process. Overall, coralyne binding was stronger than the other two alkaloids and may be related to the structural differences of the alkaloids.

Studies on the interaction of two classical DNA binding molecules proflavine and hoechst to the A- and HL-conformation of the RNA polymer with CG sequences revealed significant insights into the molecular aspects of interaction in terms of both structural and energetic of small molecules to different RNA conformations.

(c) Self structure induction in poly(A) by small molecules: an approach to RNA based drug design

For the first time our studies have revealed that the binding of coralyne and sanguinarine induced self structure in poly(A) molecules. We have reviewed the binding aspects of several small molecules to poly(A) in terms of their structures and ability to bind to poly(A) and induce self structure  and the implications.

(d) Understanding the base specificity of alkaloid-RNA interaction

The remarkably high binding affinity of coralyne to various RNA structures prompted investigation of the binding with single stranded RNAs. Results from competition dialysis, absorbance, and fluorescene revealed that c binds strongly to polypurines of poly (G) and poly(I) compared to polypyrimidines, poly(U) and poly(C). Partial intercalative binding due to stacking in between the bases was envisaged. Similar to its binding to ds RNAs and tRNA, significant heat capacity change and enthalpy-entropy compensation phenomena in the interaction process confirmed the involvement of multitude of noncovalent forces in the complexation. A complete structural and thermodynamic profile of the binding has been presented.

2. Towards synthesis of thermodynamically stable RNA oligomers: Spirocyclic Nucleosides

Several oligonucleotide analogues containing bi- and tricyclic carbohydrate mimics have displayed enhanced stabilities as compared to unmodified oligonucleotide. Substitution of the natural furanose sugar moiety results in a reduction of conformational freedom. This reduction can have an overall effect by increasing the thermodynamic stability of oligonucleotides. Paquette introduced the concept of spirocyclic restriction in nucleosides through insertion of a carbocyclic ring (a bulky substituent) at C-4′ of furanose/ thiofuranose rings. This was expected to fix the glycosyl torsional angle around the C-4′ bond, while the void space below C-4′ would be sufficient to avoid nonbonded steric superimposition.

In addition, the free radical-induced degradation of the ribose ring of nucleosides by C-4′-H abstraction can be precluded. Various other synthetic routes to C-2′-spiro, C-3′- spiro, and C-4′-spironucleosides as conformationally restricted or biased analogues have appeared in the literature. Some of these nucleosides display anti-HIV and antivirus activity. The work encouraged us to take up the synthesis of spirocycles based on five-membered heterocyclic rings from a D-glucose-derived precursor carrying two hydroxymethyl groups at C-4. One of these groups was planned to be utilized to introduce a vinyl group via oxidation and Wittig reaction, and then converted to a hydroxyethylgroup by hydroboration-oxidation reaction. Subsequent intra/intermolecular cyclization through the participation of oxygen, nitrogen, and sulfur nucleophiles was expected to furnish the desired heterocyclic systems. In the process, we also encountered newer 4,5-spirocyclic and bicyclo[3.3.0]octane systems. The products were elaborated to interesting spirocyclic and bicyclic nucleosides in addition to an unexpected 5,5,5-tricyclic conformationally locked nucleoside.

Toward this aim we have generated few analogues of spironucleosides, which could be elaborated further to RNA oligonucleotides (a few structures are shown) for various physical studies.

1.1.7. Designing Lipid based Transfection Vectors for Packaging and Target Specific Delivery of Small RNA.

 

Signal transducers and activators of transcription (STAT) proteins are a family of transcription factors first characterized for their role in cytokine signaling. However, as additional members of this protein family were discovered, they caught the attention of cancer biologists as potential mediators of growth control and possibly oncogenic events. In particular, it became recognized that STAT3 is stimulated by classic growth-promoting signals, such as activated growth factor receptors.  However, STAT3 is not targeted by any natural miRNAs.  We designed artificial miRNAs against STAT3 to test if they can be used to modulate Stat3 functions. The artificial miRNA was designed and cloned as a pre-miRNA and the clone was used in combination with DNA delivery agents developed by IICT.  This work is being extended further by designing artificial miRNA against other cancer related genes like Hsp90 and delivering them using a glucocorticoid receptor mediated delivery system developed by Dr. Rajkumar Banerjee, IICT.  We have now established that the artificial miRNA results in reduced expression of Hsp90 in cell lines.  In vivo testing is underway.

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