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Department of Bioorganic Chemistry

Design, Synthesis & Evaluation of Potential Antisense Oligonucleotides (AONs) and siRNA

All PDF files with the numbers refer to paper numbers in the original list of publications.

Recently much attention has been focused to develop antisense oligonucleotides (AONs) as therapeutic agents (Figure 1). Antisense technology is based on the fact that, once AONs are targeted to mRNA, they can down regulate the gene expression by multiple mode of mechanism, which includes the efficient binding of the AON to the target and/or by the degradation of the target in the heteroduplex by the enzyme RNase H [PDF 317]. Towards this direction variousmodifications of sugar, base and phosphate backbone of AONs have been attempted (see the Reviews below). But in most of the cases the modified AONs fails to activate RNase H, which is considered to be an important pathway of antisense action.
Figure 1. The catalytic RNase H promoted cleavage of the target mRNA through the formation of antisense Oligonucleotide/RNA Hybrid Duplex.  Kinetic scheme of the RNase H hydrolysis is shown in the bottom part of the cartoon, where D is AON (antisense Oligo); R is the target RNA; Kd1 is the equilibrium constant of dissociation of the heteroduplex DR; Kd2 is equilibrium constant of dissociation of the substrate-enzyme complex DRE.

Phosphorothioates, which had emerged as the first generation of antisense drugs, fails to gather momentum because of its non-antisense effects: high affinity to heparin-binding proteins and low sequence specificity (see: Stein's paper). It has been emerged that to act as a successful antisense drug, AONs should full fill the following criteria:
(i) specific target recognition by Watson-Crick base pairing,
(ii) good structural mimicry to the natural DNA-RNA,
(iii) activation of RNase H to promote the target mRNA cleavage
(iv) enhanced cellular uptake, and
(iv) enhanced resistance to various nucleases.

Our interest in this area has prompted us to design, synthesize and evaluate AONs which recruit RNase H. In this regard our interest is two-fold:
(i) We have recently shown that conjugation of various chromophores at the 5'-end [Nucleosides & Nucleotides 18, 2785-2818 (1999)] of AON can enhance the binding affinity to target RNA without altering the global helical conformation or flexibility of the AON/RNA duplex, keeping the RNase H eliciting power and nuclease resistance in tact. We are exploring these conjugated AONs as potential antisense candidates (see below).
(ii) We are also actively involved in the utility of Locked Nucleic Acids, as RNase H compatible antisense drugs (see below).

(1) Tethering of Various Chromophores/ hydrophobic moieties at 3' and or 5' end of AONs.

We have tethered various chromophores having different geometry, bulk, electron density, polarisability as well as hydrophobicity like phenazine, phenazinium, anthracene, pyrene [PDF 269,286], dipyridophenazine-DPPZ- [PDF 306], [Ru (phenanthroline) 2 DPPZ)2+] [PDF 319] to the 3'and/or 5' end of AONs and their antisense potential has been evaluated. All of them showed enhanced binding affinity to the target, augmented stability towards nucleases and recruitment of RNase H in a faster or comparable rate to the non-conjugated counterpart [PDF 314].

Groups having different hydrophobicities like cholestrol, cholic acid and cholic acid triacetate have also been conjugated to the 3'-end of the AONs, keeping in mind their applicability for easy delivery into cells. A detailed kinetic analysis of RNase H hydrolysis has been performed where various 3'-conjugated AONs were compared with the non-conjugated native counter part [PDF 332]. It has thus emerged that 3'-DPPZ tethered AON has the highest value of Vmax/Km ratio, thereby showing its high capability to recruit the RNase H.

(2) Design and Synthesis and evaluation of Various Conformationally Locked Nucleic Acids.

Conformationally locked Nucleic Acids having natural phosphate backbones have attracted much attention recently (See the recent Reviews below), because of its unprecidential binding affinity to the target. Among the various sugar modified locked nucleic acids there is only one qualitative report [PNAS, 97, 5633-38 (2000)] regarding the activation of RNase H, where the target is cleaved relatively at a reduced rate compared to the unmodified counterpart. We are working on development of novel compounds as well as on the procedures that allowed synthesis and supply of sufficient amounts of chemically modified antisense oligonucleotides (AONs) and short interfering RNAs (siRNAs) constructs to permit their evaluation as gene silencing agents in vivo and in vitro. The aim is to achieve gene down-regulation at the RNA level by antisense mechanism or the RNA interference action (RNAi) relying upon deployment of our in-house developed chemically modified AONs and siRNAs as potential therapeutic agents against various diseases including viral infections, cancer, inflammatory diseases etc. The chemical modification of the antisense or siRNA strand were performed to enhance the (A) target affinity, (B) stability in the blood serum and (C) tissue specific delivery and (D) RNase H recruitment for the antisense action in order to achieve intended gene silencing and to improve overall pharmacokinetic properties. A short list of achievement during the report period (2007-2010) is as follows:We have synthesized and evaluated various North-type constrained  1',2'- and 2',4'-modified nucleotides incorporated into AONs for their antisense potential:

1',2'- and 2',4'-Conformationally locked sugar modified nucleosides
PDF 363, 364
PDF 313, 315, 329, 333
carba-LNA and carba-ENA
PDF 366, 369
Carba-LNA, Carba-ENA
carba-LNA: PDF 372, 379, 387, 393, 396, 397;
carba-ENA: PDF 377, 399.
double constrained
Double Sugar and Phosphate Backbone-Constrained Nucleotides
(Sp)-D2-CNA-dT 17a (A) and (Rp)-D2-CNA-dT 17b (B)
PDF 381
Methylene-bridged hexopyranosyl nucleoside (BHNA)
PDF 378, 380

(A) 1',2'-modifications:  oxetane- and azetidine-types

Oxetane modifications

We have reported the synthesis and antisense properties of the conformationally constrained oxetane-C and -T [PDF 313, 315, 329, 333, 336 (C)] containing oligonucleotides, which have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells. This series had been completed by synthesis of the oxetane-A and oxetane-G nucleosides [350 (G, A), 360] as well as be their incorporations into antisense oligonucleotides (AONs), and by comparison of their structural and antisense properties with those of the T and C modified AONs (including the thermostability and RNase H recruitment capability of the AON/RNA hybrid duplex by Michaelis-Menten kinetic analyses, their resistance in the human serum, as well as in the presence of exo and endonucleases).

Azetidine modification

The North-East type conformationally constrained nucleosides have been achieved also in the case of 1',2'-azetidine type of modification.
The synthesis of novel 1',2'-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) ''azetidine'' pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks for automated solid-phase oligonucleotide synthesis is reported [PDF 363, 364]. The novel bicyclonucleoside ''azetidine'' monomers were synthesized by two different strategies starting from the known sugar intermediate 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose. Conformational analysis performed by molecular modeling (ab initio and MD simulations) and NMR, showed that the azetidine-fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in the range of 44.5º to 53.8º and sugar puckering amplitude (fm) of 29.3º to 32.6º for the azetidine modified T, U, C, and 5Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes show that the azetidine fused nucleosides [PDF 363, 364] display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues [PDF 314, 315, 329, 333].

(B) 2',4'-modifications:  aza-ENA, carba-LNA, carba-ENA

Aza-ENA modification

The 2'-deoxy-2'-N,4'-C-ethylene bridged thymidine ''aza-ENA-T'' [PDF 366, 369] has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7o < P < 27o, 44o < fm < 52o). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and the equatorial N-H in 12a. The latter is thermodynamically preferred while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is so far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly-modified 15-mer antisense oligonucleotides (AON). Their AON/RNA duplexes showed a Tm increase of 2.5 to 4 °C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3'-degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.

For further details on aza-ENA see the original papers [PDF 366, 369373, 374]

Carba-LNA and Carba-ENA modifications

Carba-LNA and carba-ENA

Figure 1. Developed by us family of novel 2',4'-fused 5- (LNA-type) and 6-(ENA-type) membered carbocyclic nucleotides

Our most recently devised, developed and synthesized category of the conformationally locked nucleotides (2007-2010) consists of novel conformationally-constrained 2',4'-fused carbocyclic nucleotide analogs of LNA and ENA thus extending our arsenal of chemical tools to approach intended goals of gene-silencing research pursued in our research. PDF 314, 315] Until 2007 the in-house developed family of the conformationally constrained nucleotides consisted of 1',2'-fused oxetane- and azetidine-modified nucleotides as well as 2',4'-fused aza-ENA nucleotide [see our papers oxetane-T, -C, -A, -G [PDF 314, 315, 329, 333, 335 (C), 350 (G, A), 360], azetidine-T [PDF 363, 364], aza-ENA-T,-A [PDF 366, 369, 373, 374]. The oxetane and azetidine modification have been successfully incorporated into the purine and pyrimidine modified nucleotides as well as aza-ENA pyrimidine [Chattopadhyaya et al. J. Am. Chem. Soc. 128 (47), 15173 (2006) PDF 366]. The main synthetic efforts during 2007-2010 period has been concentrated on development of novel 2',4'-fused carbocyclic LNA- and ENA analogs (Figure 1) and discovering their biological action as antisense and siRNA oligos [for these carba-LNA/ENA works see  carba-LNA: PDF 372, 379, 387, 393, 396, 397; carba-ENA: PDF 377, 399], review papers PDF 390, 401

   Yet we have tested our novel carba-LNA/ENA modified AONs and siRNAs for the duplex thermal and blood serum stability  and down regulation potential against cognate RNAs of Huntington’s disease and HIV (see Figure 2 for comparison of the blood serum stability of LNA, carba-LNA, carba-ENA and aza-ENA modified AONs). We have also established worldwide collaborations which allowed us to utilize our new chemical modifications for testing in the antisense and RNA interference actions against target RNA in the whole cell based assays. We are now developing in vivo and animal models using our synthetic delivery stabilitysystems. In this process, we have learnt how our novel chemically modified AONs and siRNAs with, conformationally constrained nucleotides (oxetane, aza-ENA, carbocylic-LNA and -ENA or hexitol or LNA modified oligos) have worked in comparison with other commercial derivatives, 2’-aminoethoxy, 2’-methoxy or 2’-fluoro modified sugars, seco-RNA derivatives, ANA, HNA by screening to explore the tolerated site of modifications in siRNA construct. We have reported this comparative study with 21 different types of chemical modifications on siRNA efficiency and cell viability using a total of 2160 modified siRNAs [Chattopadhyaya et al. Nucl Acids Research, 2009, 37, 2867, PDF 382]. We showed that the siRNA duplex should preferably be destabilized by modifications in the SS 3’-end rather than at the SS 5’-end in order to facilitate AS incorporation into RISC. Chemically modified siRNA overhangs are also found to favour AS incorporation into RISC providing various conditions of SS and SA chemico-physical properties are met. Thus SS should contain disfavoured overhangs such as UNA, HM and LNA-LNA, whereas the AS should contain favoured overhang motifs such as LNA-LNA-RNA ns. These guidelines for enhancing siRNA activity through chemical modifications are further being utilized. We are seeking to further exploit the RNAi technology and its applications to continue development of chemically-modified nuclease stable siRNAs and their mimics to improve delivery inside the cell with favorable pharmacokinetics with the aim to develop therapeutics. Further development is also needed to improve delivery of modified siRNA in vivo.

For further details see the original papers:
oxetane-T, -C, -A, -G [PDF 314, 315, 329, 333, 335 (C), 350 (G, A), 360]
azetidine-T [PDF 363, 364]
aza-ENA-T, -A [PDF 366, 369, 373, 374 ]
carba-LNA: [PDF 372, 379, 387, 393, 396, 397]
caba-ENA: [PDF 377
, 399]
review papers: [PDF 390, 401, 317]

Our carba-LNA and carba-ENA compounds have shown remarkable endo- and exo- nuclease stability and high target activity (see comparison below in Figure 3).


Figure 3. Comparison of the target affinity (thermal stability) and nuclease resistance of carba-LNA and carba-ENA families synthesized in our lab with those of the LNA. All the carba-LNA compounds (marked in red) have shown very good target affinity, several of them are good as LNA modification. As for nuclease resistance, type II, type III, type V and Type VII showed very much better nuclease resistance than LNA and carba-LNA.  Taking into account technological importance of both properties, type II, type VI and type VII modifications could be excellent candidates as antisense therapeutic agents.

Double Sugar and Phosphate Backbone-Constrained Nucleotides

Two diastereomerically pure carba-LNA dioxaphosphorinane nucleotides [(Sp)- or (Rp)-D2-CNA], simultaneously conformationally locked at the sugar and the phosphate backbone, have been designed and synthesized [PDF 381]. Structural studies by NMR as well as by ab initio calculations showed that in (Sp)- and (Rp)-D2-CNA the sugar is locked in extreme North-type conformation with P =11° and Φm =54°; (ii) the six-membered 1,3,2-dioxaphosphorinane ring adopts a half-chair conformation and the phosphate backbone is double-locked (for details see [PDF 381]). It has been found that F- ion can catalyze the isomerization of pure (Sp)-D2-CNA or (Rp)-D2-CNA to give an equilibrium mixture (K = 1.94). It turned out that at equilibrium concentration the (Sp)-D2-CNA isomer is preferred over the (Rp)-D2-CNA isomer by 0.39 kcal/mol.

Double constrained
The chemical reactivity of the six-membered dioxaphosphorinane ring in D2-CNA was found to be dependent on the internucleotidic phosphate stereochemistry. Thus, both (Sp)- and (Rp)-D2-CNA dimers (17a and 17b) were very labile toward nucleophile attack in concentrated aqueous ammonia [t1/2 = 12 and 6 min, respectively] to give carba-LNA-6′,5′-phosphodiester (21) ≈ 70-90%, carba-LNA-3′,5′-phosphodiester (22) ≈ 10%, and carba-LNA-6′,3′-phosphodiester (23) <10%. In contrast, the (Sp)-D2-CNA was about 2 times more stable than (Rp)-D2-CNA under hydrazine hydrate/pyridine/AcOH (pH = 5.6) [t1/2 = 178 and 99 h, respectively], which was exploited in the deprotection of pure (Sp)-D2-CNA-incorporated antisense oligodeoxynucleotides (AON). Thus, after removal of the solid supports from the (Sp)-D2-CNA-modified AONs by BDU/MeCN, they were treated with hydrazine hydrate in pyridine/AcOH to give pure AONs in 35-40% yield, which was unequivocally characterized by MALDI-TOF to show that they have an intact six-membered dioxaphosphorinane ring. The effect of pure (Sp)-D2-CNA modification in the AONs was estimated by complexing to the complementary RNA and DNA strands by the thermal denaturation studies. This showed that this cyclic phosphotriester modification destabilizes the AON/DNA and AON/RNA duplex by about -6 to -9 °C/modification. Treatment of (Sp)-D2-CNA-modified AON with concentrated aqueous ammonia gave carba-LNA-6′,5′-phosphodiester modified AON (∼80%) plus a small amount of carba-LNA-3′,5′-phosphodiester-modifiedAON(∼20%). It is noteworthy that carba-LNA-3′,5′-phosphodiester modification stabilized the AON/RNA duplex by +4 °C/modification (J. Org. Chem. 2009, 74, 118), whereas carba-LNA-6′, 5′-phosphodiester modification destabilizes both AON/RNA and AON/DNA significantly (by -10 to -19 °C/modification), which, as shown in our comparative CD studies, that the cyclic phosphotriester modified AONs as well as carba-LNA-6′,5′-phosphodiester modified AONs are much more weakly stacked than carba-LNA-3′,5′-phosphodiester-modified AONs.

For further details see the original publication: PDF 381

(D) Methylene-bridged hexopyranosyl nucleoside (BHNA)

A new member of hexopyranosyl nucleoside family, methylene-bridged hexopyranosyl nucleoside (BHNA), has been synthesized through generation of carbon radical at C6′ in [6′S-Me, 7′S-Me]-carba-LNA-T nucleoside, followed by rearrangement to C4′ radical which was quenched by hydrogen atom to give BHNA [PDF 378, 380]. The stereoelectronic requirement for this unusual radical rearrangement has been elucidated by chemical model building and ab intio calculations to show that the coplanarity of the single electron occupied p-orbital at C6' with σ*O4'-C4' plays an important role for the rearrangement reaction to take place. The solution structure of BHNA has also been studied using NMR as well as by ab initio calculations. The new six-membered pyranosyl ring in BHNA, unlike other known hexopyranosyl nucleosides, adopts a twist conformation, with base moiety occupying the axial position while 3′-hydroxymethyl and 4′-hydroxyl occupying the equatorial position.

This hyper-constrained BHNA nucleotide has been incorporated into the antisnese oligonucleotides (AON) [PDF 378] which have shown more preference for binding toward the complementary RNA (Tm loss by ca 5°C) than that with the complementary DNA (Tm loss by 10°C), vis-à-vis corresponding native duplex. The origin of reduction of Tm of the duplexes formed by the BHNA incorporated AON and the complementary RNA or DNA has further been investigated by thermal denaturation study with the single-mismatched DNA or RNA, CD spectroscopy, RNase H digestion study, as well as by molecular model building. These studies have shown that the introduction of BHNA causes only a limited local conformational perturbation in the AON/RNA heteroduplex, whereas it affects the global conformation in the AON-DNA duplex. BHNA incorporated AONs also show improved stability in the human blood serum, which may prove to have some potential therapeutic application.

For further details on methylene-bridged hexopyranosyl nucleoside (BHNA) and its antisense properties see PDF 378, 380

(3) Conformational analysis of antisense oligonucleotide/RNA duplexes as substrates for RNase H

Two complementary methods - (a) high-field NMR, and (b)  ab initio (for the individual modified AONs) and MM/MD (for AON/DNA duplexes) simulations in the aqueous environment - are used to draw a correlation between the structure of the native and oxytane-modified DNA(AON)/RNA hybrid and its properties as a substrate for the RNase H, as well as to point the crucial structural requirements for the modified AONs to preserve their RNase H potency. Thus, we are investigating conformational changes in a set of hybrid AON/RNA duplexes (10 and 15 mers), consisting of a series of analogous single bicyclic psiconucleoside modified AONs with fixed North conformation and the complementary target RNA.

To shade light on the recognition, interaction, intermolecular hydrolysis, and in general the mechanism of the RNase H promoted cleavage reaction of the AON/RNA hybrid duplex, a model docking of the RNase H to the substrate (i.e. hybrid duplex) is to be performed following the dynamics of their interaction (click on this link to see a model structure of the enzyme docked to AON/RNA duplex). Since it is not possible to obtain experimental crystal structure of this reactive complex, the theoretical modeling seems to be a sound way to investigate the processes involved in this interaction and the conformational readjustments that result from the ternary complex formation before the onset of the hydrolysis. The role of Mg2+  ion in this cleavage reaction is important to address at the catalytic center of this reaction.

Reviews on the Antisense technology:

1) Uhlmann, E.; Peyman, A: Antisense oligonucleotides: A new therapeutic principle. Chemical Reviews 1990, 90: 543-584.
2) Mesmaekar, A.D.; Haner, R.; Martin, P.; Moser, E.H. Antisense oligonucleotides. Acc. Chem. Res. 1995, 28:366-374
3) Frier, S.M.; Altmann, K-H. .The ups and downs of nucleic acid duplex stability:structure stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 1997, 25:4429-4443
4) Crooke, S. T. Progress in Antisense Technology: The End of the Beginning.2000, Methods in Enzymology, 313, 3-45.
5) Zamaratski, E., Pradeepkumar, P. I., Chattopadhyaya, J. A critical survey of the structure- function of antisense oligonucleotide/RNA hetroduplex as a substrate for RNase H. 2001, .J Biochem. Biophys. Methods, 48, 189-208. [PDF 317].
6) Stein, C. A.; The experimental use of antisense oligonucleotides: a guide for the perplexed. J. Clin. Invest. 2001, 108, 641-644.
7) Kvaerno, L., Wengel, J. Antisense molecules and furanose conformations-is it really that simple? Chem. Commun., 2001, 1419-1424.
8) Herdewijn, P. Conformationally restricted carbohydrate-modified nucleic acids and antisense technology. Biochimica et Biophysica Acta. 1999, 1489:167-179.

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