Synthetic siRNA targeting human papillomavirus 16 E6: a perspective on in vitro nanotherapeutic approaches.

High-risk human papillomaviruses infect skin and mucosa, causing approximately 5% of cancers worldwide. In the search for targeted nanotherapeutic approaches, siRNAs against the viral E6 transcript have been molecules of interest but have not yet seen successful translation into the clinic. By reviewing the past approximately 15 years of in vitro literature, we identify the need for siRNA validation protocols which concurrently evaluate ranges of key treatment parameters as well as characterize downstream process restoration in a methodical, quantitative manner and demonstrate their implementation using our own data. We also reflect on the future need for more appropriate cell culture models to represent patient lesions as well as the application of personalized approaches to identify optimal treatment strategies.

C2'-F Stereoconfiguration As a Puckering Switch for Base Stacking at the Dinucleotide Level.

Fluorine configuration at C2' of the bis(2'-fluorothymidine) dinucleotide is demonstrated to drive intramolecular base stacking. 2'-ß F-Configuration drastically reduces stacking compared to the 2'-α series. Hence, base stacking emerges as being tunable by the C2'-F stereoconfiguration through dramatic puckering variations scrutinized by NMR and natural bond orbital analysis. Accordingly, 2'-ß F-isomer photoreactivity is significantly reduced compared to that of the 2'-α F-isomer.

Carbocyclic C-C Bond Formation: Intramolecular Radical Ring Closure to Yield Diastereomerically Pure (7'S-Me- or 7'R-Me-) Carba-LNA Nucleotide Analogs.

In light of the impressive gene-silencing properties of carba-LNA modified oligo DNA and RNA, both in antisense RNA and siRNA approaches, which have been confirmed as proof-of-concept for biochemical applications in post-transcriptional gene silencing, we envision the true potential of carba-LNA modifications to be revealed soon. Herein we provide detailed protocols for synthesis of carba-LNA-A, -G, -5-Me C, and -T nucleosides on a medium/large scale (gram scale), as well as important guidelines for incorporation of these modified carba-LNAs into DNA or RNA oligonucleotides. Creation of a stereoselective C-C bond during the 5-exo radical intramolecular cyclization involves trapping of a C2' radical intermediate intramolecularly by the vicinal double bond of a C4'-tethered ─CH2 -CH═CH2 group. All diastereomers of substituted carba-LNAs are now available in pure form. The present procedure allows carba-LNA to be commercialized for medicinal or biotechnological purposes. © 2017 by John Wiley & Sons, Inc.

How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pKa 9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg2+ ion availability at the cleavage site causing sequence-specific hydrolysis of the phosphodiester bond of scissile phosphate. The enhancement of the total rate of cleavage of the complementary mRNA strand by up to 25%, presented in this work, provides opportunities to engineer a single modification site in appropriately substituted AONs to design an effective antisense strategy based on the nucleolytic stability of the AON strand versus RNase H capability to cleave the complementary RNA strand.

A minute amount of s-puckered sugars is sufficient for (6-4) photoproduct formation at the dinucleotide level.

The di-2'-α-fluoro analogue of thymidylyl(3',5')thymidine, synthesized to probe the effect of a minimum amount of S conformer on the photoreactivity of dinucleotides, is endowed with only 3% and 8% of S sugar conformation at its 5'- and 3'-end, respectively. This analogue gives rise to the (6-4) photoproduct as efficiently as the dithymine dinucleotide (74% and 66% at the 5'- and 3'-end, respectively) under 254 nm. Our results suggest that the 5'-N, 3'-S conformer gives rise to the (6-4) photoproduct.

Distal two-bond versus three-bond electronegative oxo-substituent effect controls the kinetics and thermodynamics of the conversion of a C-nitroso function to the corresponding oxime in the conformationally locked pentofuranose (bicyclo[2.2.1]heptane) system.

We report the high-yielding and scalable diastereospecific synthesis of isomeric bicyclo[2.2.1]heptane-7- and -8-oximes and their corresponding C-nitroso derivatives, which are the key intermediates for the synthesis of carbanucleosides. Neither the (C7-R)-nitroso- nor (C8-S)-nitrosobicycloheptane system requires any external base in DMSO-d6 to afford the corresponding oxime, and no reverse isomerization from the oxime to the C-nitroso compound was observed. The conversion of the (C8-S)-nitroso compound to the E/Z-oximes was ∼8 times faster (at 40 °C) than that of the (C7-R)-nitroso derivative. The mechanism involves first-order reaction kinetics for the conversion of either the (C7-R)- or (C8-S)-nitroso derivative to the corresponding E/Z-oximes. The lower rate of conversion of the (C7-R)-nitroso compound to the corresponding oximes compared with that of the (C8-S)-nitroso derivative is attributed to the fact that the acidic H8 ionizing center is two bonds away from the OPMB group on C1 in the latter whereas H7 is three bonds away from the C1 OMe group in the former, making the effect of the electron-withdrawing group on C1 stronger in the latter.

New antiprotozoal agents: their synthesis and biological evaluations.

Here we report identification of new lead compounds based on quinoline and indenoquinolines with variable side chains as antiprotozoal agents. Quinolines 32, 36 and 37 (Table 1) and indenoquinoline derivatives 14 and 23 (Table 2) inhibit the in vitro growth of the Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma brucei rhodesiense subspecies and Leishmania infantum with IC50=0.25 µM. These five compounds have superior activity to that of the front-line drugs such as benznidazole, nifurtimox and comparable to amphotericin B. Thus these compounds constitute new 'leads' for further structure-activity studies as potential active antiprotozoal agents.

Unusual strain-releasing nucleophilic rearrangement of a bicyclo[2.2.1]heptane system to a cyclohexenyl derivative.

We report an unusual strain-releasing reaction of 1-mesyloxy-8,7-dimethylbicyclo[2.2.1]heptane (3) by a base-promoted substitution at the chiral C3 followed by spontaneous concerted ring opening involving the most strained C2-C3-C4 bonds (with bond angle 94°) and the C2 bridgehead leading to anti-endo elimination of the C1-mesyloxy group by the conjugate base of adenine or thymine to give two diastereomeric C3'(S) and C3'(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene: 3 → T-4a + T-4b and 3 → A-5a + A-5b. These products have been unambiguously characterized by detailed 1D and 2D NMR (J-coupling constants and nOe analysis), mass, and UV spectroscopy. Evidence has been presented suggesting that the origin of these diastereomeric C3'(S) and C3'(R) derivatives of 1-thyminyl and 9-adeninyl cyclohexene from 3 is most probably a rearrangement mechanism of a trigonal bipyramidal intermediate formed in the S(N)2 displacement-ring-opening reaction.

Steric effects in the tuning of the diastereoselectivity of the intramolecular free-radical cyclization to an olefin as exemplified through the synthesis of a carba-pentofuranose scaffold.

Two free-radical cyclization reactions with the radical at the chiral C4 of the pentose sugar and the intramolecularly C1-tethered olefin (on radical precursors 8 and 17) gave a new diastereospecific C4-C8 bond in dimethylbicyclo[2.2.1]heptane 9, whereas the new C4-C7 bond in 7-methyl-2-oxabicyclo[2.2.1]heptanes 18a/18b gave trans and cis diastereomers, in which the chirality of the C4 center is fully retained as that of the starting material. It has been shown how the chemical nature of the fused carba-pentofuranose scaffolds, dimethylbicyclo[2.2.1]heptane 9 vis-a-vis 7-methyl-2-oxabicyclo[2.2.1]heptanes 18a/18b (C7-Me in the former versus 2-O- in the latter), dictates the stereochemical outcome both at the Grignard reaction step as well as in the free-radical ring-closure reaction. The formation of pure 1,8-trans-bicyclo[2.2.1]heptane 9 from 8 suggests that the boat-like transition state is favored due to the absence of steric clash of the bulky 1(S)-O-p-methoxybenzyl (PMB) and 7(R)-Me substituents (both in the α-face) with that of the 8(R)-CH(2)(•) radical in the ß-face. The conversion of 17 → 18a-7(S) and 18b-7(R) in 6:4 ratio shows that the participation of both the chair- and the boat-like transition states is likely.

New parasite inhibitors encompassing novel conformationally-locked 5'-acyl sulfamoyl adenosines.

We describe the design, synthesis and biological evaluation of conformationally-locked 5'-acyl sulfamoyl adenosine derivatives as new parasitic inhibitors against Trypanosoma and Leishmania. The conformationally-locked (3'-endo, North-type) nucleosides have been synthesized by covalently attaching a 4'-CH(2)-O-2' bridge () across C2'-C4' of adenosine in order to reduce the conformational flexibility of the pentose ring. This is designed to decrease the entropic penalty for complex formation with the target protein, which may improve free-energy of stabilization of the complex leading to improved potency. Conformationally-locked 5'-acyl sulfamoyl adenosine derivatives (16-22) were tested against parasitic protozoans for the first time in this work, and showed potent inhibition of Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma rhodesiense and Leishmania infantum with IC(50) = 0.25-0.51 µM. In particular, the potent 5'-pentanyl acyl sulfamoyl adenosine derivative 17 (IC(50) = 0.25 µM) against intracellular L. infantum amastigotes and Trypanosoma subspecies is interesting in view of its almost insignificant cytotoxicity in murine macrophage host cells (CC(50) >4 µM) and in diploid human fibroblasts MRC-5 cell lines (CC(50) 4 µM). This work also suggests that variable alkyl chain length of the acyl group on the acylsulfamoyl side chain at 5' can modulate the toxicity of 5'-O-sulfamoylnucleoside analogues. This conformationally-locked sulfamoyl adenosine scaffold presents some interesting possibilities for further drug design and lead optimization.

Design and divergent synthesis of aza nucleosides from a chiral imino sugar.

Several novel nucleoside analogues as potential inhibitors of glycosidases and purine nucleoside phosphorylase (PNP) have been synthesized via selective coupling of an appropriate nucleobase at different positions of an orthogonally protected imino sugar as a common precursor. This synthetic strategy offers a straightforward protocol for the assembly of imino sugar containing nucleosides, establishing a new repertoire of molecules as potential therapeutics.

Molecular structure of the core-modified siRNA duplexes containing diastereomeric pair of [C6'(R)-OH]- versus [C6'(S)-OH]-carba-LNAs suggests a model for RNAi action.

Molecular structures of native and a pair of modified small interfering RNA-RNA duplexes containing carbocyclic [6 '-(R)-OH/7 '-(S)-methyl]- and [6 '-(S)-OH/7 '-(S)-methyl]-carba-LNA-thymine nucleotides, which are two diastereomeric analogs of the native T nucleotide, incorporated at position 13 in the antisense (AS) strand of siRNA, have been simulated using molecular mechanics/dynamics techniques. The main aim of the project has been to find a plausible structural explanation of why modification of siRNA at T(13) position by the [6 '(R)-O-(p-Toluoyl)-7 '(S)-methyl]-carba-LNA-Thymine [IC(50) of 3.32 ± 0.17 nM] is ca 24 times more active as an RNA silencing agent against the target HIV-1 TAR RNA than the [6 '(S)-O-(p-Toluoyl)-7 '(S)-methyl]-counterpart [IC(50) of 79.8 ± 17 nM] [1]. The simulations reveal that introduction of both C6 '(R)-OH and C6 '(S)-OH stereoisomers does not lead even to local perturbation of the siRNA-RNA duplex structures compared to the native, and the only significant difference between 6 '(S)- and 6 '(R)-diastereomers found is the exposure of the 6 '-OH group of the 6 '(R)-diastereoisomer toward the edge of the duplex while the 6 '-hydroxyl group of the 6 '(S)-diastereoisomer is somewhat buried in the minor groove of the duplex. This rules out a hypothesis about any possible local distortion by the nature of chemical modification of the siRNA-target the RNA duplex, which might have influenced the formation of the effective RNA silencing complex (RISC) and puts some weight on the hypothesis about the 6 '-hydroxy group being directly involved with most probably Ago protein, since it is known from exhaustive X-ray studies [2, 3] that the core residues are indeed involved with hydrogen bonding with the internucleotidyl phosphates. Further systematic investigation is in progress to map the position-dependent functional and nonfunctional interactions of the modified [6 '(R or S)-O-(p-Toluoyl)-7 '(S)-methyl]-carba-LNA-T with the Ago2 protein of the RISC.

Carba-LNA-5MeC/A/G/T modified oligos show nucleobase-specific modulation of 3'-exonuclease activity, thermodynamic stability, RNA selectivity, and RNase H elicitation: synthesis and biochemistry.

Using the intramolecular 5-exo-5-hexenyl radical as a key cyclization step, we previously reported an unambiguous synthesis of carba-LNA thymine (cLNA-T), which we subsequently incorporated in antisense oligonucleotides (AON) and investigated their biochemical properties [J. Am. Chem. Soc.2007, 129 (26), 8362-8379]. These cLNA-T incorporated oligos showed specific RNA affinity of +3.5-5 °C/modification for AON:RNA heteroduplexes, which is comparable to what is found for those of LNAs (Locked Nucleic Acids). These modified oligos however showed significantly enhanced nuclease stability (ca. 100 times more) in the blood serum compared to those of the LNA modified counterparts without compromising any RNase H recruitment capability. We herein report the synthesis of 5-methylcytosine-1-yl ((Me)C), 9-adeninyl (A), and 9-guaninyl (G) derivatives of cLNA and their oligonucleotides and report their biochemical properties as potential RNA-directed inhibitors. In a series of isosequential carba-LNA modified AONs, we herein show that all the cLNA modified AONs are found to be RNA-selective, but the magnitude of RNA-selectivity of 7'-R-Me-cLNA-G (cLNA-G) (ΔT(m) = 2.9 °C/modification) and intractable isomeric mixtures of 7'-(S/R)-Me-cLNA-T (cLNA-T, ΔT(m) = 2.2 °C/modification) was found to be better than diastereomeric mixtures of 7'-(S/R)-Me-cLNA-(Me)C with trace of cENA-(Me)C (cLNA-(Me)C, ΔT(m) = 1.8 °C/modification) and 7'-R-Me-cLNA-A (cLNA-A, ΔT(m) = 0.9 °C/modification). cLNA-(Me)C modified AONs however exhibited the best nuclease stability, which is 4-, 7-, and 20-fold better, respectively, than cLNA-T, cLNA-A, and cLNA-G modified counterparts, which in turn was more than 100 times stable than that of the native. When the modification sites are appropriately chosen in the AONs, the cLNA-A, -G, and -(Me)C modified sites in the AON:RNA hybrids can be easily recognized by RNase H, and the RNA strand of the hybrid is degraded in a specific manner, which is important for the design of oligos for therapeutic purposes. The cLNA-(Me)C modified AON/RNA, however, has been found to be degraded 4 times faster than cLNA-A and G modified counterparts. By appropriately choosing the carba-LNA modification sites in AON strands, the digestion of AON:RNA can be either totally repressed or be limited to cleavage at specific sites or at a single site only (similar to that of catalytic RNAzyme or DNAzyme). Considering all physico- and biochemical aspects of cLNA modified oligos, the work suggests that the cLNA modified antisense oligos have the potential of being a promising therapeutic candidate due to their (i) higher nucleobase-specific RNA affinity and RNA selectivity, (ii) greatly improved nuclease stability, and (iii) efficient RNase H recruitment capability, which can induce target RNA cleavage in a very specific manner at multiple or at a single site, in a designed manner.

Synthesis and antimycobacterial activity of prodrugs of indeno[2,1-c]quinoline derivatives.

Recently we have reported anti-TB properties of a new class of conformationally-constrained indeno[2,1-c]quinolines, which are although considerably active (MIC 0.39-0.78 µg/mL) suffered from intense solubility problems. We thought of improving their bioavailability by prodrugs approach. Accordingly esters of the "Lead" indeno[2,1-c]quinolines 1, 15 and 27 derivatives were synthesized and their prodrug nature at the physiological pH were confirmed. Prodrugs were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv by MABA assay to show that they have 2- to 4-fold improved anti-TB activities, increased aqueous solubility and superior selectivity index over their respective parent compounds. MIC of these prodrugs was in the range of <0.20-6.0 µg/mL, and in general, no cytotoxicity was observed in VERO cells.

Allele-selective inhibition of ataxin-3 (ATX3) expression by antisense oligomers and duplex RNAs.

Spinocerebellar ataxia-3 (also known as Machado-Joseph disease) is an incurable neurodegenerative disorder caused by expression of a mutant variant of ataxin-3 (ATX3) protein. Inhibiting expression of ATX3 would provide a therapeutic strategy, but indiscriminant inhibition of both wild-type and mutant ATX3 might lead to undesirable side effects. An ideal silencing agent would block expression of mutant ATX3 while leaving expression of wild-type ATX3 intact. We have previously observed that peptide nucleic acid (PNA) conjugates targeting the expanded CAG repeat within ATX3 mRNA block expression of both alleles. We have now identified additional PNAs capable of inhibiting ATX3 expression that vary in length and in the nature of the conjugated cation chain. We can also achieve potent and selective inhibition using duplex RNAs containing one or more mismatches relative to the CAG repeat. Anti-CAG antisense bridged nucleic acid oligonucleotides that lack a cationic domain are potent inhibitors but are not allele-selective. Allele-selective inhibitors of ATX3 expression provide insights into the mechanism of selectivity and promising lead compounds for further development and in vivo investigation.

Allele-selective inhibition of mutant huntingtin expression with antisense oligonucleotides targeting the expanded CAG repeat.

Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

Synthesis of 2',4'-propylene-bridged (carba-ENA) thymidine and its analogues: the engineering of electrostatic and steric effects at the bottom of the minor groove for nuclease and thermodynamic stabilities and elicitation of RNase H.

2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH(2)/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C═N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON:RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance but 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH(2) substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase H1 with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.

Synthesis and structure of azole-fused indeno[2,1-c]quinolines and their anti-mycobacterial properties.

Prompted by our discovery of a new class of conformationally-locked indeno[2,1-c]quinolines as anti-mycobacterials, compounds 2a and 3a (Fig. 1; MIC < 0.39 µg mL(-1) and 0.78 µg mL(-1), respectively)(14) with a freely rotating C2-imidazolo substituent, we herein describe the synthesis of pentacyclic azole-fused quinoline derivatives 4 and 5, in which we have restricted the rotation of the C2-imidazolo moiety by fusing it to the adjacent quinoline-nitrogen to give a five-membered fused azole heterocycle. The idea of locking the flexibility of the system by conformational constraint was simply to reduce its entropy, thereby reducing the overall free-energy of its binding to the target receptor. Out of 22 different azole-fused indeno[2,1-c]quinoline derivatives, seven structurally distinct compounds, 9, 15, 17, 25, 27, 28 and 29, have shown 79-99% growth inhibition of Mycobacterium tuberculosis H37Rv at a fixed dose of 6.25 µg mL(-1). The efficacies of these compounds were evaluated in vitro for 8/9 consecutive days using the BACTEC radiometric assay upon administration of single dose on day one. Of these, two compounds, 9 and 28, inhibited growth of M. tuberculosis very effectively at MIC < 0.39 µg mL(-1) (0.89 µM and 1 µM, respectively). These active compounds 9, 15, 17, 25, 27, 28 and 29 were screened for their cytotoxic effect on mammalian cells (human monocytic cell line U937), which showed that the human cell survival is almost unperturbed (100% survival), except for compound 25, hence these new compounds with new scaffolds have been identified as potent anti-mycobacterials, virtually with no toxicity. Thus these "hit" molecules constitute our important "leads" for further optimization by structure-activity relationship against TB.

Free-radical ring closure to conformationally locked α-L-carba-LNAs and synthesis of their oligos: nuclease stability, target RNA specificity, and elicitation of RNase H.

A new class of conformationally constrained nucleosides, α-L-ribo-carbocyclic LNA thymidine (α-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" α-L-ribo-configured tetracyclic thymidine (6,7'-methylene-bridged-α-L-carba-LNA-T) in which both the sugar puckering and glycosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These α-L-carba-LNA analogues have been subsequently transformed to corresponding phosphoramidites and incorporated into isosequential antisense oligonucleotides (AONs), which have then been examined for the thermal denaturation of their duplexes, nuclease stability, and RNase H recruitment capabilities. Introduction of a single 6',7'-substituted α-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T(m) reduction by 2-3 °C as compared to the native heteroduplex, whereas the parent 2'-oxa-α-L-LNA-T modification at the identical position in the AON strand has been found to lead to an increase in the T(m) by 3-5 °C. This suggests that the 6' and 7' substitutions lead to much reduced thermal stability for the modified heteroduplex, especially the hydrophobic 7'-methyl on α-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted α-L-carba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-α-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted α-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the ß-D-LNA-T or ß-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNase H recruitment capabilities elevate the α-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.