| 1. |
|
|
| 2. |
- Barman, Jharna, et al.
(författare)
-
Non-identical electronic characters of the internucleotidic phosphates in RNA modulate the chemical reactivity of the phosphodiester bonds
- 2006
-
Ingår i: Organic and biomolecular chemistry. - 1477-0520 .- 1477-0539. ; 4:5, s. 928-941
-
Tidskriftsartikel (refereegranskat)abstract
- We here show that the electronic properties and the chemical reactivities of the internucleotidic phosphates in the heptameric ssRNAs are dissimilar in a sequence-specific manner because of their non-identical microenvironments, in contrast with the corresponding isosequential ssDNAs. This has been evidenced by monitoring the delta H8(G) shifts upon pH-dependent ionization (pK(a1)) of the central 9-guaninyl (G) to the 9-guanylate ion (G(-)), and its electrostatic effect on each of the internucleotidic phosphate anions, as measured from the resultant delta P-31 shifts (pKa(2)) in the isosequential heptameric ssRNAs vis-`a-vis ssDNAs: [d/r( 5'-Cp(1)Ap(2)Q(1)p(3)Gp(4)Q(2)p(5)Ap(6)C-3'): Q(1) = Q(2) = A (5a/5b) or C (8a/8b), Q(1) = A, Q(2) = C (6a/6b), Q(1) = C, Q(2) = A (7a/7b)]. These oligos with single ionizable G in the centre are chosen because of the fact that the pseudoaromatic character of G can be easily modulated in a pH-dependent manner by its transformation to G(-) (the 2'-OH to 2-O- ionization effect is not detectable below pH 11.6 as evident from the N1-Me-G analog), thereby modulating/titrating the nature of the electrostatic interactions of G to G- with the phosphates, which therefore constitute simple models to interrogate how the variable pseudoaromatic characters of nucleobases under different sequence context (J. Am. Chem. Soc., 2004, 126, 8674-8681) can actually influence the reactivity of the internucleotide phosphates as a result of modulation of sequence context-specific electrostatic interactions. In order to better understand the impact of the electrostatic effect of the G to G- on the tunability of the electronic character of internucleotidic phosphates in the heptameric ssRNAs 5b, 6b, 7b and 8b, we have also performed their alkaline hydrolysis at pH 12.5 at 20 degrees C, and have identified the preferences of the cleavage sites at various phosphates, which are p(2), p(3) and p(4) (Fig. 3). The results of these alkaline hydrolysis studies have been compared with the hydrolysis of analogous N1-Me-G heptameric ssRNA sequences 5c, 7c and 8c under identical conditions in order to establish the role of the electrostatic effect of the 9-guanylate ion (and the 2'-OH to 2-O- ionization) on the internucleotidic phosphate. It turned out that the relative alkaline hydrolysis rate at those particular phosphates ( p2, p3 and p(4)) in the N1-Me-G heptamers was reduced from 16-78% compared to those in the native counterparts [Fig. 4, and ESI 2 (Fig. S11)]. Thus, these physico-chemical studies have shown that those p2, p3 and p4 phosphates in the native heptameric RNAs, which show pK(a2) as well as more deshielding ( owing to weaker P-31 screening) in the alkaline pH compared to those at the neutral pH, are more prone to the alkaline hydrolysis because of their relatively enhanced electrophilic character resulting from weaker P-31 screening. This screening effect originates as a result of the systematic charge repulsion effect between the electron cloud in the outermost orbitals of phosphorus and the central guanylate ion, leading to delocalization of the phosphorus pp charge into its d pi orbitals. It is thus likely that, just as in the non-enzymatic hydrolysis, the enzymatic hydrolysis of a specific phosphate in RNA by general base-catalyss in RNA-cleaving proteins (RNase A, RNA phosphodiesterase or nuclease) can potentially be electrostatically influenced by tuning the transient charge on the nucleobase in the steric proximity or as a result of specific sequence context owing to nearest-neighbor interactions.
|
|
| 3. |
- Bramsen, Jesper B., et al.
(författare)
-
A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity
- 2009
-
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 37:9, s. 2867-2881
-
Tidskriftsartikel (refereegranskat)abstract
- The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.
|
|
| 4. |
|
|
| 5. |
- Honcharenko, Dmytro, et al.
(författare)
-
Modulation of Pyrene Fluorescence in DNA Probes Depends upon the Nature of the Conformationally Restricted Nucleotide
- 2008
-
Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 73:7, s. 2829-2842
-
Tidskriftsartikel (refereegranskat)abstract
- The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.
|
|
| 6. |
- Li, Qing, et al.
(författare)
-
Free-Radical Ring Closure to Conformationally Locked alpha-L-Carba-LNAs and Synthesis of Their Oligos : Nuclease Stability, Target RNA Specificity, and Elicitation of RNase H
- 2010
-
Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 75:18, s. 6122-6140
-
Tidskriftsartikel (refereegranskat)abstract
- A new class of conformationally constrained nucleosides, alpha-L-ribo-carbocyclic LNA thymidine (alpha-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" alpha-L-ribo-configured tetracyche thymidine (6,7'-methylene-bridged-alpha-L-carba-LNA-T) in which both the sugar puckering and glyeosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These alpha-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 alpha-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T-m reduction by 2-3 degrees C as compared to the native heteroduplex, whereas the parent 2'-oxa-alpha-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 degrees 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 alpha-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted alpha-L-earba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-alpha-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted alpha-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the beta-D-LNA-T or beta-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNasc H recruitment capabilities elevate the alpha-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.
|
|
| 7. |
- Li, Qing, 1981-, et al.
(författare)
-
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
- 2010
-
Ingår i: Journal of Organic Chemistry. - U. S. A : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 75:18, s. 6122-6140
-
Tidskriftsartikel (refereegranskat)abstract
- 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 β-D-LNA-T or β-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.
|
|
| 8. |
- Liu, Yi, et al.
(författare)
-
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
- 2010
-
Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 75:21, s. 7112-7128
-
Tidskriftsartikel (refereegranskat)abstract
- 2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH2/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 hut 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH2 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 HI with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.
|
|
| 9. |
- Upadhayaya, RamShankar, et al.
(författare)
-
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
- 2011
-
Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 76:11, s. 4408-4431
-
Tidskriftsartikel (refereegranskat)abstract
- 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 oligonudeotides (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 degrees 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 (C-Me), 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) (Delta T-m = 2.9 degrees C/modification) and intractable isomeric mixtures of 7'-(S/R)-Me-cLNA-T (cLNA-T, Delta T-m = 2.2 degrees C/modification) was found to be better than diastereomeric mixtures of 7'-(S/R)-Me-cLNA-C-Me with trace of cENA-C-Me (cLNA-C-Me, Delta T-m = 1.8 degrees C/modification) and 7'-R-Me-cLNA-A (cLNA-A, Delta T-m = 0.9 degrees C/modification). cLNA-C-Me 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 -C-Me 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-C-Me 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.
|
|
| 10. |
- Wang, Shu Min, 1963, et al.
(författare)
-
Novel Dilute Bismides for IR Optoelectronics Applications
- 2013
-
Ingår i: Asia Communications and Photonics Conference, ACP. - Washington, D.C. : OSA. - 2162-108X.
-
Konferensbidrag (refereegranskat)abstract
- III-V-Bi compounds reveal a number of attractive physical properties promising for novel IR optoelectronic applications [1,2] and have received considerable attention as witnessed by the dedicated international workshops on this topic in the consecutive past four years. The isoelectronic nature of Bi atoms in III-Vs induces strong interactions with the energy bands of host materials leading to large band-gap reduction, less temperature sensitive band-gap and large spin-orbit split band. So far the most studied material is Ga(N)AsBi, while other dilute bismides have also been reported recently. In this paper, we shall briefly review several novel bismides: GaSbBi, InSbiBi, InAsBi, InPBi and InGaAsBi, and the Bi surfactant effect from our group, all grown by molecular beam epitaxy (MBE).
|
|
