Syntheses of 4′-thioribonucleosides and thermodynamic stability and crystal structure of RNA oligomers with incorporated 4′-thiocytosine

A facile synthetic route for the 4′-thioribonucleoside building block 4′SN (N = U, C, A and G) with the ribose O4′ replaced by sulfur is presented. Conversion of l-lyxose to 1,5-di-O-acetyl-2,3-di-O-benzoyl-4-thio-d-ribofuranose was achieved via an efficient four-step synthesis with high yield. Conversion of the thiosugar into the four ribonucleoside phosphoramidite building blocks was accomplished with additional four steps in each case. Incorporation of 4′-thiocytidines into oligoribonucleotides improved the thermal stability of the corresponding duplexes by ∼1°C per modification, irrespective of whether the strand contained a single modification or a consecutive stretch of 4′SC residues. The gain in thermodynamic stability is comparable to that observed with oligoribonucleotides containing 2′-O-methylated residues. To establish potential conformational changes in RNA as a result of the 4′-thio modification and to better understand the origins of the observed stability changes, the crystal structure of the oligonucleotide 5′-r(CC4′SCCGGGG) was determined and analyzed using the previously solved structure of the native RNA octamer as a reference. The two 4′-thioriboses adopt conformations that are very similar to the C3′-endo pucker observed for the corresponding sugars in the native duplex. Subtle changes in the local geometry of the modified duplex are mostly due to the larger radius of sulfur compared to oxygen or appear to be lattice-induced. The significantly increased RNA affinity of 4′-thio-modified RNA relative to RNA, and the relatively minor conformational changes caused by the modification render this nucleic acid analog an interesting candidate for in vitro and in vivo applications, including use in RNA interference (RNAi), antisense, ribozyme, decoy and aptamer technologie

Syntheses of 4′-thioribonucleosides and thermodynamic stability and crystal structure of RNA oligomers with incorporated 4′-thiocytosine

A facile synthetic route for the 4′-thioribonucleoside building block (4′S)N (N = U, C, A and G) with the ribose O4′ replaced by sulfur is presented. Conversion of l-lyxose to 1,5-di-O-acetyl-2,3-di-O-benzoyl-4-thio-d-ribofuranose was achieved via an efficient four-step synthesis with high yield. Conversion of the thiosugar into the four ribonucleoside phosphoramidite building blocks was accomplished with additional four steps in each case. Incorporation of 4′-thiocytidines into oligoribonucleotides improved the thermal stability of the corresponding duplexes by ∼1°C per modification, irrespective of whether the strand contained a single modification or a consecutive stretch of (4′S)C residues. The gain in thermodynamic stability is comparable to that observed with oligoribonucleotides containing 2′-O-methylated residues. To establish potential conformational changes in RNA as a result of the 4′-thio modification and to better understand the origins of the observed stability changes, the crystal structure of the oligonucleotide 5′-r(CC(4′S)CCGGGG) was determined and analyzed using the previously solved structure of the native RNA octamer as a reference. The two 4′-thioriboses adopt conformations that are very similar to the C3′-endo pucker observed for the corresponding sugars in the native duplex. Subtle changes in the local geometry of the modified duplex are mostly due to the larger radius of sulfur compared to oxygen or appear to be lattice-induced. The significantly increased RNA affinity of 4′-thio-modified RNA relative to RNA, and the relatively minor conformational changes caused by the modification render this nucleic acid analog an interesting candidate for in vitro and in vivo applications, including use in RNA interference (RNAi), antisense, ribozyme, decoy and aptamer technologies

Are babies conceived during Ramadan born smaller and sooner than babies conceived at other times of the year? A Born in Bradford Cohort study

Background It is not known whether infants exposed to intermittent maternal fasting at conception are born smaller or have a higher risk of premature birth than those who are not. Doctors are therefore unsure about what advice to give women about the safety of Ramadan fasting. This cohort study aimed to investigate these questions in Muslim mother–infant pairs to inform prenatal care. Methods Routinely collected data accessed from maternity records were the source for information. Mothers were considered exposed if they were Muslim and Ramadan overlapped with their infant conception date, estimated to be 14 days after the last menstrual period. Infants were included as exposed if their estimated conception date was in the first 21 days of Ramadan or 7 days prior to Ramadan. Results After adjusting for gestational age, maternal age, infant gender, maternal body mass index at booking, smoking status, gestational diabetes, parity and year of birth, there was no significant difference in birth weight between infants born to Muslim mothers who were conceived during Ramadan (n=479) and those who were not (n=4677) (adjusted mean difference =24.3 g, 95% CI −16.4 to 64.9). There was no difference in rates of premature births in exposed and unexposed women (5.2% vs 4.9%; OR=1.08, 95% CI 0.71 to 1.65). Conclusions Healthy Muslim women considering becoming pregnant prior to, or during Ramadan, can be advised that fasting does not seem to have a detrimental effect on the size (weight) of their baby and it appears not to increase the likelihood of giving birth prematurely

Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs

RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3\u27-CH2-CO-NH-5\u27 amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5\u27-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications

Recognition of O6-benzyl-2′-deoxyguanosine by a perimidinone-derived synthetic nucleoside: a DNA interstrand stacking interaction

The 2′-deoxynucleoside containing the synthetic base 1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-1H-perimidin-2(3H)-one] (dPer) recognizes in DNA the O6-benzyl-2′-deoxyguanosine nucleoside (O6-Bn-dG), formed by exposure to N-benzylmethylnitrosamine. Herein, we show how dPer distinguishes between O6-Bn-dG and dG in DNA. The structure of the modified Dickerson-Drew dodecamer (DDD) in which guanine at position G4 has been replaced by O6-Bn-dG and cytosine C9 has been replaced with dPer to form the modified O6-Bn-dG:dPer (DDD-XY) duplex [5′-d(C1G2C3X4A5A6T7T8Y9G10C11G12)-3′]2 (X = O6-Bn-dG, Y = dPer) reveals that dPer intercalates into the duplex and adopts the syn conformation about the glycosyl bond. This provides a binding pocket that allows the benzyl group of O6-Bn-dG to intercalate between Per and thymine of the 3′-neighbor A:T base pair. Nuclear magnetic resonance data suggest that a similar intercalative recognition mechanism applies in this sequence in solution. However, in solution, the benzyl ring of O6-Bn-dG undergoes rotation on the nuclear magnetic resonance time scale. In contrast, the structure of the modified DDD in which cytosine at position C9 is replaced with dPer to form the dG:dPer (DDD-GY) [5′-d(C1G2C3G4A5A6T7T8Y9G10C11G12)-3′]2 duplex (Y = dPer) reveals that dPer adopts the anti conformation about the glycosyl bond and forms a less stable wobble pairing interaction with guanin

Prolinyl phosphoramidates of nucleotides with increased reactivity

Nucleoside monophosphates (NMPs) are the subunits of RNA. They are incorporated into growing complementary strands when sequences are copied in enzyme‐free reactions using organic leaving groups at the phosphates. Amino acids are rarely considered as leaving groups, but proline can act as a leaving group when N‐linked to NMPs, so that prolinyl NMPs hydrolyze in aqueous buffer at 37 °C, with half‐life times as short as 2.4 h, and they act as monomers in enzyme‐free primer extension. Still, their level of reactivity is insufficient for practical purposes, requiring months for some extensions. Herein we report the synthesis of eight substituted prolinyl AMPs together with seven related compounds and the results of a study of their reactivity. A δ‐carboxy prolinyl NMP was found to be converted with a half‐life time of just 11 min in magnesium‐free buffer, and a δ‐isopropyl prolinyl NMP was shown to react sevenfold faster than its prolinyl counterpart in enzyme‐free genetic copying of RNA. Our results indicate that both anchimeric and steric effects can be employed to increase the reactivity of aminoacidyl nucleotides, i.e. compounds that combine two fundamental classes of biomolecules in one functional entity.Deutsche ForschungsgemeinschaftVolkswagen Foundatio

A cluster-randomised feasibility trial of a children's weight management programme:the Child weigHt mANaGement for Ethnically diverse communities (CHANGE) study

Background: Community-based programmes for children with excess weight are widely available, but few have been developed to meet the needs of culturally diverse populations. We adapted an existing children's weight management programme, focusing on Pakistani and Bangladeshi communities. We report the evaluation of this programme to assess feasibility of programme delivery, acceptability of the programme to participants from diverse communities, and feasibility of methods to inform a future trial. Methods: A cluster-randomised feasibility trial was undertaken in a large UK city. Children's weight management programmes (n = 24) were randomised to be delivered as the adapted or the standard programme (2:1 ratio). Routine data on participant attendance (n = 243) at the sessions were used to estimate the proportion of families completing the adapted and standard programmes (to indicate programme acceptability). Families planning to attend the programmes were recruited to participate in the feasibility study (n = 92). Outcome data were collected from children and parents at baseline, end of programme, and 6 months post-programme. A subsample (n = 24) of those attending the adapted programme participated in interviews to gain their views of the content and delivery and assess programme acceptability. Feasibility of programme delivery was assessed through observation and consultation with facilitators, and data on costs were collected. Results: The proportion of Pakistani and Bangladeshi families and families of all ethnicities completing the adapted programme was similar: 78.8% (95% CI 64.8-88.2%) and 76.3% (95% CI 67.0-83.6%) respectively. OR for completion of adapted vs. standard programme was 2.40 (95% CI 1.32-4.34, p = 0.004). The programme was feasible to deliver with some refinements, and participant interview data showed that the programme was well received. Study participant recruitment was successful, but attrition was high (35% at 6 months). Data collection was mostly feasible, but participant burden was high. Data collection on cost of programme delivery was feasible, but costs to families were more challenging to capture. Conclusions: This culturally adapted programme was feasible to deliver and highly acceptable to participants, with increased completion rates compared with the standard programme. Consideration should be given to a future trial to evaluate its clinical and cost-effectiveness. Trial registration: ISRCTN81798055, registered: 13/05/2014

Influence of Microstructural Effect on Microvickers Hardness Properties of SiO2-Na2O-CaO (SNC) Waste Based Glass-ceramic

There are a lot of waste materials consist of silicate based such as coal combustion ash, slag from steel production, fly ash, mud, as well as glass cullet or mixtures to produce glass-ceramics. This research work using clam shell (CS) ash and soda-lime-silica (SLS) waste glass powder for fabricating novel SiO2-Na2O-CaO (SNC) glass-ceramic. The samples were composed of SLS (50%), Na2CO3 (30%), and CS (20%) in weight percentage via conventional melt-quenching technique and solid-state sintering technique. The samples were investigated via X-Ray Diffractometer (XRD), Field emission microscope (FESEM), and microvickers hardness tester. The samples were sintered at 550-950 °C to investigate the influence of microstructural effect on microvickers hardness properties at applied force 0.5 and 1.0 kgf. The optimal Vickers hardness properties at sintering temperature 850 °C due to high crystallization of SiO2 phase from the residual glass and CaO content enhanced the viscosity flow, high compactness of particles arrangement and densification of sample

Influence of Microstructural Effect on Microvickers Hardness Propertiesof SiO2-Na2O-CaO (SNC) Waste Based Glass-ceramic

There are a lot of waste materials consist of silicate based such as coal combustion ash, slag from steel production, fly ash, mud, as well as glass cullet or mixtures to produce glass-ceramics. This research work using clam shell (CS) ash and soda-lime-silica (SLS) waste glass powder for fabricating novel SiO2-Na2O-CaO (SNC) glass-ceramic. The samples were composed of SLS (50%), Na2CO3 (30%), and CS (20%) in weight percentage via conventional melt-quenching technique and solid-state sintering technique. The samples were investigated via X-Ray Diffractometer (XRD), Field emission microscope (FESEM), and microvickers hardness tester. The samples were sintered at 550-950 °C to investigate the influence of microstructural effect on microvickers hardness properties at applied force 0.5 and 1.0 kgf. The optimal Vickers hardness properties at sintering temperature 850 °C due to high crystallization of SiO2 phase from the residual glass and CaO content enhanced the viscosity flow, high compactness of particles arrangement and densification of sample