scholarly journals Feasibility of cRGD conjugation at 5′-antisense strand of siRNA by phosphodiester linkage extension

2021 ◽  
Vol 25 ◽  
pp. 603-612
Author(s):  
Xinyang Zhou ◽  
Yufei Pan ◽  
Lijia Yu ◽  
Jing Wu ◽  
Zheng Li ◽  
...  
Keyword(s):  
Antisense Strand ◽  
Phosphodiester Linkage

scholarly journals Effect of 2′-5′/3′-5′ phosphodiester linkage heterogeneity on RNA interference

2020 ◽  
Vol 48 (9) ◽  
pp. 4643-4657
Author(s):  
Maryam Habibian ◽  
S Harikrishna ◽  
Johans Fakhoury ◽  
Maria Barton ◽  
Eman A Ageely ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Rna Interference ◽  
Structure Function ◽  
Antisense Strand ◽  
New Class ◽  
Phosphodiester Linkage ◽  
Mrna Targets ◽  
Sense Strand ◽  
Added Benefit ◽  
Dynamics Simulations

Abstract We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2′-5′ linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5′-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5′p-rN1-(2′-5′)-N2 unit in the antisense strand does not alter the ‘clover leaf’ bend and sugar puckers that are critical for anchoring the 5′-phosphate to Ago 2 MID domain. Importantly, 2′-5′-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2′-5′/3′-5′-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.

Convenient Syntheses of Oligoribonucleotides Having 2′-5′ and/or 3′-5′ Phosphodiester Linkage

1990 ◽  
Vol 19 (6) ◽  
pp. 959-962 ◽  
Author(s):  
Hiroaki Ozaki ◽  
Akio Nakamoto ◽  
Takeo Shimidzu
Keyword(s):  
Phosphodiester Linkage

scholarly journals Functional Comparison of HBZ and the Related APH-2 Protein Provides Insight into Human T-Cell Leukemia Virus Type 1 Pathogenesis

2016 ◽  
Vol 90 (7) ◽  
pp. 3760-3772 ◽  
Author(s):  
Amanda R. Panfil ◽  
Nathan J. Dissinger ◽  
Cory M. Howard ◽  
Brandon M. Murphy ◽  
Kristina Landes ◽  
...  
Keyword(s):  
T Cell ◽  
Leukemia Virus ◽  
Antisense Strand ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
T Cell Leukemia Virus

ABSTRACTHuman T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are highly related retroviruses that transform T cellsin vitrobut have distinct pathological outcomesin vivo. HTLV-1 encodes a protein from the antisense strand of its proviral genome, the HTLV-1 basic leucine zipper factor (HBZ), which inhibits Tax-1-mediated viral transcription and promotes cell proliferation, a high proviral load, and persistencein vivo. In adult T-cell leukemia/lymphoma (ATL) cell lines and patient T cells,hbzis often the only viral gene expressed. The antisense strand of the HTLV-2 proviral genome also encodes a protein termed APH-2. Like HBZ, APH-2 is able to inhibit Tax-2-mediated viral transcription and is detectable in most primary lymphocytes from HTLV-2-infected patients. However, unlike HBZ, the loss of APH-2in vivoresults in increased viral replication and proviral loads, suggesting that HBZ and APH-2 modulate the virus and cellular pathways differently. Herein, we examined the effect of APH-2 on several known HBZ-modulated pathways: NF-κB (p65) transactivation, transforming growth factor β (TGF-β) signaling, and interferon regulatory factor 1 (IRF-1) transactivation. Like HBZ, APH-2 has the ability to inhibit p65 transactivation. Conversely, HBZ and APH-2 have divergent effects on TGF-β signaling and IRF-1 transactivation. Quantitative PCR and protein half-life experiments revealed a substantial disparity between HBZ and APH-2 transcript levels and protein stability, respectively. Taken together, our data further elucidate the functional differences between HBZ and APH-2 and how these differences can have profound effects on the survival of infected cells and, ultimately, pathogenesis.IMPORTANCEHuman T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are highly related retroviruses that have distinct pathological outcomes in infected hosts. Functional comparisons of HTLV-1 and HTLV-2 proteins provide a better understanding about how HTLV-1 infection is associated with disease and HTLV-2 infection is not. The HTLV genome antisense-strand geneshbzandaph-2are often the only viral genes expressed in HTLV-infected T cells. Previously, our group found that HTLV-1 HBZ and HTLV-2 APH-2 had distinct effectsin vivoand hypothesized that the differences in the interactions of HBZ and APH-2 with important cell signaling pathways dictate whether cells undergo proliferation, apoptosis, or senescence. Ultimately, these functional differences may affect how HTLV-1 causes disease but HTLV-2 generally does not. In the current study, we compared the effects of HBZ and APH-2 on several HTLV-relevant cellular pathways, including the TGF-β signaling, NF-κB activation, and IRF-1 transactivation pathways.

scholarly journals Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation

2018 ◽  
Author(s):  
Peter Kindgren ◽  
Ryan Ard ◽  
Maxim Ivanov ◽  
Sebastian Marquardt
Keyword(s):  
Rna Polymerase Ii ◽  
Freezing Tolerance ◽  
Biological Significance ◽  
Antisense Strand ◽  
Fitness Costs ◽  
Profound Impact ◽  
Non Coding Rna ◽  
Cold Sensitive ◽  
Long Non Coding Rna ◽  
Transcriptional Direction

SummaryMost DNA in the genomes of higher organisms does not encode proteins, but is transcribed by RNA polymerase II (RNAPII) into long non-coding RNA (lncRNA). The biological significance of most lncRNA is largely unclear. Here, we identify a lncRNA (SVALKA) in a cold-sensitive region of the Arabidopsis genome. Mutations in SVALKA affect the timing of maximal CBF1 expression and freezing tolerance. RNAPII read-through transcription of SVALKA results in a cryptic lncRNA overlapping CBF1 on the antisense strand, termed asCBF1. asCBF1 transcription is anti-correlated with CBF1 expression. Our molecular dissection reveals that CBF1 is suppressed by RNAPII collision stemming from the SVALKA-asCBF1 lncRNA cascade. The SVK-asCBF1 cascade provides a mechanism to tightly control CBF1 expression and timing that could be exploited to maximize freezing tolerance with mitigated fitness costs. Inversion of the transcriptional direction of a lncRNA cascade relative to the genes in a co-regulated cluster provides an elegant inbuilt negative feedback for cluster expression. Our results provide a compelling example of local gene regulation by lncRNA transcription having a profound impact on the ability of plants to appropriately acclimate to suboptimal environmental conditions.

scholarly journals Probing E. coli SSB Protein-DNA topology by reversing DNA backbone polarity

2020 ◽  
Author(s):  
Alexander G. Kozlov ◽  
Timothy M. Lohman
Keyword(s):  
Dna Binding ◽  
Crystal Structures ◽  
Nearest Neighbor ◽  
Genome Maintenance ◽  
Binding Properties ◽  
Ssdna Binding ◽  
E Coli ◽  
Phosphodiester Linkage ◽  
The Individual ◽  
Dna Binding Properties

AbstractE. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homo-tetramers bind ssDNA in two major modes differing in occluded site size and cooperativity. The (SSB)35 mode in which ssDNA wraps on average around two subunits is favored at low [NaCl] and high SSB to DNA ratios and displays high “unlimited”, nearest-neighbor cooperativity forming long protein clusters. The (SSB)65 mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (> 200 mM) and displays “limited” low cooperativity. Crystal structures of E. coli SSB and P. falciparum SSB show ssDNA bound to the SSB subunits (OB-folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)70 constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse polarity (RP) phosphodiester linkage, either 3’-3’ or 5’-5’. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)70 with a 3’-3’ polarity switch shows decreased affinity for EcSSB in the (SSB)65 mode and lower cooperativity in the (SSB)35 mode. However, (dT)70 in which every phosphodiester linkage is reversed, does not form a completely wrapped (SSB)65 mode, but rather binds EcSSB in the (SSB)35 mode, with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)65 mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB-folds if there is a change in ssDNA wrapping topology of the RP ssDNA.Statement of SignificanceSingle stranded (ss) DNA binding (SSB) proteins are essential for genome maintenance. Usually homo-tetrameric, bacterial SSBs bind ssDNA in multiple modes, one of which involves wrapping 65 nucleotides of ssDNA around all four subunits. Crystal structures of E. coli and P. falciparum SSB-ssDNA complexes show ssDNA bound with different backbone polarity orientations raising the question of whether these SSBs maintain strict backbone polarity in binding ssDNA. We show that both E. coli and P. falciparum SSBs can still form high affinity fully wrapped complexes with non-natural DNA containing internal reversals of the backbone polarity. These results suggest that both proteins maintain a strict backbone polarity preference, but adopt an alternate ssDNA wrapping topology.

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