Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R(9)F(2) was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R(9) conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R(9)F(2) and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide

Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

The trans-activation response (TAR) RNA stem–loop that occurs at the 5′ end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem–loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R(6)-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP–PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP–PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition

A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification

RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3′ end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed ‘crook’ siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics

Parallel synthesis and splicing redirection activity of cell-penetrating peptide conjugate libraries of a PNA cargo

A novel method for the parallel synthesis of peptide-biocargo conjugates was developed that utilizes affinity purification for fast isolation of the conjugates in order to avoid time consuming HPLC purification. The methodology was applied to create two libraries of cell-penetrating peptide (CPP)-PNA705 conjugates from parallel-synthesized peptide libraries. The conjugates were tested for their ability to induce splicing redirection in HeLa pLuc705 cells. The results demonstrate how the novel methodology can be applied for screening purposes in order to find suitable CPP-biocargo combinations and further optimization of CPPs.</p

Systematic screening of LNA/2′-O-methyl chimeric derivatives of a TAR RNA aptamer

AbstractWe synthesized and evaluated by surface plasmon resonance 64 LNA/2′-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2′-O-methyl nucleoside analogues where one or two LNA units are located on the 3′ side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay

Synthesis and Splice-Redirecting Activity of Branched, Arginine-Rich Peptide Dendrimer Conjugates of Peptide Nucleic Acid Oligonucleotides

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide−PNA conjugates showed poor activity compared to a linear (R-Ahx-R)4−PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide−PNA conjugates showed activity similar to that of the corresponding linear peptide−PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence

Sequential NMR assignments of labile protons in DNA using two-dimensional nuclear-overhauser-enhancemnt spectroscopy with three jump-and-return pulse sequences

Two-dimensional nuclear Overhauser enhancement (NOESY) spectra of labile protons were recorded in H2O solutions of a protein and of a DNA duplex, using a modification of the standard NOESY experiment with all three 90&#176; pulses replaced by jump-and-return sequences. For the protein as well as the DNA fragment the strategically important spectral regions could be recorded with good sensitivity and free of artifacts. Using this procedure, sequence-specific assignments were obtained for the imino protons, C2H of adenine, and C4NH2 of cytosine in a 23-base-pair DNA duplex which includes the 17-base-pair OR3 repressor binding site of bacteriophage &#955;. Based on comparison with previously published results on the isolated OR3 binding site, these data were used for a study of chain termination effects on the chemical shifts of imino proton resonances of DNA duplexes

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10 μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated

Improved cell-penetrating peptide–PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle

Steric blocking peptide nucleic acid (PNA) oligonucleotides have been used increasingly for redirecting RNA splicing particularly in therapeutic applications such as Duchenne muscular dystrophy (DMD). Covalent attachment of a cell-penetrating peptide helps to improve cell delivery of PNA. We have used a HeLa pLuc705 cell splicing redirection assay to develop a series of PNA internalization peptides (Pip) conjugated to an 18-mer PNA705 model oligonucleotide with higher activity compared to a PNA705 conjugate with a leading cell-penetrating peptide being developed for therapeutic use, (R-Ahx-R)4. We show that Pip–PNA705 conjugates are internalized in HeLa cells by an energy-dependent mechanism and that the predominant pathway of cell uptake of biologically active conjugate seems to be via clathrin-dependent endocytosis. In a mouse model of DMD, serum-stabilized Pip2a or Pip2b peptides conjugated to a 20-mer PNA (PNADMD) targeting the exon 23 mutation in the dystrophin gene showed strong exon-skipping activity in differentiated mdx mouse myotubes in culture in the absence of an added transfection agent at concentrations where naked PNADMD was inactive. Injection of Pip2a-PNADMD or Pip2b-PNADMD into the tibealis anterior muscles of mdx mice resulted in ∼3-fold higher numbers of dystrophin-positive fibres compared to naked PNADMD or (R-Ahx-R)4-PNADMD

With a biomechanical treatment in knee osteoarthritis, less knee pain did not correlate with synovitis reduction

© 2017 The Author(s). Background: Braces are used to treat pain in patellofemoral joint osteoarthritis (PFJOA). In a trial, we previously reported pain improvement after 6-weeks brace use. The pain reduction did not correlate with changes in Magnetic Resonance Imaging (MRI) assessed Bone Marrow Lesion volume or static synovial volume. Studies show that changes in the synovium on dynamic contrast enhanced (DCE) MRI are more closely associated with symptom change than static synovial volume changes. We hypothesised change in synovitis assessed using dynamic imaging could explain the reduction in pain. Method: One hundred twenty-six men and women aged 40-70 years with painful radiographically confirmed PFJOA were randomised to either brace wearing or no brace for 6-weeks. Pain assessment and DCE-MRI were performed at baseline and 6 weeks. DCE data was analysed using Tofts's equation. Pain measures included a VAS of pain on nominated aggravating activity (VAS NA ), and the KOOS pain subscale. Paired t-tests were used to determine within person change in outcome measures and Spearman's correlation coefficients were used to determine the correlation between change in pain and change in the DCE parameters. Results: Mean age of subjects was 55.5 years (SD = 7.5) and 57% were female. There was clear pain improvement in the brace users compared to controls (VAS NA - 16.87 mm, p = < 0.001). There was no significant change to the dynamic synovitis parameters among brace users nor was pain change correlated with change in dynamic synovitis parameters. Conclusion: The reduction in knee pain following brace wearing in patients with PFJOA is not explained by changes in synovitis. Trial registration: Trial registration number UK. ISRCTN50380458 /Registered 21.5.2010