Modied comb-shaped antisense oligonucleotides may secure us against many important viral diseases including AIDS

To inhibit HIV replication and infection, we have designed novel linear single stranded modied antisense nucleic acid oligonucleotides ending with or without chain terminating bases (Which resemble the shape of the comb). They were targeting specically the HIV-1 clone pNL4-3 strong promoter pre PBS region to stop cDNA synthesis within or before the R region, preventing the viral reverse transcriptase (RT) jumping to the 3' end and continue copying the virus. The main advantages of our comb shaped oligonucleotides are their specicity and extreme protection against resistance by known viral mutations. Promising results were obtained for two 15-mer compounds at one tenth azidothymidine concentration. As a result we claim that when adapted properly, the comb shaped antivirals can be used to target the genomic RNA of a number of serious viruses such as for example Ebola, SARS-CoV-2, Inuenza, Dengue, hepatitis C, Chikungunya and Zika as they are all using polymerases to copy their genomic RNA 1-8 . Their genomic RNA could be destroyed through the human or viral endonucleases instead of the viral RT RNAseH site when their polymerases are stopped at specic sites.


Main Text
In 1987 the FDA approved Zidovudine, the rst therapy against HIV which was a nucleoside reverse transcriptase inhibitor. By 1996, department of health and human services (DHHS) and the world health organization (WHO) recommended combining anti-HIV medicines; this combined treatment is called antiretroviral therapy 9 .
Anti-HIV drugs are organized into ve classes based on the stage of the HIV life cycle they inhibit. As of 2019, there were 28 individual agents and 13 xed dosed combination drugs targeting viral reverse transcriptase (RT), protease and integrase, as well as the cellular entry co-receptor CCR5 10 .
Thirteen individual agents are reverse transcriptase inhibitors belonging to the nucleoside or nucleotide reverse-transcriptase inhibitors (NRTIs) category which lack 3' hydroxyl group on their ribose or ribose mimic moiety including Emtricitabine 11 , Lamivudine 12 , Abacavir 13 , Di-adenosine, Stavudine, Zidovudine and Zalcitabine which has been discontinued 14 , 15,16 or to the non-nucleoside or nucleotide reversetranscriptase inhibitors (NNRTIs) which bind into a hydrophobic pocket close to the polymerase active site and inhibit polymerization, including Rilpivirine, Etravirine,Delavirdine, Doravirine,Efavirenz and Nevirapine 17 .
All anti-HIV drugs are associated with a number of short-and long-term side effects 18 . Chronic HIV infection is associated with increased risk co morbidities. Lapses in medication adherence can lead to viral rebound and disease progression 19 also HIV develops rapidly mutations that affect virus susceptibility to treatment 20 .
For decades the possibility to use nucleic acids as antiviral therapeutics has been studied. In theory antisense oligonucleotides, ribozymes, DNAymes, and aptamers can trigger sequence speci c inhibition of particular mRNA transcripts including the viral genomes. However di culties with their e ciency, offtarget effects, toxicity, delivery and stability halted their development and use in the clinic 21 .
GEM (gene expression modulator) 91 is a 25-mer phosphorothioate (PS) oligonucleotide complementary to the HIV-1 gag initiation site with multiple inhibitory mechanisms 22 . The non-sequence dependent inhibition of virus entry and reverse transcription were due to a poly-anionic effect of the PS backbone. GEM 91 intracellular RNase H cleavage was ine cient as its free uptake was almost restricted to endosomal vesicles within the cytosol 23 . PS oligonucleotides nuclear delivery was e ciently improved with cationic lipids inducing higher antisense activity 24 . Our design provides compounds intended to stop cDNA synthesis before or at the R region of HIV-1 clone pNL4-3 (GenBank accession No. M19921.2). they prevent RT from continuing cDNA synthesis from the 3' end as no or small part of the R region is going to be transcribed, the last 3' base in these compounds could be a chain terminating base. It is di cult for RT RNAse H site or nucleases to break these compounds down and continue transcription and synthesis as they are modi ed to resist breaking in all breakable positions.
All mutations that affect RT, protease, integrase as well as envelop gene can't affect them.as they target the pre-PBS region which is a highly conserved region. Moreover, any possible mutations such as SNPs will not affect them as they are polynucleotides. The suspected lower resistance may allow such compounds to have virucidal activity when a suitable dose is used.
Our compounds have a good stability, water solubility and sequence speci city and could be more improved to be druggable and may constitute a nucleus of a new type of antiviral compounds.

Methods
Four Comb shaped antisense compounds overlapping the strong promoter region of HIV-1 (oligos 1-4) and two scrambled controls (oligo 5 and 6) were custom synthesized by Biosynthesis incorporation, Texas, USA. At the time of experiment they were brie y centrifuged and re-suspended in non-DEPC-treated water at a concentration of 1mM. Sequences are given in  Digoxigenin-and biotin-labeled nucleotides were incorporated into the RT-synthesized DNA molecule. Then, the detection and quanti cation of synthesized DNA (as a parameter for RT activity) was done using a sandwich ELISA protocol: Biotin-labeled DNA binds to the surface of micro-plate (MP) modules that have been precoated with streptavidin. In the next step, an antibody to digoxigenin conjugated to peroxidase (anti-DIG-POD) binds to the digoxigenin-labeled DNA. In the nal step, the peroxidase substrate ABTS was added. The peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product 1, 2 . The intensity of the color product was then determined using an ELISA reader and is directly correlated to the level of RT activity in the sample (Figure 2).
After PCR ampli cation, the DNA was puri ed using the Qiaquick PCR puri cation kit (Qiagen), then digested using restriction enzymes KpnI and NotI, and cloned into a linearized pcDNA3.1 vector (prepared using KpnI and NotI) using standard cloning techniques 3 and DH5α competent cells 4 . The resulting construct was con rmed by the colony PCR technique and DNA sequencing. Single bacterial colonies carrying the HIV-1-promoter-pcDNA3.1 vector were used to initiate an overnight liquid culture to amplify the plasmid DNA which was isolated and puri ed using Qiagen Maxi-Prep kit.

RNA Transcription
The plasmid (HIV-1-promoter-pcDNA3.1) was linearized using XhoI (NEB), then puri ed by phenol/chloroform extraction and ethanol precipitation and dissolved in nuclease-free water. The mMESSAGEmMACHINE T7 Ultra transcription kit (Invitrogen) 5 , was used to generate the replicon RNAs in the correct orientation from the linearized vector according to manufacturer's instruction. The synthesized RNA was puri ed by phenol:chloroform extraction and isopropanol precipitation. (reaction mixture) were then added. This is termed 'the reaction mixture.' Next, we added the test oligos at 0.5, 5 &50 μM concentration to the reaction mixture and incubated them for 1 h at 37°C. We also incubated the reaction mixture without any oligos, as our no-inhibitor control.
Reaction mixture plus oligos with no HIV-1-RT was used as negative controls. These samples were then transferred to wells of the kit's Micro Plate module. The plate was covered and incubated for 1 h at 37°C and washed ve times with wash buffer. Anti-DIG-POD (200 μl working dilution) was added and incubated for 1 h at 37°C. The plate was again washed ve times with wash buffer. ABTS substrate solution (200 μl) was added and incubated at room temperature for 30 min until a green color developed.
The plate was then read at 405 nm in an ELISA reader. Lysis buffer without RT was used as a negative control and azidothymidine (0.5mM AZT) with RT was used as a positive control.

Results
The resulting colorimetric signal intensity is directly proportional to the RT activity. Therefore, the inhibitory activity of the tested RT inhibitors can be expressed as the percent inhibition relative to a sample without inhibitor or the no-inhibitor control.