3-4 Advances in RNA Sensing by the Immune System Separation of siRNA Unwanted Effects from RNA Inter

Creative Biolabs is a contract research organization based in New York that specializes in antibody discovery and biomanufacturing solutions. They recently discussed the molecular basis of RNA immunofencers and the role of TLR7 and TLR8 in immune response. They found that 2-feed modified RNA can prevent immune activation and may be used as a drug for immune-related diseases. They also discussed the gene silencing pathway and the role of small interfering RNAs. It was observed that small interfering RNAs with 2 bases 3 feet overhangs can trigger innate immunity. MicroRNAs, which are involved in gene regulation, are transcribed from endogenous genes and can escape immune activation. The sensing effect of RNA helicases retinoic acid inducible gene 1 on foreign RNA can be detected by transfecting small interfering RNAs into cells. Chloroquine can inhibit the immunostimulatory activity of chemically synthesized small interfering RNAs. RNA helicases retinoic Welcome to Creative Biolabs, 100% of the effort, 100% of the service. As a dynamic contract research organization, we are based in New York and serve the whole world. Our seasoned scientists are skilled in antibody discovery, antibody engineering, and biomanufacturing solutions. Good evening, dear friends. Thank you for tuning in again. In the last program, we talked about the molecular basis of single stranded and double stranded RNA immunofencers. The main innate immune response of immune cells to chemically synthesize small interfering RNAs seems to be mediated by TLR7 or TLR8 or both. Importantly, studies have also shown that replacing uridine with 2-feed modified uridine can prevent immune activation. Our focus was on how to separate gene silencing from immune stimulation. Today, David will tell us how to inhibit 2-feed modified RNA and the molecular basis of RNA sensing by RNA helicases retinoic acid inducible gene 1. Welcome, David. Thank you for inviting me. I'm very excited to be here. So why don't we start from why we need to inhibit 2-feed modified RNA? Unmodified RNA, rather than 2-feed modified RNA, was found to be an effective trigger for innate immunity, which raises the question whether the structural differences could be related to the binding of TLR7 or TLR8. Two problems we are looking at here. Which step will be affected by the 2-feed modification? And why can't 2-feed modified RNA trigger immune activation? To find the answer to the first question, we need to evaluate whether 2-feed modified RNA can antagonize immune stimulating RNA to trigger TLR7 or TLR8 signaling. So what did the results show? Can 2-feed modified RNA have this effect? Studies on transfected human monocytes showed that 2-feed oxymethyl modified RNA at very low concentrations could eliminate the activation of TLR7 by immune stimulating RNA. And they can also effectively inhibit immune activation through a variety of immune stimulating sequences, including bacterial and mitochondrial RNA. What about chemically modified RNA? What changes can it cause? Chemically modified RNA can antagonize immune stimulating RNA and activate the immunosuppressive enzyme. According to our data, 2-feed modified immune stimulating RNA can play the role of TLR7 or TLR8 antagonist by inhibiting TLR7 signal induced by immune stimulating RNA in mouse and human cells. So it seems 2-feed modified RNA can be used as a drug for immune related diseases. Considering that 2-feed deoxyuridine or thymidine modified small interfering RNA cannot bind to TLRs, inhibitory 2-feed modified RNA should represent a new class of drugs, which may be useful in the treatment of autoimmunity triggered by TLR7 and TLR8 signals. What RNAs are involved in the gene silencing pathway? As we talked about earlier, the gene silencing pathway is initiated by the production of small RNAs containing 20 to 30 nucleotides. The sequences of these small RNAs are complementary to some regulatory transcripts. There are three main types of small regulatory RNAs. Small interfering RNAs, microRNAs, and Peewee domain interacting RNAs. Now to produce small interfering RNAs and microRNAs, we need to rely on the endonucleolytic processing of double stranded RNA precursors by Dicer. This is an N-nuclease belonging to the RNASA3 family yielding small interfering RNA duplexes of 21 to 25 nucleotides with two bases 3 feet overhangs. Is small interfering RNA detectable by viral sensor RNA helicases retinoic acid inducible gene 1? That's a good question. So in some of the recent studies, it was observed that the introduction of small interfering RNA through electroporation into the cytoplasm of peripheral blood mononuclear cells does not induce the production of inflammatory cytokines and interferon 1. But the same sequence can induce the production of inflammatory cytokines and type 1 interferon through lipid delivery. So you see, the conclusion given was that the synthesized small interfering RNA could not be detected by the newly identified viral RNA sensor RNA helicases retinoic acid inducible gene 1. And do we know why? Unfortunately, their data can't really explain the why at this time. Oh, but I'm sure as more studies are going to be focused on this, the mystery will soon be revealed. What about any special functions of small interfering RNAs with two bases 3 feet overhangs? Any more studies on these? I'm happy to report for this, yes. Recent structural studies have shown that the synthesized small interfering RNA double strand with two bases 3 feet overhangs not the small interfering RNAs with blunt ends can trigger innate immunity. The researchers showed that RNA helicases retinoic acid inducible gene 1 combined small interfering RNAs with or without two base 3 feet overhangs. However, only those with blunt ends could trigger its ATPase activity. This observation implies that endogenous short hairpin RNAs or micro RNA harboring the dicer signature 2 base 3 feet overhangs are not perfect stimulators of RNA helicases retinoic acid inducible gene 1. Therefore, the 5 feet end structure between short hairpin RNAs and non-self double stranded RNAs, such as viral RNA, is crucial for self-recognition and non-self-recognition. We have known that the main forms of double stranded RNAs naturally produced in mammalian cells are endogenous micro RNAs. What can you tell us about the micro RNAs? These micro RNAs constitute a large class of non-coding small RNAs, which are involved in the gene regulation of various organisms from plants to mammals. At present, more than 1000 potential human micro RNAs have been identified, many of which have been verified by experiments. Where do these micro RNAs come from? Generally, micro RNAs are transcribed from endogenous genes by RNA polymerase 2 as long RNA precursors called primary micro RNAs. They are then transported from the nucleus to the cytoplasm through export protein 5 and its 2 bases 3 feet overhangs is recognized by dicer, leading to the generation of mature micro RNAs that can escape RNA helicases retinoic acid inducible gene 1 activation. And during this same study, the scientists have also found that innate immunity was not activated when a synthetic 21 nucleotide single stranded sequence was delivered to the cytoplasm by electroporation. These RNAs do not contain 2 bases 3 feet overhangs because they are single stranded. Do we know how to detect the sensing effect of RNA helicases retinoic acid inducible gene 1 on foreign RNA? To do that, T7 transcribed small interfering RNAs or chemically synthesized small interfering RNAs need to be transfected into adherent peripheral blood mononuclear cells. One of the best way to achieve this is using a T7 high yield transcription kit to do the in vitro transcription. Will chloroquine affect these two kinds of small interfering RNA? Chloroquine can inhibit the immunostimulatory activity of chemically synthesized small interfering RNA, but it cannot eliminate the immunostimulatory activity of small interfering RNA synthesized by T7. In addition, the specific inhibitor of protein kinase receptors cannot inhibit the immune stimulation of small interfering RNA synthesized by T7. So which are the cytoplasmic factors that can sense in vitro transcribed RNA but not chemically made small interfering RNAs? Many cytokines can recognize RNA transcribed in vitro. Among them, and again we mentioned this, RNA helicases retinoic acid inducible gene 1 is the most studied. Researchers have shown that it can recognize single stranded RNA carrying 5 feet triphosphate, which is a specific signal of virus and RNA transcribed in vitro. More recently, the recognition of triphosphorylated RNA was found to be mediated by the C-terminal regulatory domain of RNA helicases retinoic acid inducible gene 1. I just realized something interesting. Why do certain endogenous RNAs with 5 feet triphosphates escape the recognition of RNA helicases retinoic acid inducible gene 1? Right, we don't know why currently. In mammals, cellular RNA is treated to mask or remove the 5 feet triphosphate fraction. Naturally occurring 2 feet modification may protect cellular RNA carrying feet triphosphate from detection by RNA helicases retinoic acid inducible gene 1. Thank you for sharing with us your insight, David. That's the end of today's program. Thank you for listening. We will continue our mRNA discussion next time.