Nucleic Acid Membraneless Organelles

BackgroundNucleic acid nanostructures include DNA and RNA nanostructures.In recent years,researchers have successfully constructed a series of exquisite nucleic acid nanostructures with extensive functions.In general,complicated structures need multi–component nucleic acids,but large number of unique nucleic acid strands bring two problems:1)the synthesis is quite expensive;2)the handling in experiment is tedious and easy to introduce errors.It would be highly desirable to produce nucleic acid nanostructures with small number of unique nucleic acid strands and simple assembly process.According to genetic central dogma,current established methods can silence the target gene at different levels.Gene silencing that occurs at the post–transcriptional level is termed as post–transcriptional gene silencing.Most of the currently established post–transcriptional gene silencing methods are silencing in the cytoplasm,and rare methods are used to directly interfere with RNA in the nucleus.Molecularly self–assembled nucleic acid nanostructures with excellent programmability and biocompatibility targetting post–transcriptional gene silencing will possibly establish a safe and effective new method.Nucleic acid membraneless organelles refer to membraneless aggregates composed of nucleic acids in the cell.At present,few studies have been addressed on the formation mechanism and function of nucleic acid membraneless organelles in vitro and in vivo,and therefore,the combination of the fields of both nucleic acid nanostructures and membraneless organelles can promote their development.Objectives1 To conveniently construct DNA and RNA nanostructures with single component and extensive functions.2 Establish a new method of gene silencing that can inhibit mRNA translation in the nucleus.3 Combining the fields of both nucleic acid nanostructures and membraneless organelles to promote their development.Methods and Results1 Design,assembly and characterization of DNA membraneless organellesAccording to the characteristics of the palindrome,two sets of DNA strands with different numbers of palindromic domains（with and without tail）were designed by Tiamat software.The secondary structures and the folding information of the two sets of DNA strands were identified through online website（https://sg.idtdna.com and http://unafold.rna.albany.edu）.The DNA membraneless organelles DNA microparticles（DNAμPs）containing different numbers of palindromic domains were assembled by one–pot,one–step process and analyzed by native polyacrylamide gel electrophoresis（PAGE）,agarose gel electrophoresis,and laser scanning confocal microscope（LSCM）.All assembled DNA samples were large aggregates with high molecular weights,and the aggregates formed are larger with increasing number of palindromic domains.Both sets of DNA can form micron–sized particles.Both sets of DNAμPs have phase separation properties by centrifugation.Assembly yields of two sets of DNAμPs were semi–quantified by centrifugation.The assembly yields for all DNAμPs were estimated to be roughly the same,96%.The tailed DNAμPs with more palindromic domains had higher polymerization yield and would result in larger homopolymers.The assembly yields were 20%for 4 PB,and 90%for 7 PA,respectively.The effect of DNA concentration on the assembly of DNAμPs with tail was observed by LSCM.The effect of Mg²⁺concentration on the assembly of DNAμPs with tail was investigated by LSCM and centrifugation method.We found that lower DNA concentration led to less and smaller DNAμPs.When the concentration decreased to lower than 0.63μM,we could hardly observe any DNAμP.A higher Mg²⁺concentration led to more and larger DNAμPs and a higher assembly yield.At 22.5 mM Mg²⁺,DNAμPs started to fuse with each other into large aggregates.At 3 mM Mg²⁺,we could hardly observe any DNAμPs.2 Study on the effect of DNA membraneless organelles on immunostimulation of macrophages and capture of single–stranded DNAWe linked a CpG motif（CpG 1826）to the multidomained,polymerizable DNA strands,successfully designed and assembled four immunostimulatory CpG–DNAμPs with different numbers of palindromic domains.Enzyme–linked immunosorbent assay（ELISA）revealed that CpG–DNAμPs can stimulate RAW 264.7 macrophages to secrete proinflammatory cytokines and immune responses.Cell counting kit–8（CCK8）experimental data showed that CpG–DNAμPs does not affect the survival rate of RAW 264.7 macrophages.Native PAGE analysis revealed that DNAμPs with tail can capture single–stranded DNA in vitro.At 37°C,the capture efficiency of all DNAμPs was estimated to be roughly the same,50%.At 22°C,the capture efficiency was 88%for 7 PA and nearly 100%for the other three types of DNAμPs.3 Capturing intracellular oncogenic miR–21 with DNA membraneless organelles for miRNA–based cancer therapyLink antisense oligonucleotide 21（ASON 21）to DNAμPs to design DNAμP21（n P21,n=1–7）with different number of palindromic domains.One–pot,one–step method was used to assemble each DNAμP21,and LSCM was used to record the morphology of DNAμP21.7 P21,6 P21,5 P21,4 P21,and 3 P21 were micrometer–sized particles,but 2 P21 and 1 P21 could hardly form particles.Native PAGE analysis revealed that DNAμP21 can capture miR–21.The capture efficiency of 7 P21is about 40%,and about 85%for 6 P21,5 P21 and 4 P21.For all other DNAμP21,the efficiency is nearly 100%.LSCM imaging revealed that DNAμP21 was uptaken by MCF–7 cells.The Cy3–tagged DNAμP21（red）aggregated as large particles surrounding the nucleus（blue）.Real–time quantitative polymerase chain reaction（qPCR）results found that DNAμP21 can effectively knockdown intracellular oncogenic miRNA levels.Compared with the transfection reagent group,the levels of miR–21 in each DNAμP21 group were down–regulated significantly,and the down–regulating effect on miR–21 levels gradually increased when the number of palindromic domains of DNAμP21 decreased.CCK 8 method found that DNAμP21can inhibit MCF–7 breast cancer cell proliferation.With the decrease of the palindromic domain,the effect of DNAμP21 on MCF–7 breast cancer cell proliferation inhibition was gradually strengthened.The effect of 21–1 P on MCF–7breast cancer cell proliferation inhibition was gradually strengthened with longer administration time,reaching 30%cell proliferation at 72 h.The effect of 21–1 P on MCF–7 breast cancer cell proliferation inhibition was gradually strengthened with dosage increase,and the cell proliferation was of 30%at 2400 nM.4 Design,assembly and characterization of RNA membraneless organellesThe DNA sequences with multiple palindromic domains were transcribed to the RNA sequences with multiple palindromic domains.To identify the secondary RNA structures and the folding information,we used online software（https://sg.idtdna.com and http://unafold.rna.albany.edu）.7 C,6 C,5 C and 4 C were successfully obtained by transcription in vitro.Agarose gel electrophoresis,native PAGE and LSCM were used to investigate the effects of annealing process and Mg²⁺concentration on the assembly of RNA membraneless organelles–RNA microaggregates（RNAμAs）.The results show that Mg²⁺concentration is beneficial for the assembly of RNAμAs,and the high temperature will hinder the assembly of RNAμAs.RNAμAs with multiple palindromic domains can be used for self–assembly in vivo.5 Study on gene silencing effect of RNA membraneless organellesTarget DNA were amplified from the recombinant plasmids,including GFP–pcDNA3.1,7 C+GFP–pcDNA3.1 and NC+GFP–pcDNA3.1.The mRNA of GFP,7C+GFP and NC+GFP were obtained by transcription in vitro.Compared with GFP mRNA and NC+GFP mRNA,the mobility of 7 C+GFP mRNA in the gel is reduced.The results show that RNAμAs can reduce the gel mobility of GFP mRNA.48 h after administration of recombinant plasmids,GFP expression in Hela cells was detected by LSCM,flow cytometry and western blotting.The results show that RNAμAs can reduce the expression of GFP protein in Hela cells,and the silencing efficiency was about 50%.Cytotoxicity of RNAμAs was estimated using cell counting kit–8（CCK8）assay.Effect of RNAμAs on the expression of internal reference proteins GAPDH and Bata Tubulin in Hela cells was investigated by Western blot.The results show that RNAμAs did not affect the Hela cell survival and intracellular function.Conclusions1 Single component DNA membraneless organelles–DNAμPs with simple design and convenient assembly were constructed successfully.The DNAμPs can transport functional nucleic acid drugs into cells,thereby enhancing the immune response and inhibiting cancer cell proliferation.2 Single component RNA membraneless organelles,RNAμAs with simple design and convenient assembly strategy was constructed successfully.These RNAμAs can be intracellularlly self–assembled.3 A new method for post–transcriptional gene silencing has been established.In this experiment,RNAμAs were used to capture the mRNA of the target protein,thereby preventing the mRNA from entering the cytoplasm and hindering the translation of the target protein.4 The fields of nucleic acid nanostructure self–assembly and of membraneless organelles were fused for multidisciplinary development.