WO2020023741A1 - Large scale production of rna particles - Google Patents

Abstract

RNA nanotechnology has shown promise for the delivery of siRNA, miRNA, or other therapeutics to multiple types of cancers while showing little accumulation in healthy organs while displaying specific targeting. However, the yield and cost of RNA production has been a bottle neck for the advancement of the RNA field in both research and clinical translation. Disclosed herein is a large scale RNA production method using rolling circle transcription (RCT) form dsDNA that uses encoded ribozymes to self-cleave the RNA concatamers to release target oligonucleotide sequences. The released co-transcribed multiple short RNA oligomers self-assembled into RNA nanoparticles, which can then be purified using industrial scale gel electrophoresis column.

Description

LARGE SCALE PRODUCTION OF RNA PARTICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 62/703,347, filed July 25, 2018, which is hereby incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant Nos. U01 CA151648 and U01 CA207946 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled“321501-2310 Sequence Listing_ST25” created on July 25, 2019. The content of the sequence listing is incorporated herein in its entirety.

BACKGROUND

The field of RNA research has been expanded dramatically due to the finding that RNA plays a major role in the regulation of cellular activities. Once thought as a molecule that only transfers information between DNA and protein, RNA has now been found to partake in activities such as catalysis (Westhof E. (2012) Methods Mol Biol, 848:1-4; Cech T.R., et al. (1981) Cell, 27:487-496; Guerrier-Takada C., et al. (1983) Cell, 35:849-857), gene regulation, translation regulation, RNA splicing, and so forth (Lieberman J., et al. (2013) Cell, 153:9-10), RNAs, including RNA nanoparticles (Guo P. (2010) Nature

Nanotechnology, 5:833-842; Jasinski D., et al. (2017) ACS Nano, 1 1 :1 142-1 164; Afonin K.A., et al. (2014) Nano Lett., 14:5662-5671), small therapeutic RNAs such as siRNA (Yang D., et al. (2002) Proc. Natl. Acad. Sci. U. S. A, 99:9942-99), ribozyme (Hampel A. (1998) TProg. Nucleic Acid Res Mol Biol., 58:1-39), miRNA (Dennis C. (2002) Nature, 420:732), riboswitch (Tucker B.J., et al. (2005) Curr. Opin. Struct. Biol., 15:342-348), aptamers (Zhou J., et al. (2012) Front Genet., 3:234), and other chemicals targeting RNA, have been predicted to be the third milestone in drug development, following chemical drugs and protein drugs (Jasinski D., et al. (2017) ACS Nano, 1 1 :1 142-1 164).

RNA nanotechnology, with its concept proven in 1998 by showing the assembly of RNA dimers, trimers, tetramers and hexamers from engineered RNA oligoes (Guo P., et al. (1998) Mol. Cell., 2:149-155), has been a field that continues to emerge rapidly. The versatility, complexity, and diversity of RNA make it an attractive biomaterial to construct nanostructures with defined shape, structure, and physical or biological properties (Afonin K.A., et al. (2014) Nano Lett., 14:5662-5671 ; Afonin K.A., et al. (2016) Nano Lett, 16:1746- 1753; Khisamutdinov E.F., et al. (2014) ACS Nano., 8:4771-4781 ; Grabow W.W., et al. (2014) Accounts of Chemical Research, 47:1871-1880; Lee J.B., et al. (2012) Nat. Mater., 1 1 :316-322). Many issues of RNA instability have been overcome to achieve a nanoparticle platform with potential for treatment of disease, especially cancers (Guo P. (2010) Nature Nanotechnology, 5:833-842; Liu J., et al. (201 1) ACS Nano, 5, 237-246; Binzel D.W., et al. (2014) Biochemistry, 53:2221-2231 ; Binzel D.W., et al. (2016) RNA, 22:1710-1718).

Targeting aptamers and functional RNAs such as siRNA, miRNA, ribozymes, and riboswitches are available to construct diverse multi-functional nanoparticles (Afonin K.A., et al. (2012) Nano. Lett., 12:5192-5195; Boerneke M.A., et al. (2016) Angew. Chem Int. Ed Engl., 55:4097-4100; Leontis N.B., et al. (2014) Science, 345:732-733; Afonin K.A., et al. (2014) Acc. Chem. Res., 47:1731-1741 ; Liu Y., et al. (2009) Biol. Chem., 390:137-144; Esposito C.L., et al. (201 1) PLoS. One., 6:e24071). Development of RNA nanotechnology has been accelerated by the finding of an unusually stable three-way junction (3WJ) RNA motif from the packaging RNA (pRNA) of the phi29 DNA packaging motor (Shu D., et al.

(201 1) Nature Nanotechnology, 6:658-667). The 3WJ has been used for targeted delivery of therapeutic modalities to multiple cancer types and has been used as a scaffold for the construction of RNA nanoparticles with controllable size and shape (Lee T.J., et al. (2015) Oncotarget, 6:14766-14776; Cui D., et al. (2015) Scientific reports, 5:10726; Khisamutdinov E.F., et al. (2016) Advanced Materials, 28:100079-100087; Khisamutdinov E., et al. (2014) Nucleic Acids Res., 42:9996-10004; Jasinski D., et al. (2014) ACS Nano, 8:7620-7629).

The yield of RNA nanoparticle construction has proven a bottle neck for the advancement of the RNA field due to somewhat complicated nanoparticle assembly methods. For example, assembly of a small three-stranded RNA nanoparticle requires synthesis of three DNA templates, followed by purification of ssRNA monomers, assembly of the particles, and finally an additional particle purification.

SUMMARY

RNA nanotechnology has shown promise for the delivery of siRNA, miRNA, or other therapeutics to multiple types of cancers while showing little accumulation in healthy organs while displaying specific targeting. However, the yield and cost of RNA production has been a bottle neck for the advancement of the RNA field in both research and clinical translation. Disclosed herein is a large scale RNA production method using rolling circle transcription (RCT) from circular dsDNA templates that uses encoded ribozymes to self-cleave the RNA concatamers to release target oligonucleotide sequences. The released co-transcribed multiple short RNA oligomers self-assembled into RNA nanoparticles, which can then be purified using industrial scale gel electrophoresis columns.

Disclosed herein is a circular dsDNA polynucleotide that includes a nucleic acid sequence encoding a nucleic acid sequence encoding an RNA particle flanked by selfcleaving ribozymes operably linked to an expression control sequence.

Also disclosed is a system for large-scale synthesis of an RNA particle that involves the disclosed circular dsDNA polynucleotide and a buffered medium comprising an RNA polymerase and ribonucleotide triphosphates (NTPs), wherein rolling circle transcription (RCT) of the dsDNA template by the RNA polymerase produces an RNA concatamer, wherein self-cleavage of the ribozymes produces one or more RNA fragments that self- assemble to form the RNA particle.

Also disclosed is a method for large-scale synthesis of an RNA particle that involves incubating the circular dsDNA polynucleotide under conditions that promote rolling circle transcription (RCT) of the dsDNA polynucleotide and self-cleavage of the self-cleaving ribozymes to produce one or more RNA fragments that self-assemble to form the RNA particle; and purifying the RNA nanoparticle on an electrophoresis column.

The disclosed circular dsDNA polynucleotide can encode any RNA, such as a therapeutic RNA, that self-assembles into a particle. In some embodiments, the RNA particle is a RNA nanoparticle. In some embodiments, the RNA particle is a siRNA, ribozyme, miRNA, riboswitch, or aptamer.

In some embodiments, the RNA particle is self-assembled from a single RNA molecule. However, in some embodiments, the RNA particle is self-assembled from two or more RNA subunits, including 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits. In these embodiments, the nucleic acid sequence encoding the RNA particle comprises a nucleic acid sequence encoding each of the two or more RNA subunits, wherein each nucleic acid encoding a RNA subunit comprises a ribozyme-cleavable sequence.

In some embodiments, the expression control sequence is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS

FIG. 1 is a scheme for Circular dsDNA Assembly. Assembly scheme for the construction of double stranded circular DNA encoding for T7 RNA promoter (red), selfcleaving ribozymes (green), and product RNA sequence (orange).

FIG. 2 illustrates circular dsDNA Assembly. Assembly of circular dsDNA encoded for a single pRNA 3WJ strand surrounded by the ribozyme for cleavage. Representative gel image of the assembly process. 1 : ssLin DNA; 2: ssCirc DNA, 3: dsLin DNA; 4: dsCirc DNA +Nick; 5: dsCirc DNA +T4 Ligation to close nick

FIG. 3 illustrates RCT Assessment. Inactive ribozymes leads to concatamerized RNA, while cleavage results in short oligomers. 1 : Lin ssDNA; 2: Circ ssDNA; 3: Lin dsDNA; 4: Circ dsDNA +Nick; 5: Circ dsDNA; 6: Lin dsDNA; 7: Circ dsDNA +Nick; 8: Circ dsDNA. Lanes 3-5 use inactivated ribozyme sequences, lanes 6-8 use active ribozyme sequences.

FIGs. 4A and 4B show in situ or one-pot co-transcriptional nanoparticles. FIG. 4A shows circular and linear transcription for dsDNA constructs encoding for release of the 3WJ ssRNA oligomers. 1 : 3WJ-A-Lin; 2: 3WJ-A-Circ; 3: 3WJ-B-Lin; 4: 3WJ-B-Circ; 5: 3WJ-C-Lin; 6: 3WJ-C-Circ; 7: 5’/3’Rbz-IN; 8: 3’Rbz-ln; 9: 5’Rbz-IN; 10: 5’Rbz Only; 11 : 3’Rbz Only. FIG. 4B shows assembly of the 3WJ occurs co-transcriptionally by mixing equimolar amounts of dsDNA encoding for the release of 3WJ-A, B, and C strands. RCT-3WJ is assembled from purified RNA fragments from RCT reaction.

FIGs. 5A to 5C show assembly of one-Strand RNA Nanoparticle with functionalities. FIG. 5A is a schematic for modified design of self-cleaving ribozyme. FIG. 5B shows circular dsDNA assembled to code for the 3WJ and MG-3WJ nanoparticles assembled from one long piece of RNA. FIG. 5C shows PAGE analysis of transcription of one piece RNA nanoparticles. 1 : RCT-3WJ; 2: 3WJ-Loop; 3: MG-3WJ-Lin; 4: MG-3WJ-Circ.

FIGs. 6A to 6D show linear vs Circular DNA Transcription Kinetics. FIGs. 6A and 6B shows gel analysis of transcription. Bands were integrated and plotted. FIGs. 6C and 6D show monitoring of transcription using MG fluorescence (specific to nanoparticle folding) and SYBR Green II (RNA specific).

FIGs. 7A and 7B show large scale purification of RNA by gel-electrophoresis Column. FIG. 7A shows MG fluorescence and absorbance at 260 nm were used to analyze fractions after purification. FIG. 7B shows PAGE analysis demonstrating purity before and after gel purification.

FIG. 8A shows PAGE analysis shows active RCT constructs along with size controls of inactivated ribozyme constructs. Size controls allow confirmation of ribozyme cleavage and release of target RNA oligomers. FIG. 8B shows a typical experiment run to determine cleavage efficiency of self-cleaving ribozymes. The target sequence (green box) intensity was added with the cleaved ribozyme (blue box) intensity and then divided by the total band intensity (red + blue + green box) per well. A plot on the right shows the ribozyme cleavage efficiency over time, comparing first generation design (RCT-1.0) to the second generation design (RCT-1.1). The construct containing 3WJ-b sequence is shown here. While a better curve was desired for ribozyme cleavage kinetics, ribozymes self-cleave as they are being transcribed, making it difficult to obtain time points of low percent cleavage. FIG. 8C shows an increase in ribozyme efficiency is attributed to increasing the length of the duplex in the “closing” region of the ribozyme sequence, shown in red boxes.

FIG. 9 shows PAGE analyzing the assembly of circular dsDNA constructs containing the T7 promoter used for transcription reactions.

FIG. 10A shows ribozyme cleaved 3WJ ssRNA oligomers were compared to chemically synthesized sequences identical to those of the target sequence. Evidenced by identical migration rate, we can conclude that the cleaved RNA oligomers are the same size as chemically synthesized controls. FIG. 10B shows RCT cleaved 3WJ ssRNA oligomers were purified by PAGE band isolation. After elution from gel pieces assembly was tested on native PAGE. A stepwise assembly from monomer to dimer and finally trimer complex demonstrate the ssRNA from RCT reactions are indeed the correct sequences.

FIGs 11 A and 11 B show plots and linear fitting of DNA template concentration, x-axis, versus RNA output, as monitored by SYBR Greenll fluorescence. FIG. 11 C shows values of slope and intercept, along with their standard errors and R-Squared values of the fits.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the

publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be

independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise.

As used herein, "self-assembly" refers to the ability of nucleic acids (and, in some instances, preformed nucleic acid nanostructures (e.g., crystals)) to anneal to each other, in a sequence- specific manner, in a predicted manner and without external control. In some aspects, nucleic acid nanostructure self-assembly methods include combining nucleic acids (e.g., single- stranded nucleic acids, or oligonucleotides) in a single vessel and allowing the nucleic acids to anneal to each other, based on sequence complementarity. In some aspects, this annealing process involves placing the nucleic acids at an elevated temperature and then reducing the temperature gradually in order to favor sequence- specific binding. Various nucleic acid nanostructures or self-assembly methods are known and described herein.

As used herein, the term“nanoparticle” is meant to refer to a particle between 1 nm up to 1 ,000 nm in diameter. The nanoparticle can be between 5 nm and 30, 10 nm and 50 nm, between 10 nm and 40 nm, between 10 nm and 30 nm, between 10 nm and 20 nm, and 10 nm and 15 nm. The RNA can be obtained from any source, for example bacteriophages phi 29, HIV, Drosophila, the ribosome, or be a synthetic RNA.

RNA is most commonly synthesized in vitro using run-off transcription of a linear dsDNA template. In vitro run-off transcription is limited by yield, time efficiency, homogeneity, and purity due to delayed RNA polymerase binding and initiation (Maslak M., et al. (1993) Biochemistry, 32:4281-4285; Chamberlin M., et al. (1973) J Biol Chem, 248:2235-2244; Chamberlin M., et al. (1973) J Biol Chem, 248:2245-2250). Rolling circle transcription (RCT) is increasing in popularity due to its unique capabilities (Lee J.B., et al. (2012) Nat. Mater.,

1 1 :316-322; Guo P (2012) Rolling Circle Mol Ther-Nucleic Acids, 1 :e36; Mohsen M.G., et al. (2016) Acc. Chem Res, 49:2540-2550; Mezger A., et al. (2014) PLoS ONE, 9:e1 1 1874; Li X., et al. (2015) Chem Commun. (Camb.), 51 :1 1976-1 1979; Roh Y.H., et al. (2015) Angew. Chem. Int. Ed Engl., 55:3347-3351 ; Shopsowitz K.E., et al. (2014) Small, 10:1623-1633; Hsu B.B., et al. (2014) Biomacromolecules, 15:2049-2057; Han D., et al. (2014) Nature

Communications, 5:4367; Kim H., et al. (2015) Sci Rep., 5:12737). Many functional RNA motifs, such as aptamers, miRNA, siRNA, and ribozymes can be continually synthesized in a normal RCT reaction (Roh Y.H., et al. (2015) Angew. Chem. Int. Ed Engl., 55:3347-3351 ; Shopsowitz K.E., et al. (2014) Small, 10:1623-1633; Hsu B.B., et al. (2014) Biomacromolecules, 15:2049-2057). Short single stranded (ss) circular DNAs encoding for ribozymes that self-process into unit length functional ribozymes have been synthesized. These ribozymes show biological functionality in trans, cleaving HIV-1 RNA targets

(Daubendiek S.L., et al. (1997) Nat Biotechnol, 15:273-277; Diegelman A.M., et al. (1998) Nucleic Acids Res, 26:3235-3241). RCT has been used to synthesize siRNA loaded microsponges that show successful gene knockdown in vivo (Lee J.B., et al. (2012) Nat. Mater., 1 1 :316-322; Roh Y.H., et al. (2015) Angew. Chem. Int. Ed Engl., 55:3347-3351 ; Shopsowitz K.E., et al. (2014) Small, 10:1623-1633). Showing the versatility of RCT, nanowires (Zheng H.N., et al. (2014) Chem Commun. (Camb.), 50:2100-2103), millimeter sized RNA membranes (Han D., et al. (2014) Nature Communications, 5:4367), mRNA nanoparticles (Kim H., et al. (2015) Sci Rep., 5:12737), and tandem repeats of fluorogenic RNA aptamers have all been synthesized (Furukawa K., et al. (2008) Bioorg. Med. Chem Lett, 18:4562-4565).

RCT’s increased transcription efficiency over traditional run-off transcription could help to increase the production yield of RNA oligomers and RNA nanoparticles (Daubendiek S., et al. (1995) J. Am. Chem. Soc., 1 17 (29):7818-7819). Additionally, in vitro transcription is not limited by length and one-strand or multi-strand nanoparticle assembly can occur co- transcriptionally, reducing the total number of steps required for RNA nanoparticle preparation (Afonin K.A., et al. (2012) Nano. Lett., 12:5192-5195; Afonin K.A., et al. (2014) Nucleic Acids Res., 42:2085-2097). However, previous methods of circular DNA preparation for RCT are not amenable for ssDNA templates displaying stable secondary structure.

Bacterial RNA polymerases are sensitive to secondary structure, falling off template DNA when encountering stable DNA hairpins and loops (Ducani C., et al. (2014) Nucleic Acids Res, 42:10596-10604).

Previous methods to create defined RNA oligomers using RCT include the use of ssDNA oligomers and RNAse H during RCT, allowing site-specific cleavage (Wang X., et al. (2015) Mol Ther. Nucleic Acids, 4:e215). However, it could be possible to improve upon current technologies by avoiding the addition of enzymes or DNA oligos to catalyze RNA cleavage. Encoding sequence specific self-cleaving ribozymes (Cech T.R. (1989) RNA chemistry. Nature, 339:507-508; Murray J.B., et al. (1998) Cell, 92:665-673; Ruffner D.E., et al. (1990) Biochemistry, 29:10695-10702) in the DNA template, alongside RNA nanoparticle sequences, would allow simpler experimental processes and more widespread application of the RCT process to synthesize defined sequence RNA oligomers (Ruffner D.E., et al. (1990) Biochemistry, 29:10695-10702; McCall M.J., et al. (1992) Proc Natl Acad Sci U. S A, 89:5710-5714; Ruffner D.E., et al. (1989) Gene, 82:31-41). Additionally, in vivo expression of artificial RNA oligomers and RNA nanoparticles could be possible. Ribozymes have a stable secondary structure and when combined in the same ssDNA template as an RNA nanoparticle sequence, the AG of the ssDNA template is quite low, thus not conducive to RCT using ssDNA templates.

Disclosed herein is a large scale RNA production method using rolling circle transcription (RCT) from circular dsDNA that uses encoded ribozymes to self-cleave the RNA concatamers to release target oligonucleotide sequences. The released co-transcribed multiple short RNA oligomers self-assembled into RNA nanoparticles, which can then be purified using industrial scale gel electrophoresis column.

Self-cleaving RNA motifs include naturally-occurring ribozymes, ribozymes from plant pathogens, viroids, derivatives and modified forms of the naturally-occurring ribozymes, and synthetic ribozymes. They generally range between 50 and 150 nucleotides in length.

A growing number of ribozyme families are found in nature, including: hairpin, hammerhead, hepatitis delta virus (HDV)-like, glmS, Neurospora Varkud satellite, twister, the recently discovered twister sister, pistol, and hatchet motifs. All rely on a combination of catalytic strategies to complete self-scission in an active site formed by the secondary and tertiary structures unique to each family. For these ribozymes, cleavage involves a nucleophilic attack by a 2' oxygen on an adjacent phosphodiester bond, yielding a 2'-3' cyclic phosphate and a 5'-hydroxyl product.

In an embodiment, hammerhead ribozyme (5’- CUGAUGAGUCCGUGAGGACGAAAC-3’, SEQ ID NO:1) is used for self-cleavage of RNA concatemer to RNA product.

The disclosed approach can be used to produce any RNA molecule that self- assembles to form a stable particle. In some embodiments, ssRNA, pRNA-3WJ based RNA nanoparticles, RNA nanoparticle multi-way junctions, RNA nanoparticle branched motifs, and RNA nanoparticle dendrimers, composed of one or more RNA oligos. RNA particle products can be of length 1 - 200 nucleotide or more, with complementary sequences to itself or another RNA oligo for the self-assembly of RNA nanoparticle or RNA motif.

In some embodiments, RNA particle will self-assemble during rolling circle transcription based on strong folding stability with melting temperatures >50 °C. Therefore, the disclosed systems and methods can be used to produce any RNA particles with a folding stability of at least 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 °C.

The disclosed systems can be used in a rolling circle transcription reaction to produce RNA particles. This process can be conducted either in either cellular or acellular in vitro systems. This is because stable RNA particles are able to form immediately upon transcription. In some embodiments, the process is conducted in a buffered medium comprising RNA polymerase and ribonucleotides under conditions suitable for rolling circle transcription. The amount of RNA particles produced from the reaction is a function of reaction volume, dsDNA and buffered medium concentrations, and time. Reactions will come to completion upon depletion of ribonucleotides and loss of activity of RNA polymerase; however, reactions can be scaled to any volume to produce RNA particles of desired amount, provided reaction conditions are met. Reactions can take place in batch reactor or continuous stirred tank reactor on a volume scale as small as 100 pL to any size including >10 L.

Once peak RNA particle production is achieved, the reaction can be stopped, and the RNA particles can be isolated and purified from the reaction mixture. In some embodiments, this involves the use of an electrophoresis column. In some embodiments, electrophoresis column is composed of polyacrylamide gel for the separation of RNA particle by size.

Electrophoresis column can be varied polyacrylamide concentration for varying levels of resolution and purification. Electrophoresis column can be varied in buffer condition and temperature to allow for purification of single RNA particle or RNA particle composed of more than one RNA oligo.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1 :

Disclosed herein is a method for the construction of circular dsDNA templates that code for self-cleaving ribozymes and RNA oligomers. Upon in vitro transcription, the ribozymes self-cleave with high efficiency, producing large amounts of target RNA. The phi29 pRNA-3WJ was assembled from its three component strands both co-transcriptionally and by self-assembly after RNA oligomer purification. Single stranded RNA nanoparticles were synthesized via RCT by addition of loops to link adjacent 3WJ strands. The malachite green fluorogenic RNA aptamer (MGA) was fused to one helix of the 3WJ and malachite green (MG) fluorescence was monitored in real time during in vitro transcription. Fluorescent signals from transcription reactions using circular dsDNA were 8-10 times higher compared to identical sequences in linear dsDNA, indicative of the increased production of RNA nanoparticle by RCT reaction. Materials and Methods

Sequence Design

Sequences for product oligomers were derived from the pRNA-3WJ nanoparticle as reported previously (Shu D., et al. (201 1) Nature Nanotechnology, 6:658-667). Additional nucleotides were added to the 5’ and 3’ ends of the native 3WJ sequences, 5’-GAC-3’ and 5’-GUC-3’ respectively, aiding in ribozyme stability and cleavage efficiency. For the synthesis of single stranded RNA nanoparticles, loops (5’-GAGA-3’) were used to join adjacent 5’ and 3’ ends of the multi-strand RNA nanoparticles. Ribozyme sequences were adapted from previously solved structures of the hammerhead ribozyme (Ruffner D.E., et al. (1990) Biochemistry, 29:10695-10702). For ribozyme efficiency assays, ribozyme sequences were mutated to abolish activity as previously described (Ruffner D.E., et al. (1990)

Biochemistry, 29:10695-10702). The malachite green aptamer sequence was adapted from previously published sequences (Baugh C., et al. (2000) J. Mol. Biol., 301 :1 17-128). All sequences are summarized in Table 3.

DNA and RNA Preparation

RNA oligomers were prepared in vitro using T7 RNA polymerase from linear and circular dsDNA containing the T7 promoter. Linear dsDNA was prepared by PCR using ssDNA primers purchased from Integrated DNA Technologies (IDT). Circular dsDNA was prepared from ultramer oligomers purchased from IDT. The assembly method of circular dsDNA is detailed in Figure 1. ssDNA anti-sense to the RNA strand (including T7 promoter, ribozymes, and product RNA) was self-cyclized intramolecularly using Epicentre CircLigase following the manufacturer’s protocol. Remaining linear ssDNA was removed by addition of DNA Exonuclease I. ssDNA complementary to the cyclized ssDNA was annealed by thermal denaturation at 85°C for five minutes followed by slow cooling to 4°C at a rate of -2°C/minute at 4 micromolar (mM) concentration. Following annealing, T4 DNA ligase closed the nick in the circular dsDNA. Ligations were performed at a DNA concentration of 2.5 pM in 1X DNA ligase buffer, 5% (w/v) PEG4000, and a ligase concentration of 0.25 U/pL. The ligation mixture was incubated at 25°C for 4 hours followed by heating at 65°C to heat denature the enzyme. Control constructs without T4 ligation were also assembled.

RNA transcriptions were completed using T7 RNA polymerase isolated from E. coli containing plasmid encoding for the polymerase through the use of a His tag in the protein. All transcriptions, linear and circular, were completed at 37°C with the following final concentrations of reagants: 40 mM HEPES-KOH (pH 7.5), 12 mM MgCI₂, 30 mM DTT, 1 mM Spermidine, 5 mM rNTPs (rATP, rCTP, rGTP, and rUTP) along with DNA template and T7 RNA polymerase. Reactions were incubated for varying times depending on the desired experiment but complete reactions were carried out for 4 hr.

Gel Analysis and Quantification

Assembly of linear and circular dsDNA constructs and RNA products was confirmed using 10% (29:1) PAGE in a buffer containing 50 mM TRIS, 100 mM NaCI, and 5 mM EDTA, and DNA or RNA was stained using ethidium bromide (EB) solution or MG dye solution (final concentration of 5 uM MG Dye, HEPES pH=7.4, 100 mM KCI, 5 mM MgCI₂).

Ribozyme Cleavage Efficiency

Time course experiments were run to analyze ribozyme cleavage efficiency. A typical transcription reaction was quenched at specific time points by addition of DNase. Equal aliquots of each time point were then analyzed on PAGE and gel band intensity was integrated using ImageJ software (Collins T.J. (2007) Biotechniques, 43:25-30). Ribozyme cleavage occurred during the transcription process, thus are at 37°C and in T7 RNA polymerase transcription buffer. Cleavage efficiency was calculated by dividing band intensity of the cleaved fractions by total band intensity per lane. Cleavage percentage versus time was then plotted using OriginPro.

Gel Analysis of Transcription Kinetics

dsDNA constructs, both linear and circular, were transcribed following typical T7 in vitro transcription protocols with a 250 nM final DNA concentration. At 0.5, 1 , 2, and 4 hours, transcriptions reactions were quenched using DNase. Equal aliquots of transcription from each time point were analyzed on PAGE and product RNA bands were integrated using ImageJ software. Gels were stained separately for total RNA (EB) and MG fluorescence. Gel band intensity versus time was then plotted using OriginPro. Gel assays were carried out in triplicate.

Fluorescence Monitoring of RNA Transcription

To monitor transcription in real time, MG dye or SYBR Greenll were added at a final concentration of 5 mM to transcription reactions. Solutions were incubated at 37°C in 96 well microplates and fluorescent signal was monitored every 15 minutes using a BioTek Synergy 4 Microplate Reader. MG signal was read from excitation and emission wavelengths of 590 nm and 630 nm, respectively. SYBR Greenll fluorescence was read from excitation and emission wavelengths of 496 nm and 520 nm, respectively. OriginPro was used to plot fluorescent signal versus time. Transcription analysis was performed at DNA concentrations of 10, 100, and 250 nM.

Large-Scale Purification Using Gel Electrophoresis Column Transcription reactions were purified on a BioRad model 491 Prep Cell using continuous-elution gel electrophoresis following the manufacturer’s standard protocol. 20 mL of 8% polyacrylamide gel were prepared in a buffer containing 50 mM TRIS, 100 mM NaCI, and 5 mM EDTA. The gel was polymerized and to a column height of 5.5 centimeters and pre-run at 300 V for 1 hour. Large-scale, 1 mL transcriptions with final DNA concentration of 250 nM, transcriptions were diluted to 2 mL with 2X gel loading dye and loaded onto the column. After two hours of electrophoresis at 300 V, fractions were collected at a rate of .25 mL/min for 4 minutes for a total fraction volume of 1 mL. A total of 60 fractions were collected for four hours. Fractions were analyzed by adding 20 pL of 10X MG binding buffer (final concentration of 5 uM MG Dye, HEPES pH=7.4, 100 mM KCI, 5 mM MgCI₂) to 180 pL of each fraction. Fluorescence was analyzed as described previously. RNA concentrations was measured by reading absorbance at 260 nm.

Results and Discussion

Sequence Design and Optimization for Ribozyme Cleavage Using Linear dsDNA

The goal of this example was to produce short RNA oligomers that would then selffold into RNA nanoparticles. To induce oligomer release, self-cleaving ribozymes were incorporated into the DNA templates. To enhance ribozyme cleavage efficiency, sequence optimization was carried out experimentally by gel analysis using linear dsDNA. The optimized sequences were then incorporated in circular constructs, as described in a later section.

Linear dsDNA containing the T7 promoter, the 5’ and 3’ ribozymes, the RNA sites for ribozyme self-cleavage, and the product RNA were constructed using PCR (Figure 1). The sequences of RNA products for ribozyme optimization were 3WJ-a, 3WJ-b, and 3WJ-c, which then assemble to form the pRNA-3WJ (Shu D., et al. (201 1) Nature Nanotechnology, 6:658-667). Hammerhead ribozyme sequences were chosen as they are well characterized and display high cleavage efficiency (Murray J.B., et al. (1998) Cell, 92:665-673; Ruffner D.E., et al. (1990) Biochemistry, 29:10695-10702; McCall M.J., et al. (1992) Proc Natl Acad Sci U. S A, 89:5710-5714; Blount K.F., et al. (2002) Biochem. Soc. Trans., 30:1 1 19-1 122). While the ribozyme core sequence must be conserved to maintain cleavage, non-core sequences can be modified, aiding in self-cleavage optimization (Ruffner D.E., et al. (1990) Biochemistry, 29:10695-10702; McCall M.J., et al. (1992) Proc Natl Acad Sci U. S A, 89:5710-5714).

The 5’ and 3’ disabled ribozyme, the 5’ disabled ribozyme, the 3’ disabled ribozyme, and ribozyme only were used as RNA size controls (Figure 8A), in which ribozyme was disabled and cleavage activity was disabled by sequence mutation (Ruffner D.E., et al. (1990) Biochemistry, 29:10695-10702). The product RNA strands were chemically synthesized for size and assembly controls. Cleavage efficiency was calculated by comparing the product RNA band intensity to the total intensity per lane over the two-hour after the initiation of transcription (Figure 8B). ImageJ software was used to integrate the gel band intensity. Cleavage kinetics assays were done using truncated versions of the dsDNA. For example, 5’ ribozyme efficiency was assayed using dsDNA template with no 3’ ribozyme. The cleavage efficiency of each ribozyme, 5’ and 3’ of the product RNA, was calculated independently (Figure 8B).

A two base-pair (bp)“clamping” duplex led to a cleavage efficiencies ranging from 36% to 65% cleavage. Upon lengthening the“clamping” duplex to five bp (Figure 8C) to enhance the stability of the ribozyme sequence, cleavage efficiencies increased to 65% to 78%. When full length constructs with both active ribozymes were tested, cleavage efficiencies were more than 80% (Table 1).

Circular dsDNA Construction

RCT offers many advantages over traditional in vitro transcription methods including higher transcription rate (Furukawa K., et al. (2008) Bioorg. Med. Chem Lett, 18:4562-4565; Daubendiek S., et al. (1995) J. Am. Chem. Soc., 1 17 (29):7818-7819), template DNA economy, and the potential for in vivo expression of artificial RNA sequences and RNA nanoparticles (Shu D., et al. (2013) Nucleic Acids Res., 42:e10). Previously published RCT method for circular DNA preparation applied a short splint DNA to a longer phosphorylated ssDNA followed by DNA ligation, resulting in circular DNA with a double stranded RNA promoter region and a single stranded region anti-sense to the desired RNA sequence (Lee J.B., et al. (2012) Nat. Mater., 1 1 :316-322; Guo P (2012) Rolling Circle Mol Ther-Nucleic Acids, 1 :e36; Mohsen M.G., et al. (2016) Acc. Chem Res, 49:2540-2550; Mezger A., et al. (2014) PLoS ONE, 9:e1 1 1874; Li X., et al. (2015) Chem Commun. (Camb. ), 51 :1 1976- 1 1979; Roh Y.H., et al. (2015) Angew. Chem. Int. Ed Engl., 55:3347-3351 ; Shopsowitz K.E., et al. (2014) Small, 10:1623-1633; Hsu B.B., et al. (2014) Biomacromolecules, 15:2049- 2057; Han D., et al. (2014) Nature Communications). While this method is amenable for ssDNA templates with little secondary structure (AG of self-folding close to zero or a positive value, Table 2) (Diegelman A.M., et al. (1998) Nucleic Acids Res, 26:3235-3241 ;

Daubendiek S., et al. (1995) J. Am. Chem. Soc., 1 17 (29):7818-7819; Lindstrom U.M., et al. (2002) Proc Natl Acad Sci U. S A, 99:15953-15958; Hartig J.S., et al. (2005) Chembiochem, 6:1458-1462) or stable dumbbell sequences (Lee J.B., et al. (2012) Nat. Mater., 1 1 :316-322; Roh Y.H., et al. (2015) Angew. Chem. Int. Ed Engl., 55:3347-3351 ; Furukawa K., et al. (2008) Bioorg. Med. Chem Lett, 18:4562-4565; Jang M., et al. (2015) Nat Commun., 6:7930), it was not amenable for ssDNA templates encoding for RNA with strong secondary structure such as the pRNA-3WJ.

The AG of self-folding for the ssDNA templates used in this study range from -1 1.6 kcal/mol to -27 kcal/mol (Table 2), resulting in stable secondary structures, which hinder transcription by bacterial RNA polymerases. The published methods (Lee J.B., et al. (2012) Nat. Mater., 1 1 :316-322; Guo P (2012) Rolling Circle Mol Ther-Nucleic Acids, 1 :e36; Mohsen M.G., et al. (2016) Acc. Chem Res, 49:2540-2550; Mezger A., et al. (2014) PLoS ONE,

9:e1 1 1874; Li X., et al. (2015) Chem Commun. (Camb.), 51 : 1 1976-1 1979; Roh Y.H., et al. (2015) Angew. Chem. Int. Ed Engl., 55:3347-3351 ; Shopsowitz K.E., et al. (2014) Small, 10:1623-1633; Hsu B.B., et al. (2014) Biomacromolecules, 15:2049-2057; Han D., et al. (2014) Nature Communications, 5:4367; Kim H., et al. (2015) Sci Rep., 5:12737) to prepare circular DNA template were attempted, however, stable secondary structure of the sequences hindered transcription, even in the presence of single stranded binding proteins and elevated transcription temperatures at 42°C. Removing the issue of stable secondary structure in DNA template was addressed by making the circular template entirely double stranded. Therefore, circular dsDNA was made starting with two complimentary and phosphorylated ssDNAs (Figure 1).

Phosphorylated ssDNA complimentary to the T7 promoter, 5’ and 3’ ribozymes, and product RNA sequence was self-ligated using Epicentre ssDNA Circ Ligase to form circular ssDNA. To confirm cyclization, polyacrylamide gel electrophoresis (PAGE) was used to visualize DNA bands before and after ssCirc ligation (Figure 2, Lane 1 vs 2). Upon singlestrand cyclization, an increase in migration rate was seen presumably due to the compact structure of the now self-folded ssDNA. Assembly of all dsDNA constructs can be found in Figure 9.

To form circular dsDNA, cyclized ssDNA and its phosphorylated compliment were mixed at equimolar concentrations and annealed by thermal denaturation followed by slowly cooling to 4°C over one hour. Assembly of the compliment strands resulted in a dramatic decrease in migration rate, indicating successful hybridization (Figure 2, Lane 2 vs 4/5). Compared to linear dsDNA controls, circular dsDNA migrates much slower, indicating circular conformation. After assembly, T4 DNA ligase was used to ligate the nicked circular dsDNA (Figure 2, Lane 3 vs 4/5). No apparent shift is observed by gel analysis after T4 ligation.

RCT Reaction

To show successful RCT reaction, ribozyme activity was disabled by sequence mutation (Ruffner D.E., et al. (1990) Biochemistry, 29:10695-10702). Disabling of ribozyme resulted in full template length RNA product using linear dsDNA, and long concatemeric RNA using circular dsDNA, respectively. Whereas active ribozymes will result in release of the product RNAs. The encoded RNA sequence was 3WJ-a for both active and inactive ribozyme constructs. After in vitro transcription and termination by DNase, PAGE analysis was used to visualize RNA transcripts (Figure 3). Heavy accumulation of RNA transcripts in the well of the gel indicate long RNA concatamers and successful RCT (Figure 3, Lane 5), compared to the transcription of both the linear dsDNA template (Figure 3, Lane 3) and the nicked circular dsDNA template (Figure 3, Lane 4). When ribozyme activity was restored, successful cleavage and release of product RNA strand was observed (Figure 3, Lanes 6-8), evidenced by the appearance of short RNA transcripts not seen in inactivated ribozyme constructs.

One-pot Co-Transcriptional Assembly of 3WJ Nanoparticles

To generate RNA oligomers and RNA nanoparticles with defined sequences a cleavage method was devised to release the product RNA sequence from the rest of the transcript. Ribozymes can be engineered to self-cleave, and their catalytic property was implemented to self-cleave co-transcriptionally and release specified RNA oligoes that can assemble into the pRNA-3WJ. This allows a hands-off method for transcriptional production of short and defined RNA oligomers in high yield without the use of additional enzymes, and will aid in the future scale up of this method for large-scale synthesis of RNA nanoparticles in vitro and in vivo.

RNA 3WJ or other nanoparticles have the unique ability to self-assemble co- transcriptionally under isothermal conditions (Afonin K.A., et al. (2012) Nano. Lett., 12:5192- 5195; Afonin K.A., et al. (2014) Nucleic Acids Res., 42:2085-2097). To assemble the pRNA- 3WJ co-transcriptionally, 3WJ-b and 3WJ-c sequences were incorporated in separate circular dsDNA constructs. Both constructs demonstrated cleavage and release of the product RNA (Figure 4A). Product RNA strands were equal in size to that of their chemically synthesized size controls, indicating successful cleavage and release of product sequences (Figure 10A). To confirm that the sequences of product RNA were correct, each fragment was isolated and their assembly tested. Formation of the 3WJ from isolated RNA bands indicates sequence specific cleavage and release of product RNA (Figure 10B). Additionally, it appears that low expression of the 3WJ-c strand from the transcription; however, due to the low secondary structure of the 3WJ-c strand by itself, there is low EB intercalation and staining of the strand (Binzel D.W., et al. (2016) RNA, 22:1710-1718).

pRNA-3WJ nanoparticles were assembled co-transcriptionally by mixing 3WJ-a, b, and c dsDNA constructs in an equimolar ratio followed by in vitro transcription. PAGE analysis of both linear and circular co-transcription products indicates successful assembly of 3WJ nanoparticles when compared to the assembled 3WJ from gel purified RCT product (Figure 4B).

To assemble nanoparticles from one ssRNA oligomer, circular dsDNAs encoding for the full sequence of the 3WJ, with the helix ends closed with loops or MGA sequences, were constructed (Figure 5). Including an aptamer in the sequence serves two purposes: (1) monitoring transcription kinetics by fluorescence; (2) shows accurate cleavage and correct folding of the RNA nanoparticle, as MG will not bind to MGA unless the sequences is correct. Following transcription, the RCT reaction mixture was analyzed by PAGE (Figure 5C). RCT-3WJ assembled from purified 3WJ monomers and one piece 3WJ with loops were used as size controls. The one-stranded nanoparticles migrate slower due to increased size from the incorporation of loop sequences used to connect helix ends. Gel staining with MG shows binding of the MG-3WJ nanoparticle to its fluorophore, indicating correct sequence and folding of the MG-3WJ. No MG signal from the 3WJ, which lacks the MGA, indicates specific binding of MG to MGA. Higher order concatamers are present in the RCT reactions, indicating that ribozyme cleavage is not 100%.

Real-Time Monitoring of Transcription by Fluorescence

The advantage to preparing circular dsDNA for transcription is the hypothesis that RCT will result in higher amounts of RNA nanoparticles from the same starting DNA concentrations, therefore being more efficient and faster in nanoparticle production. Thus, gel analysis was first used to compare the transcription rate of linear and circular dsDNA by analyzing transcription time points after termination by DNasel. Gels were stained for total RNA using ethidium bromide (EB) and for MG signal using MG dye (Figure 6A). Integration of gel band intensity using ImageJ indicates both faster production and higher yield of MG- 3WJ by RCT compared to that of linear transcription (Figure 6B). As the ribozyme cleavage efficiency was not 100%, gel staining represents an accurate comparison of RNA nanoparticle production since the quantified gel band was the produced RNA nanoparticle.

Fluorescence measurements were used to monitor transcription rates in real-time. MGA fluorescence was monitored by adding MG dye to a final concentration of 5 mM in the transcription mixture while total RNA production was monitored by adding RNA specific SYBR Greenll to a final concentration of 1X. Both MG and MGA are not fluorescent by themselves alone but fluorescence will appear when the MG binds to the MGA.(59;63;64) Fluorescence measurements were taken every 15 minutes and intensity was plotted versus time using OrignPro (Figure 6C, D). Among three different DNA template concentrations of 10(^), 100 (■), and 250 (· ) nM, RCT reactions (red) consistently outperformed linear transcription (black) in both nanoparticle production rate and overall transcription yield. It is important to note that identical sequences were used in all constructs in comparison experiments, the only difference being circular or linear dsDNA. RCT produced on average 10 times more RNA at the termination of the transcription reaction, consistent with previous findings on the rate of RCT (Furukawa K., et al. (2008) Bioorg. Med. Chem Lett, 18:4562- 4565; Daubendiek S., et al. (1995) J. Am. Chem. Soc., 1 17 (29):7818-7819). Interestingly, nicked circular dsDNA template without T4 ligation was similar in transcription rate to linear dsDNA at the same concentration, suggesting that T7 polymerase does not proceed to RCT with nicked circular dsDNA.

Substrate concentration dependent kinetics of total RNA transcription were analyzed by comparing SYBR Greenll fluorescence values at different time points (values from Figure 6D). Time points from 60 - 165 minutes were chosen as these were during and after the highest increase of observed fluorescence. Fluorescence values were plotted and fit linearly ( y = mx + b ) (Figure 1 1A, 1 1 B). A strong linear correlation is seen between DNA template concentrations and their fluorescence output (Figure 1 1 C). The slope (x) values for circular DNA template display an average value of 5.27, higher than those observed for linear DNA template, average equal to 1.27. These values can be correlated to fluorescence output per nM (RFU/nM) of DNA template. Thus, these results support that RCT reaction results in an increase in transcription efficiency. At these DNA template concentrations, the relationship appears to be linear.

Large-Scale Purification Post RCT

Purification of the MG-3WJ nanoparticles was carried out using a preparative version of typical gel electrophoresis. A BioRad Model 491 Gel Electrophoresis Prep Cell was used to purify large-scale transcription products of RNA nanoparticles transcribed from circular dsDNA encoding for the MG-3WJ. Fractions were analyzed for both MG fluorescent signal and absorbance at 260 nm (Figure 7 A). Three distinct peaks were seen by MG fluorescence: fractions 5-7 (peak 1), 7-23 (peak 2), and 25-32 (peak 3). PAGE analysis was used to determine the identity of each peak compared to crude transcription mixture. Peak 1 was smaller than the product RNA, peak 2 contained the product RNA, and peak 3 contained both the product RNA and larger RNA bands. Peak 2 fractions were then combined and analyzed by PAGE (Figure 7B). Of note is the large absorbance value of the first fractions, which has been attributed to remaining nucleotides from the transcription mixture, as no band was seen by gel analysis.

Here we were able to construct self-folding RNA nanoparticles by combining several technologies: rolling circle transcription, ribozyme self-cleavage, and gel column electrophoresis. By combining these techniques, RNA nanoparticle construction was simplified, in that the complete nanoparticles were produced through three simple steps of template construction, RNA transcription, and nanoparticle purification. The novelty behind this methodology of RNA nanoparticle production removes several steps producing and purifying each RNA strand within the nanoparticle, nanoparticle assembly, and nanoparticle purification. Furthermore, through the development of this method set, RNA production was proven to be produced at a higher yield than traditional linear in vitro transcription and RNA nanoparticles were shown to self-assemble with original and authentic folding. While this system is not fully optimized, it may lead to breakthroughs in current industrial techniques for RNA production leading to a much needed reduced cost in RNA nanoparticle production.

Conclusion

This study establishes a solution for the simplification of RNA nanoparticle preparation, as well as demonstrating the potential for higher yield assembly of RNA nanoparticles in vitro. The method for circular dsDNA preparation is broadly applicable to the field of RNA biology and RNA nanotechnology for the production of functional self-folding nanoparticles. To release RNA nanoparticles transcribed during RCT, self-cleaving ribozymes were coded for in the template allowing bottom-up assembly either in situ or one- pot co-transcriptional releasing of the product RNA fragments. Production was monitored in real time. RNA nanotechnology is emerging as a new drug delivery platform and shows great promise to help advance the current state of nanomedicine. The methods introduced here are a step towards the large-scale production of RNA nanoparticles and could be helpful for future clinical applications of RNA nanotechnology. As well as large-scale batch synthesis, in vivo expression and production of RNA nanoparticles could be possible using the methods introduced here.

Table 1 summarizes the cleavage efficiencies of the ribozymes in each of the sequences, broken down for 5’ and 3’ ribozyme of each sequence, as well as total cleavage efficiency of the full length constructs (those containing both 5’ and 3’ ribozymes).

Table 2 summarizes sequences used in publications with RCT reactions. The DeltaG of most templates are close to 0 or positive, indicating unstable secondary structure, which is suitable for transcription using bacterial polymerases. Besides dumbbell sequences

(denoted by a“*” before the reference), which have previously been shown as suitable substrates for transcription. Templates from this manuscript display a large negative DeltaG value, demonstrating the need for a fully double stranded circular DNA template.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A circular dsDNA polynucleotide, comprising a nucleic acid sequence encoding a nucleic acid sequence encoding an RNA particle with a folding stability of at least 50 °C flanked by self-cleaving ribozymes operably linked to an expression control sequence.

2. The circular dsDNA polynucleotide of claim 1 , wherein the RNA particle is an RNA therapeutic.

3. The circular dsDNA polynucleotide of claim 1 , wherein the RNA particle an RNA nanoparticle.

4. The circular dsDNA polynucleotide of claim 1 , wherein the RNA particle comprises a siRNA, ribozyme, miRNA, riboswitch, or aptamer.

5. The circular dsDNA polynucleotide of any one of claims 1 to 4, wherein the RNA particle is self-assembled from two or more RNA subunits.

6. The circular dsDNA polynucleotide of claim 5, wherein the nucleic acid sequence encoding the RNA particle comprises a nucleic acid sequence encoding each of the two or more RNA subunits, wherein each nucleic acid encoding a RNA subunit is flanked by a selfcleaving ribozyme.

7. The circular dsDNA polynucleotide of any one of claims 1 to 6, wherein the expression control sequence is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.

8. A system for large-scale synthesis of an RNA particle, comprising the circular dsDNA polynucleotide of any one of claims 1 to 7 and a buffered medium comprising an RNA polymerase and ribonucleotide triphosphates (NTPs),

wherein rolling circle transcription (RCT) of the dsDNA template by the RNA polymerase produces an RNA concatamer, wherein self-cleavage of the ribozymes produces one or more RNA fragments that self-assemble to form the RNA particle.

9. The system of claim 8, further comprising a large-scale electrophoresis column for the purification of RNA particle of claim 8.

10. A method for large-scale synthesis of an RNA particle, comprising

(a) incubating the circular dsDNA polynucleotide of any one of claims 1 to 7 under conditions that promote rolling circle transcription (RCT) of the dsDNA polynucleotide and self-cleavage of the self-cleaving ribozymes to produce one or more RNA fragments that self-assemble to form the RNA particle; and

(b) purifying the RNA nanoparticle on an electrophoresis column.

1 1 . The method of claim 10, wherein step (a) comprises incubating the dsDNA in vitro in a buffered medium comprising an RNA polymerase and ribonucleotide triphosphates (NTPs).