Compounds and methods for the highly-efficient synthesis and purification of capped ribonucleic acids

Abstract

The present disclosure provides modified mRNA cap compounds and methods of using the compounds in the synthesis and purification of capped mRNAs. The materials and methods yield highly pure capped mRNAs in high yields. The capped mRNAs find wide utility for therapeutic and other purposes.

Description

Patent Application 5881-00-002W01COMPOUNDS AND METHODS FOR THE HIGHLY-EFFICIENT SYNTHESIS AND PURIFICATION OF CAPPED RIBONUCLEIC ACIDSCross-Reference to Related Applications

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 734,720, filed on December 16, 2024, the disclosure of which is incorporated herein by reference in its entirety.Background of the Invention

[0002] Messenger RNA (mRNA) has emerged as a transformative platform for therapeutic and vaccine applications, with its ability to encode virtually any protein, enabling treatments for cancer, genetic disorders, and infectious diseases. Unlike DNA-based delivery systems, mRNA offers critical advantages, including (i) immediate protein expression without the risk of genomic integration, (ii) dosedependent control over protein levels, (iii) streamlined, scalable synthesis, and (iv) adaptability to a wide range of delivery platforms. These attributes have propelled mRNA into the forefront of biotechnology, underpinning the success of mRNA-based vaccines and sparking interest in broader therapeutic applications.

[0003] A technology gap that continues to limit the broader applications of mRNA is that forming fully functional mRNA in vitro remains challenging and expensive. Combining a properly designed and linearized DNA template with recombinant T7 bacteriophage RNA polymerase (T7 RNAP) and synthetic nucleoside triphosphates in the presence of an appropriate buffer has long been known to be sufficient for efficient transcription. However, the RNA product of such an in vitro transcription (IVT) reaction will not be properly recognized as functional mRNA in vivo, may be rapidly degraded and may induce a strong innateimmune response. As such, several critical factors must be addressed, including enhancing recognition by the translation apparatus, escaping nuclease-mediated degradation, and reducing detection by the innate immune sensors expressed in eukaryotic cells in order to produce mRNA suitable for in vivo use, often described as GMP-grade mRNA.

[0004] A crucial structural element of functional mRNA is the cap structure at its 5' end, typically a 7-methylguanosine (m7G) linked by a 5'-5' triphosphate bridge to the first nucleotide. This cap structure, called Cap 0, is sufficient to protect mRNA from exonuclease degradation, promote nuclear export, and facilitate translation initiation via interactions with eukaryotic translation initiation factor 4E (eIF-4E). mRNA lacking a cap structure is translationally inactive, unstable and prone to immunogenicity, making the capping process a critical step in mRNA production. For in vivo applications, further modifications such as 2'-O-methylation of the first or first two nucleotides, leading to Cap 1 or Cap 2 cap structures respectively, can enhance translation efficiency, improve stability and reduce innate immune activation.

[0005] As recognized by the 2023 Nobel Prize in Physiology or Medicine to Kariko and Weissman, another factor required to synthesize mRNA for in vivo applications such as vaccines is to perform the IVT with modified nucleotides such as pseudo-uridine, further reducing immunogenicity. Other factors that impact translation efficiency and reduce immunogenicity are related to the presence of a polyadenylated 3’ tail on each mRNA and limiting contamination with double stranded RNA and other side products or reaction components of IVT.

[0006] Current methods for manufacturing GMP-grade mRNA by IVT include co-transcriptional capping and enzymatic capping. The former uses cap analogs formed via chemical synthesis, typically consisting of m7G linked via a 5'-5' triphosphate bridge to one, two or more nucleotides, that is incorporated by T7 RNAP at the 5’ end as each transcript is initiated. In the latter, nascent or completed RNA transcripts already formed by T7 RNAP are then modified by capping enzymes that incorporate an m7G onto the 5’ ends to form a cap structure.

[0007] In co-transcriptional capping, the cap analog serves much like a primer that is incorporated when T7 RNAP initiates transcription. However, even in thepresence of cap analogs, T7 RNAP can initiate transcription on its own, leading to uncapped transcripts. This exposes an unsolved challenge in manufacturing GMP-grade mRNA for in vivo use, that purification of capped mRNA from uncapped transcripts remains impractical. Applying methods that have been demonstrated in the laboratory based on affinity or chemical tagging to enrich capped mRNA or nuclease-mediated degradation to eliminate uncapped RNA would add unrealistic cost and complexity for manufacturing GMP-grade mRNA.

[0008] To reduce the production of uncapped transcripts during IVT, cap analogs are often added at high molar ratios compared to the nucleotides. While high molar ratio increases capping efficiency, this has the undesirable effects of increasing truncated transcripts, likely reflecting when cap analogs bind along the length of the template or are mistaken by T7 RNAP as nucleotides during elongation. The resulting internal priming and early termination may result in high levels of capped but truncated transcripts. If capped transcripts could be enriched efficiently and cost-effectively, a lower percentage of capping during IVT could be tolerated. This would permit lowering the molar ratio of cap analogs in IVT, with the result that the fraction of full length transcript and overall yields would be increased.

[0009] In addition to uncapped and truncated transcripts, IVT reactions also produce other contaminating impurities such as double- stranded RNA (dsRNA). Purification of mRNA from IVT must also eliminate reaction components such as T7 RNAP, other proteins, free nucleotides, additives, and buffer chemicals. These impurities may be toxic, reduce translational efficiency, and increase immunogenicity. The resulting need to reduce this wide range of impurities in the final product poses significant challenges to manufacturing GMP-grade mRNA. Separation by size using tangential flow filtration or other methods well-suited to large-scale manufacturing fails to eliminate truncated transcripts. Other purification methods that better enrich for full length mRNA such as reverse phase chromatography have not been found to be cost-effective.

[0010] Initial studies with co-transcriptional capping using the dinucleotide cap analog m7G-5’ -PPP-5 ’-G were limited by the tendency of T7 RNAP to incorporate the cap analog in either orientation. An advance in co-transcriptional capping wasprovided by the anti-reverse cap analog, ARCA, in which m7G-5’-PPP-5’-G is modified by 3’-O-methylation of the m7G, thereby eliminating elongation in the reverse orientation. Adding ARCA to the IVT reaction at a high molar ratio along with wild-type T7 RNAP yields up to 80% of transcripts bearing a Cap 0 structure, though at cost of inhibiting overall yield and where a high proportion of the capped transcripts are prematurely terminated. Subsequent optimization over several decades has led to development and validation of IVT methods, engineered T7 RNAP enzymes, and synthetic cap analogs better suited to co-transcriptional capping to produce GMP-grade mRNA. Currently, most manufacturing of GMP-grade mRNA is performed with trinucleotide cap analogs where the m7G is linked by a 5 '-5' triphosphate bridge to a dinucleotide such as AG or GG. These cap analogs are more efficient at initiation and thus can be used at a lower molar ratio while still yielding up to 95% capped transcripts. Trinucleotide caps also offer higher yields and lower levels of truncated transcripts. The trinucleotide caps also offer advantages such as that the ribose of the first nucleotide can be 2’-O-methylatcd to produce capped mRNA with a Cap 1 structure. Similarly, trinucleotide cap analogs with the structure m7G-5’-PPP-5’-AG can be produced with adenosine methylated on the N6 position (m6A), further increasing translation. Recent advances in engineering T7 RNAP have yielded enzymes, some appropriate for manufacturing, that produce higher levels of capped transcripts while tolerating lower molar ratios of cap analogs and / or less truncated transcripts and double stranded RNA.

[0011] While these and other innovations in IVT and post-transcriptional capping are making an impact, current methods to obtain sufficiently pure mRNA for in vivo use remain resource-intensive and are particularly problematic for large-scale mRNA synthesis, especially given the stringent restrictions of the FDA and other regulators along with short timelines required for vaccine production in response to outbreaks of new variants or novel pathogens. While the alternative strategy of enzymatic capping offers up to 100% capping without reducing yield of full-length transcripts and thus a more direct path to GMP-grade mRNA, it introduces additional synthetic and purification steps, increased complexity, higher costs, and longer times that offset the advantages for most applications.

[0012] Building on the impact of the ARCA dinucleotide cap analog on co-transcriptional capping via IVT, further applications of synthetic chemistry along with validation in vitro and then in vivo have resulted in a wide range of synthetic cap structure analogs reported in the prior art. Toward improving IVT beyond formation of Cap 0 structures, Ishikawa et al. (2009) Nucleic Acids Symposium Series No. 53:129 (doi:10.1093 / nass / nrp065) describe trinucleotide cap analogs sharing the overall structure of m7G-5’-PPP-5’-AG, including those bearing modifications to bases and sugars that allow synthesis of mRNA with natural cap structures for expression in mammalian cells. These modifications include 2’-O-methylation on the adenosine to form a Cap 1 structure and adenosine methylation at the N6 positions to form m6A. These workers found that mRNA formed with the analog containing both 2’-O-methyl and N6-methyl was most efficiently translated in a reticulocyte lysate translation system.

[0013] Other chemical modifications have led to cap analogs that bear chemical tags that have been used for a wide range of purposes, including to enhance purification of the mRNA, alter stability or translation, facilitate tracking the mRNA, or identify its binding partners. As a recent example, Warminski et al. (2023) bioRxiv 2023.11.10.566532 (doi: 10.1101 / 2023.11.10.566532) describe a trinucleotide mRNA cap analog bearing an N6 benzylated adenosine. This modification was shown to facilitate mRNA purification and confer superior translational properties in vitro and in vivo.

[0014] Senthilvelan et al. (2023) Bioorg. Med. Chem. 77: 117128 (doi:10.1016 / j.bmc.2022.117128) describe a trinucleotide mRNA cap analog containing the propargyl group, allowing the mRNA to be modified at the 5’ end by click chemistry. Once incorporated by T7 RNA polymerase, mRNA capped with the analog can be efficiently translated in cells transfected with the mRNA and detected or captured by click chemistry.

[0015] Warminski et al. (2024) Adv. Sei. 2400994 (doi:10.1002 / advs.202400994) describe photoactivatable cap analogs that are incorporated into mRNA and then used for RNA-protein crosslinking in cells.

[0016] Other cap analogs have been described that bear a releasable modification to enable further manipulation of the mRNA. As an example, Klbcker et al. (2022)Nature Chemistry 14:905-913 (doi:10.1038 / s41557-022-00972-7) describe photocaged 5’ cap analogs that bear a modified m7G to block translation but which is cleaved upon light exposure, allowing their use in providing optochemical control of mRNA translation in eukaryotic cells.

[0017] Inagaki et al. (2023) Nature Communications 14:2657(doi: 10.1038 / s41467 -023-38244-8) describe mRNA cap analogs bearing a photocleavable tag that enables enrichment of the capped transcripts from the IVT reaction by adsorption onto a hydrophobic resin and then release of the mRNA by UV irradiation.

[0018] Other selective chemical modification techniques have been developed to facilitate the synthesis and purification of oligonucleotides more generally.

[0019] For example, U. S. Patent No. 5,410,068 describes compounds and methods for the attachment of functional groups to natural products, including oligonucleotides. The patent discloses a synthetic oligonucleotide comprising a 5’-O-dimethoxytrityl (DMT) group modified with an N-hydroxysuccinimidyl (NHS) group. The NHS group can be used to functionalize the oligonucleotide with compounds comprising primary amines, but the reaction is not selective nor is it reversible. The NHS group is also not stable to the conditions typically used to cleave synthetic oligonucleotides from the synthesis resin (e.g., concentrated ammonium hydroxide), so oligonucleotides released from the resin by standard cleavage conditions are no longer reactive.

[0020] U. S. Patent No. 5,586,586 describes methods and compounds useful for the purification of oligonucleotides and their analogues. The methods and compounds facilitate removal of oligonucleotides having abasic sites by formation of imine linkages with the contaminants.

[0021] PCT International Publication No. 2012 / 047639 describes methods for purifying synthetic oligonucleotides and novel capping agents. The methods comprise, for example, capping, polymerizing, and separating failure sequences or reacting full-length oligonucleotides with a compound to attach a polymerizable functional group to an end of the full-length oligonucleotides, polymerizing the full-length oligonucleotides, and removing failure sequences to recover the full-length oligonucleotides. The capping agents comprise a polymerizable functional group.

[0022] York et al. (2011) Nucl. Acids Res. 40 e4 (doi: 10.1093 / nar / gkr910) describe a highly-parallele method for the purification and functionalization of 5’-labeled oligonucleotides. The method utilizes oligonucleotides functionalized with a 5 ’-aldehyde group to generate 5’-hex-Histidine-labeled oligonucleotides that can be purified using a nickel resin. The 5’-hex-Histidine label is then exchanged for a biotin label, and the biotin- labeled oligonucleotides are used in targeted sequencing applications.

[0023] Zitterbart et al. (2021) Chem. Sei. 12:2389 and U. S. Patent No.10,954,266 describe reagents and methods for the purification of synthetic peptides using a reductively-cleavable linker system.

[0024] Given the growing demand for high-quality mRNA for therapeutic and vaccine applications, there remains a large unmet need for rapid, scalable, and efficient methods to produce highly pure, homogenously capped and full length mRNA. Such methods should minimize waste, reduce immunogenic impurities, and ensure compatibility with existing IVT workflows while maintaining the ability to produce GMP-grade mRNA suitable for human use.Summary of the Invention

[0025] The present disclosure addresses these and other needs by providing in some aspects a compound having a structure:, or a chemically acceptable salt thereof; wherein R1, R2, R4, R7, and R8is each independently -H, -alkyl, or a cleanly-releasable linker moiety; R3is -H, a nucleotide residue, or an oligonucleotide residue; and B1is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue; wherein the compound includes at leastone cleanly-releasable linker moiety; and wherein the at least one cleanly- releasable linker moiety includes a selectively-reactive linker moiety.

[0026] In some aspects, the techniques described herein relate to a compound, wherein R2is -CH3.

[0027] In some aspects, the techniques described herein relate to a compound, wherein B1is an adenine or guanine residue.

[0028] In some aspects, the techniques described herein relate to a compound, wherein R1, R2, R4, R7, and R8is each independently -H, -CH3, or the cleanly- releasable linker moiety.

[0029] In some aspects, the techniques described herein relate to a compound having a structure:or a chemically acceptable salt thereof; wherein T’ is the cleanly-releasable linker moiety.

[0030] In some aspects, the techniques described herein relate to a compound, wherein R2is -CH3.

[0031] In some aspects, the techniques described herein relate to a compound, wherein B1is an adenine or guanine residue.

[0032] In some aspects, the techniques described herein relate to a compound, wherein R1, R2, R4, R7, and R8is each independently -H or -CH3.

[0033] In some aspects, the techniques described herein relate to a compound having a structure:acceptable salt thereof; wherein R5is -H, a nucleotide residue, or an oligonucleotide residue; R6and R9is each independently -H, -alkyl, or a cleanly-releasable linker moiety; and B2is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue.

[0034] In some aspects, the techniques described herein relate to a compound, wherein R2is -CH3.

[0035] In some aspects, the techniques described herein relate to a compound, wherein B1is an adenine or guanine residue.

[0036] In some aspects, the techniques described herein relate to a compound, wherein R1, R2, R4, R6, R7, R8, and R9is each independently -H, -CH3, or the cleanly-releasable linker moiety.

[0037] In some aspects, the techniques described herein relate to a compound having a structure:. R8chemically acceptable salt thereof; wherein R5is -H, a nucleotide residue, or an oligonucleotide residue: R6and R9is each independently -H, -alkyl, or a cleanly-releasable linker moiety; and B2is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue.

[0038] In some aspects, the techniques described herein relate to a compound, wherein R1, R2, R4, R6, R7, R8, and R9is each independently -H, -CH3, or the cleanly-releasable linker moiety.

[0039] In some aspects, the techniques described herein relate to a compound having a structure:-zi -‘.10 gyoacceptable salt thereof; wherein T’ is the cleanly-releasable linker moiety.

[0040] In some aspects, the techniques described herein relate to a compound having a structure:OR6orchemically acceptable salt thereof; wherein T’ is the cleanly-releasable linker moiety.

[0041] In some aspects, the techniques described herein relate to a compound, wherein R2is -CH3.

[0042] In some aspects, the techniques described herein relate to a compound, wherein R1, R2, R4, R7, and R8is each independently -H or -CH3.

[0043] In some aspects, the techniques described herein relate to a compound, wherein the cleanly-releasable linker moiety has a structure:Lf Ph'Ph 1Ptf 'C'; wherein L’ includes the selectively-reactive linker moiety, Ph is an optionally substituted phenylene moiety, each Ph’ is independently an optionally substituted phenyl group, and C’ is a connecting group or a bond.

[0044] In some aspects, the techniques described herein relate to a compound, wherein each Ph’ is independently substituted with one or more C1-C4 alkoxy groups, one or more C1-C4 alkyl groups, or a combination of C1-C4 alkoxy groups and C1-C4 alkyl groups.

[0045] In some aspects, the techniques described herein relate to a compound,0 ^ 0O O N O N' wherein C’ includesHo o H0, orz, or is a bond.

[0046] In some aspects, the techniques described herein relate to a compound, wherein the cleanly-releasable linker moiety has a structure:wM'; wherein each M’ is independently a C1-C4 alkoxy group, a C1-C4 alkyl group, -H, or a combination thereof.

[0047] In some aspects, the techniques described herein relate to a compound, wherein at least one M’ is a methoxy group.

[0048] In some aspects, the techniques described herein relate to a compound, wherein the cleanly-releasable linker moiety has a structure:

[0049] In some aspects, the techniques described herein relate to a compound, wherein the cleanly-releasable linker moiety has a structure:

[0050] In some aspects, the techniques described herein relate to a compound, wherein the selectively-reactive linker moiety includes a reactive carbonyl group, a reactive oxyamino group, a reactive hydrazino group, a component of a click reaction, or a derivative of any thereof.

[0051] In some aspects, the techniques described herein relate to a compound, wherein the selectively-reactive linker moiety includes:N bk. No', or a derivative of any thereof.

[0052] In some aspects, the techniques described herein relate to a method of preparing a capped mRNA comprising the steps of a) providing any of the modified mRNA cap compounds described above; and b) generating an mRNA including the modified mRNA cap compound.

[0053] In some aspects, the techniques described herein relate to a method, wherein the mRNA generating step includes a co-transcriptional capping step.

[0054] In some aspects, the techniques described herein relate to a method, wherein the mRNA generating step includes a post -transcriptional capping step.

[0055] In some aspects, the techniques described herein relate to a method, further including the step of: reacting the mRNA including the modified mRNA cap compound with a solid support including a reactive oxyamino group, a reactive hydrazino group, a reactive carbonyl group, or a component of a click reaction to selectively bind the mRNA including the modified mRNA cap compound to the solid support.

[0056] In some aspects, the techniques described herein relate to a method, further including the step of washing the solid support.

[0057] In some aspects, the techniques described herein relate to a method, further including the step of releasing the selectively -bound mRNA including the modified mRNA cap compound from the solid support.

[0058] In some aspects, the techniques described herein relate to a method, wherein the releasing step cleanly releases the cleanly-releasable linker moiety from the modified mRNA cap.

[0059] In some aspects, the techniques described herein relate to a method, wherein the selectively-reactive linker moiety includes a reactive carbonyl group, a reactive oxyamino group, a reactive hydrazino group, a component of a click reaction, or a derivative of any thereof.

[0060] In some aspects, the techniques described herein relate to a method, wherein the selectively-reactive linker moiety includes:O, or a derivative of any thereof.

[0061] In some aspects, the techniques described herein relate to a compound having a structure:o o o?8HO-Ij’-O-Ij’-O-Ij’-O - 1 B1o O O kr '~7OR3OR4,or achemically acceptable salt thereof, wherein R3, R4, and R8is each independently -H, -alkyl, or a cleanly- releasable linker moiety, and B1is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue, wherein the compound comprises at least one cleanly-releasable linker moiety, and wherein the at least one cleanly- releasable linker moiety comprises a selectively-reactive linker moiety.Brief Description of the Drawings

[0062] FIG. 1 illustrates exemplary modified mRNA cap compounds of the disclosure, where the compounds include ribose rings modified with a cleanly- releasable linker moiety.

[0063] FIG. 2 illustrates exemplary modified mRNA cap compounds of the disclosure, where the compounds include nucleobases modified with a cleanly- releasable linker moiety.

[0064] FIG. 3 illustrates exemplary modified mRNA cap compounds of the disclosure, where the cleanly-releasable linker moiety comprises a carbamate or carbonate connecting group.

[0065] FIG. 4 shows the chemical activation of formyl-DMT-OH for freely releasable modification of nucleosides, nucleotides, mRNA cap structures, and other molecules and biomolecules.

[0066] FIG. 5 shows the preparation of a 2’-O-formyl DMT-acetal 3’-0Me m7G diphosphate intermediate.

[0067] FIG. 6 shows an exemplary solution phase synthesis of 2’-O-formyl DMT-acetal 3’-OMe-m7G-5’-ppp-5’-G and 2’-O-formyl DMT-acetal 3’-0Me-m7G-5’-ppp-5’-(2’-OMe A)-p-G.

[0068] FIG. 7 shows an exemplary solid phase oligonucleotide synthesis to form 2’-O-formyl-DMT-acetal 3’-OMe-m7G-5’-ppp-5’(2’-OMe A)-p-G.

[0069] FIG. 8 shows the formation of 2’-O-formyl-DMT-acetal GTP for enzymatic capping.

[0070] FIG. 9 shows the formation of N6-formyl-DMT-carbamate GTP for enzymatic capping.

[0071] FIG. 10 shows an exemplary solution phase synthesis of 4- formylbenzyl acctal-modificd dinuclcotidc and trinucleotide cap analogs.

[0072] FIG. 11 shows an exemplary solid phase oligonucleotide synthesis to form 4-formylbenzyl acetal-modified trinucleotide cap analog.Detailed Description of the Invention

[0073] The present disclosure addresses several of the critical challenges in the preparation and use of mRNAs as therapeutic agents through a novel Covalent Capture and Release (CC& R) technology. This technology enables the selective purification of capped mRNA with high specificity and yield and without residual alteration of the resulting final product. The disclosed compounds and their methods of use integrate seamlessly with current mRNA production pipelines, offering a streamlined solution to the production of high-purity, capped RNA for various applications. These approaches represent a significant advancement in mRNA manufacturing, ensuring scalability, efficiency, and quality while reducing production costs and environmental impacts.

[0074] As noted above, the mRNA cap structure plays a crucial role in protecting mRNA from exonuclease degradation, promoting export of the mRNA from the nucleus, and facilitating initiation of protein translation. Accordingly, it isimportant that the compounds disclosed herein display normal mRNA cap functionality during the transcription process, whether by co-transcriptional or post-transcriptional methods, and that any modification of the mRNA cap structure either have no effect on the normal function of the cap structure in the resulting mRNA transcription product or can be cleanly removed from the cap structure prior to therapeutic or other use of the mRNA.

[0075] According to some aspects, the compounds of the instant disclosure therefore have the following structure:Oo o oO-fj’-O-Fj’-O-fj’-O - 1 B1O O O 'C VOR3OR4,or achemically acceptable salt thereof.

[0076] In these compounds, the R1, R2, R4, R7, and R8groups can each independently be -H, -alkyl, or a cleanly-releasable linker moiety, where the cleanly-releasable linker moiety will be further described below. The alkyl group can be a straight-chain or a branched alkyl group and can optionally be a substituted alkyl group. The alkyl group is preferably a C1-C5 alkyl group.

[0077] In the above compounds, R3can be -H, a nucleotide residue, or an oligonucleotide residue.

[0078] In the above compounds, B1is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue. The nucleobase residue, protected nucleobase residue, or modified nucleobase residue found in any of the compounds disclosed herein can be any suitable nucleobase residue, whether natural or artificial, as would be understood by those of ordinary skill in the art. In preferred embodiments, the nucleobase is an adenine or guanine residue. The specific nucleobase used in a particular compound will depend on the specific desired cap structure, as is understood by those of ordinary skill in the art.

[0079] The above compounds comprise at least one cleanly-releasable linker moiety, as will be described in detail below. Furthermore, the at least one cleanly-releasable linker moiety comprises a selectively-reactive linker moiety, as will also be described in detail below.

[0080] The disclosed compounds can be the exact chemical structure shown in the drawing or can be a “chemically acceptable salt’’ of the shown structure. For example, where the structure includes an acid or base group, it should be understood that the compound can be the acid or base form, the corresponding conjugate base or conjugate acid form, or a suitable salt form of the respective conjugate base or conjugate acid. For example, the triphosphate linker shown in the structure illustrated above can be any suitable salt form of the triphosphate linker or can be a protonated form of the linker, in any combination, as would be understood by those of ordinary skill in the art.

[0081] It should also be understood that the term “residue”, as used herein, refers to the residual portion of a molecule that has been incorporated into the structure of the larger molecule. For example, in compounds having the structure illustrated above with a nucleotide residue or an oligonucleotide residue as the R3group, it should be understood that the structure would typically include a monophosphate diester group linking the compound to the 5 ’-hydroxyl of the nucleotide or oligonucleotide, respectively. Likewise, the nucleobase residue, protected nucleobase residue, or modified nucleobase residue serving as the B1group is attached to the ribose ring and the R8group through standard chemical linkages, as would be understood by those of ordinary skill in the art.

[0082] In some specific compound embodiments, R2is -CH3.

[0083] In other specific compound embodiments, B1is an adenine or guanine residue.

[0084] In still other specific compound embodiments, the R1, R2, R4, R7, and R8group is each independently -H, -CH?, or the cleanly -releas able linker moiety.

[0085] In more specific embodiments, the compounds have any of the following structures:Ooo, or a chemically acceptable salt thereof.

[0086] In these structures, T’ is the cleanly-releasable linker moiety.

[0087] In specific compound embodiments. R2is -CH3.

[0088] In other specific compound embodiments, B1is an adenine or guanine residue.

[0089] In still other specific compound embodiments, R1, R2, R4, R7, and R8is each independently -H, or -CH3.

[0090] In other more specific embodiments, the compounds can have the following structure:salt thereof.

[0091] In these compounds, R5is -H, a nucleotide residue, or an oligonucleotide residue, R6and R9is each independently -H, -alkyl, or a cleanly-releasable linker moiety, and B2is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue.

[0092] In specific compounds, R2is -CH3.

[0093] In other specific compounds, B1is an adenine or guanine residue.

[0094] In still other specific compounds, the R1, R2, R4, R6, R7, R8, and R9group is each independently -H, -CH3, or the cleanly-releasable linker moiety.

[0095] In still other more specific embodiments, the compounds can have the following structure:acceptable salt thereof;wherein R5is -H, a nucleotide residue, or an oligonucleotide residue;R6and R9is each independently -H, -alkyl, or a cleanly-releasable linker moiety; andB2is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue.

[0096] In these compounds, the R1, R2, R4, R6, R7, R8, and R9group is each independently -H, -CH3, or the cleanly-releasable linker moiety.

[0097] In yet still other more specific embodiments, the compound can have any of the following structures:-sz- ‘.10 S^OBHOR5oracceptable salt thereof.

[0098] In these structures, T’ is the cleanly-releasable linker moiety.

[0099] In specific embodiments, the R2group can be -CH3.

[0100] In other specific embodiments, the R1, R2, R4, R7, and R8group is each independently -H, or -CH3.

[0101] In other even more specific embodiments, the compound is any of the compounds illustrated in any of FIGs. 1-3.

[0102] In some embodiments, particularly where the compound is used in a post- transcriptional capping reaction, it can be advantageous for the compound to have the following structure:o o o Ho-i’-o-ij’-o-ij’-o — I B1o o o 'C?OR3OR4,or achemically acceptable saltthereof.

[0103] In these compounds, the R3, R4, and R8groups can each independently be -H, -alkyl, or a cleanly-releasable linker moiety, where the cleanly-releasable linker moiety will be further described below. The alkyl group can be a straightchain or a branched alkyl group and can optionally be a substituted alkyl group. The alkyl group is preferably a C1-C5 alkyl group.

[0104] In the above compounds, B1is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue. The nucleobase residue, protected nucleobase residue, or modified nucleobase residue found in any of the compounds disclosed herein can be any suitable nucleobase residue, whether natural or artificial, as would be understood by those of ordinary skill in the art. In preferred embodiments, the nucleobase is an adenine or guanine residue, and is most preferably a guanine residue. The specific nucleobase used in a particular compound will depend on the specific desired cap structure, as is understood by those of ordinary skill in the art.

[0105] The above compounds comprise at least one cleanly-releasable linker moiety, as will be described in detail below. Furthermore, the at least one cleanly- releasable linker moiety comprises a selectively-reactive linker moiety, as will also be described in detail below.

[0106] In more specific embodiments, the compounds have either of the following structures:O O O?Ho-ij’-o-ij’-o-ij’-o — | B1o o o 'C?OR3OT'O O O THO-Ij’-O-Ij’-O-Ij’-O B1O o OOR3OR4, or a chemically acceptable salt thereof.

[0107] In these structures, T’ is the cleanly-releasable linker moiety, and the other groups are as defined above.

[0108] In even more specific embodiments, the compounds can have either of the following structures:or a chemically acceptable salt thereof, where R3, R4, and R8is each independently -H, -alkyl, or a cleanly-releasable linker moiety. More specifically, R3, R4, and R8is each independently -H, -CH3, or the cleanly-releasable linker moiety.

[0109] In some embodiments, the compounds can have any of the following structures:, or a chemically acceptable salt thereof;wherein T’ is the cleanly-releasable linker moiety, and R3, R4, and R8are defined above. More specifically, R3, R4, and R8is each independently -H or -CH3. Cleanly-Releasable Linker Moieties

[0110] As described above, the compounds of the instant disclosure achieve at least some of their goals by including in the structure at least one cleanly-releasable linker moiety. A cleanly-releasable linker moiety is understood to be a chemical moiety that is completely removable, or nearly completely removable, from the modified mRNA cap structure under mild conditions, so that the mRNA cap structure regains its normal structure and function upon removal of the releasable moiety. Such chemical moieties, which are well known in the synthetic chemical arts, can also be referred to as “protecting groups”. See, e.g., Greene et al. (1999) Protective Groups in Organic Synthesis (3rdEd.) (J. Wiley Sons, Inc.)

[0111] Protecting groups suitable for use in the cleanly-releasable linker moieties of the instant compounds are preferably designed to protect an alcohol or a primary amino group. In addition, they are preferably chosen to be cleanly and completely removable from the modified mRNA cap under conditions that do not otherwise damage or modify an mRNA molecule. Modified mRNA caps comprising a cleanly-releasable linker moiety should also be recognized by, and display normal activity with, the enzymes and other reagents used for in vitro transcription reactions. Finally, the cleanly-releasable linker moieties should be capable of being modified by yet another chemical group — a sclcctivcly-rcactivc linker moiety — without altering the functionality of the cleanly -releas able linker moiety. I’he structure and function of the selectively-reactive linker moiety component of the cleanly-releasable linker moiety will be described in more detail below.

[0112] In some embodiments, the cleanly-releasable linker moieties of the instant compounds comprise a modified trityl protecting group. Such groups are well known and understood by those of ordinary skill in the chemical arts. In particular, such groups are widely used in the field of oligonucleotide synthesis to cleanly and releasably protect the 5’-hydroxyl group of the nucleoside phosphoramidite monomers used in the oligonucleotide synthetic cycle. For example, the dimethyoxytrityl (DMT) protecting group (also referred to simply as the trityl group) is often used for this purpose:H3COCompounds comprising modified trityl protecting groups for use in capture and release purification methods with synthetic oligonucleotides are described in U. S.Application No. 18 / 534,413, filed on December 8, 2023, now U. S. Patent No. 12,168,674 Bl, and PCT International Publication No. WO2025 / 122986 Al, the disclosure each of which is incorporated herein by reference in its entirety.

[0113] In more specific embodiments, the cleanly-releasable linker moiety can have the following general structure:Ph'Ph iPh,>xC^wherein L’ comprises a selectively-reactive linker moiety, Ph is an optionally substituted phenylene moiety, each Ph’ is independently an optionally substituted phenyl group, and C’ is a connecting group or a bond. The optional substituents of the phenylene and phenyl groups can be, for example, one or more electronwithdrawing groups, which can modulate the deprotection conditions required for release of the trityl protecting group from the modified cap after transcription and purification of an mRNA. The optional substituents can, in some embodiments, be one or more C1-C4 alkoxy groups, one or more C1-C4 alkyl groups, or any combination of these substituents.

[0114] The C’ connecting group in the above structure is an optional linker that can advantageously provide additional spacing between the cap structure and the trityl group. Under some circumstances, the additional spacing may improve the interactions between the modified cap structure and an RNA polymerase and thus improve transcription of RNAs prepared using the modified cap. The C connecting group is also ideally chosen so that it is fully releasable from the modified cap structure without leaving a chemical residue. In other words, the desired, unmodified cap structure is preferably generated after release of the cleanly-releasable linker moiety.

[0115] In some embodiments C’ comprises a carbonate residue, a carbamate residue, or an optionally substituted straight-chain or branched C1-C10 alkylene, wherein each carbon atom is optionally substituted with a heteroatom, or is a bond.o o N OMore specifically, C’ comprisesH

[0116] In some embodiments, the cleanly-releasable linker moiety can have the following structure:wherein L’ comprises the selectively-reactive linker moiety, each M’ is independently a C1-C4 alkoxy group, a C1-C4 alkyl group, a hydrogen, or a combination of these groups, and C’ is a connecting group or is a bond. In more specific embodiments, at least one M’ is a methoxy group.

[0117] In some embodiments, the cleanly-releasable linker moiety has the structure:H3CO

[0118] More specifically, the cleanly-releasable linker moiety can have the structure:H3CO

[0119] In some embodiments, the cleanly-releasable linker moiety comprises a so-called stimulus-cleavable linker moiety. Such linker moieties have been developed recently in the context of rationally-designed drug delivery systems (DDSs), where a drug of interest is covalently modified so as to decrease exposureof healthy organs or cells to the drug before it is delivered to the target site and to improve the pharmacokinetic parameters of the drug, including bioavailability and plasma clearance. See, e.g., Xue et al. (2021) Chem Soc. Rev. 50, 4872. Stimulus-cleavable linker moieties remain stable in the absence of a specific stimulus, for example during circulation of a modified drug in vivo, but are cleanly cleaved upon exposure of the modified drag to a specific stimulus. Exemplary stimuli can be either an exogenous stimulus (e.g., light, temperature, magnetic field, high energy radiation, ultrasound, and the like) or an endogenous stimulus (e.g., an enzyme activity having an elevated level at a target site or an abnormal microenvironment in a diseased tissue). In either case, the presence of the stimulus results in release of the active form of the drag at a desired location.

[0120] Exemplary stimulus-cleavable linker moieties include photo-cleavable linkers, pH-cleavable linkers, linkers cleavable by reactive oxygen species (ROS), and redox-cleavable linkers. Such linkers are described in detail by Xue et al. (2021) Chem Soc. Rev. 50, 4872, which is incorporated by reference herein in its entirety. Accordingly, in some embodiments, the cleanly-releasable linker moiety of the instant mRNA cap compounds comprises a photo-cleavable linker, a pH-cleavable linker, a linker cleavable by a reactive oxygen species (ROS), or a redox-cleavable linker.In some embodiments, the cleanly-releasable linker moiety can comprise an additional or alternative spacer feature known as a self-immolative spacer moiety or self-immolative polymer moity. Such spacer moieties are covalent chemical linkers that are designed to degrade spontaneously in response to a specific stimulus, for example the release of a stimulus-cleavable linker moiety or another component of the cleanly-releasable linker moiety. See, e.g., Gavriel et al. (2022) Polym. Chem. 13, 3188: Gong et al. (2024) Annu. Rev. Mater. Res. 54, 47: Lei et al. (2025) ChemMedChem 20, e202500262, which is each incorporated by reference herein in its entirety.Selectively-Reactive Linker Moieties

[0121] As described above, the compounds of the instant disclosure also comprise a selectively-reactive linker moiety as part of the cleanly-releasable linker moiety. The selectively-reactive linker moiety facilitates capture of anmRNA containing this group by a complementary selectively-reactive linker moiety on a capture support. Although a variety of selectively-reactive linker moiety pairs can be usefully employed in the instant compounds, capture supports, and the associated methods of preparation and use, the moieties are preferably chosen so that they selectively react with one another as efficiently and specifically as possible, in order to form a covalent linkage, and in some cases a reversibly-cleavable covalent linkage.

[0122] Examples of reversibly-cleavable covalent linkages usefully formed by the selectively-reactive linker moiety pairs of the instant disclosure include hydrazones, oximes, and other suitable Schiff base moieties. The reactions between the selectively-reactive linker moiety pairs are preferably complete, or nearly complete, at low molar concentrations of reactants in aqueous solution, and with rapid reaction kinetics. Specifically, in some embodiments, a first selectively-reactive linker moiety can comprise a reactive carbonyl group, or a derivative thereof, and the complementary second selectively-reactive linker moiety can comprise a reactive amino group, or a derivative thereof. Alternatively, the first selectively-reactive linker moiety can comprise a reactive amino group, or a derivative thereof, and the complementary second selectively-reactive linker moiety can comprise a reactive carbonyl group, or a derivative thereof.

[0123] In preferred embodiments, the reactive carbonyl group of the first or second selectively-reactive linker moiety is an aliphatic or aromatic aldehyde or ketone, or a derivative thereof, and the reactive amino group of a complementary first or second selectively-reactive linker moiety is an aliphatic or aromatic hydrazide, an aliphatic or aromatic hydrazine, an aliphatic or aromatic hydroxylamine, or a derivative thereof. Non-limiting examples of such selectively-reactive linker moieties can be found, for example, in U. S. Patent No.7,102,024, which is incorporated by reference herein in its entirety for all purposes.

[0124] For example, hydrazone conjugation moieties can be formed by the reaction of a hydrazino group, or a protected hydrazino group, with a carbonyl moiety. Exemplary hydrazino groups include aliphatic, aromatic, or heteroaromatic hydrazine, semicarbazide, carbazide, hydrazide, thiosemicarbazide,thiocarbazide, carbonic acid dihydrazine, or hydrazine carboxylate groups, as illustrated in the following structures:Hydrazine Hydrazide SemicarbazideThiosemicarbazide Carbazide ThiocarbazideCarbonic acid dihydrazine Hydrazine carboxylate

[0125] Oxime conjugation moieties can be formed by the reaction of an oxyamino group, or a protected oxyamino group, with a suitable carbonyl moiety, including any of the above-mentioned carbonyl groups or their derivatives. An exemplary oxyamino group has the following structure:OA^Xx'°xNH2H

[0126] The hydrazino and oxyamino groups can be protected by formation of a salt of the hydrazino or oxyamino group, including but not limited to, mineral acid salts, such as but not limited to hydrochlorides and sulfates, and salts of organic acids, such as but not limited to acetates, haloacetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates, or any amino or hydrazino protecting group known to those of skill in the art (see, e.g., Greene et al. (1999) Protective Groups in Organic Synthesis (3rdEd.) (J. Wiley Sons, Inc.)).

[0127] In preferred embodiments of the instant disclosure, a reversibly covalent linkage is formed by the reaction of an oxyamino-containing component and an aromatic aldehyde-containing component in the presence of an aniline catalyst (see, e.g., Dirksen et al. (2006) Angew. Chem. 45:7581-7584 (doi:10.1002 / anie.200602877).

[0128] Specific hydrazine labeling reagents, labeling methods, and uses of the labeled products are disclosed in PCT International Publication Nos.WO 01 / 70685 A2; WO 02 / 10431 A2; WO 02 / 10432 A2; WO 02 / 57422 A2;WO 2008 / 140452 Al; WO 2011 / 100493 Al; WO 2012 / 071428 A2;WO 2013 / 177046 Al; WO 2016 / 127149 A2; and WO 2018 / 017606 Al; and U. S. Patent Application Publication No. 2008 / 0221343 Al, each of which is incorporated by reference herein in its entirety for all purposes.

[0129] As mentioned above, in some embodiments, the selectively-reactive linker moiety used in the instant CC& R technology is a component of a “click” reaction, for example the copper-catalyzed reaction of an azide-substituted component with an alkyne-substituted component to form a triazole conjugation moiety. See Kolb et al. (2001) Angew. Chem. Int. Ed. Engl. 40:2004; Evans (2007) Aus. J. Chem. 60:384. Copper-free variants of this reaction, for example the strain-promoted azide-alkyne click reaction, may also be used to form the high-efficiency conjugation moiety. See, e.g., Baskin et al. (2007) Proc. Natl Acad. Sci. U. S. A. 104:16793-97. Other click reaction variants include the reaction of a tetrazine- substituted component with either an isonitrile-substituted component (Stockmann et al. (2011) Org. Biomol. Chem. 9:7303) or a strained alkenesubstituted component (Karver et al. (2011) Bioconjugate Chem. 22:2263).

[0130] The basic features of a click reaction are well understood by those of ordinary skill in the art. See Kolb et al. (2001) Angew. Chem. Int. Ed. Engl.40:2004. Useful click reactions include generally but are not limited to [3+2] cycloadditions, such as the Huisgen 1,3-dipolar cycloaddition, and in particular the Cu(I)-catalyzed stepwise variant, thiol-ene click reactions, Diels-Alder reactions and inverse electron demand Diels-Alder reactions, [4+1] cycloadditions between isonitriles (isocyanides) and tetrazines, nucleophilic substitutions, especially to small strained rings like epoxy and aziridine compounds, carbonyl-chemistry-like formation of ureas, and some addition reactions to carbon-carbon double bonds. Any of the above reactions may be used without limitation to prepare complementary selectively-reactive linker moieties for use in the instant CC& R technologies.

[0131] The above-described selectively- reactive linker moieties (e.g., a reactive carbonyl group, a reactive hydrazino group, a reactive oxyamino group, a component of a click reaction, or their derivatives) can be incorporated into the compounds of the disclosure, for example as described herein. Complementary selectively-reactive linker moieties can likewise be incorporated into suitable solid supports using appropriate chemical linkers, and the modified capture supports can be used in methods of purifying mRNA comprising the modified mRNA cap structure, for example as described herein. Hydrazine-based and carbonyl-based bifunctional crosslinking reagents for use in the conjugation and immobilization of biomolecules are described in U. S. Patent No. 6,800,728. The use of high-efficiency bisaryl-hydrazone linkers to form oligonucleotide conjugates in various detection assays and other applications is described in PCT International Publication No. WO 2012 / 071428. Each of the above references is hereby incorporated by reference herein in its entirety for all purposes.

[0132] It should also be understood that the selectively-reactive linker moiety in these compounds can be any of the above-described selectively-reactive linker moieties. More specifically, the selectively-reactive linker moiety can comprise a reactive carbonyl group, a reactive amino group, a component of a click reaction, or a derivative of any of these groups. The specific choice of selectively-reactive linker moiety will depend on the counterpart selectively-reactive linker moiety that is associated with the capture support to be used in the capture and release process, as would be understood by those of ordinary skill in the art upon review and understanding of the instant disclosure.

[0133] Three exemplary cleanly-releasable linker moieties comprising three different selectively-reactive linker moieties are as follows:where the cleanly-releasable linker moiety is a modified trityl group in each case, and where the selectively-reactive linker moiety of the cleanly-releasable linker moiety is a formyl group, an oxyamino group, and a hydrazide group, respectively.

[0134] In some embodiments, the selectively-reactive linker moiety can further comprise a protecting group that is readily released from the selectively-reactive linker moiety either before or during the capture and release process. For example, a formyl group can be protected by derivatizing it as a dimethyl acetal, as shown in the left compound below, and an oxyamino group can be derivatized with a trifluoroacetyl protecting group, as shown in the right compound below:Methods of Preparing a Capped mRNA

[0135] In another aspect are provided methods of preparing an mRNA comprising an mRNA cap structure. These methods generally comprise the step of providing a modified mRNA cap compound comprising a cleanly-releasable linker moiety that further comprises a selectively-reactive linker moiety, for example any of the compounds described above. An mRNA comprising the modified mRNA cap structure is then generated by in vitro transcription from a DNA template. The mRNA comprising the modified mRNA cap structure is then purified using a solid support comprising a complementary selectively-reactive linker moiety, for example a reactive oxyamino group, a reactive hydrazino group, a reactive carbonyl group, or a component of a click reaction. As has been described in detail above, the selectively-reactive linker moieties of the modified mRNA cap structure are chosen to be complementary to the reactive groups associated with the capture support. For example, if the selectively-reactive linker moiety of the modified mRNA cap structure comprises a reactive carbonyl group, such as a suitable formyl group, the capture support preferably comprises a reactive oxyamino group or a reactive hydrazino group, as would be understood by those of ordinary skill in the art in view of the instant disclosure. Alternatively, if the selectively-reactive linker moiety of the modified mRNA cap structure comprises a reactive oxyamino group or a reactive hydrazino group, the capture support preferably comprises a reactive carbonyl group, such as a suitable formyl group. Furthermore, if the selectively-reactive linker moiety of the modified mRNA cap structure comprises areactive azide, the capture support could comprise a reactive alkyne or strained alkyne group. Alternatively, if the selectively-reactive linker moiety of the modified mRNA cap structure comprises a reactive alkyne or strained alkyne group, the capture support could comprise a reactive azide group.

[0136] After the mRNA with the modified mRNA cap structure has been captured by reaction with the suitably-reactive capture support, the capture support can be washed, for example to remove unreacted reagents and any other impurities.

[0137] After the captured mRNA has been washed, it can be released from the capture support, either by a soft release, which reverses the capture reaction without cleaving the cleanly-releasable linker moiety from the mRNA cap, or by a hard release, which completely removes the cleanly-releasable linker moiety and the attached selectively-reactive linker moiety and generates a purified mRNA with an unmodified mRNA cap structure. In some embodiments, a soft release of the mRNA with the modified mRNA cap structure can be performed either by cleaving a linker that joins the cleanly-releasable linker moiety to the selectively-reactive linker moiety, by cleaving a linker that joins the complementary selectively-reactive linker moiety to the capture support, or by dispersing or solubilizing the capture support. For example, the linker joining the cleanly-releasable linker moiety to the selectively-reactive linker moiety or the complementary selectively-reactive linker moiety to the capture support can be an enzymatically cleavable linker, a photocleavable linker, a disulfide bridge, an azo compound, a phenacyl ester, an ort / ro-nitrobenzyl derivative, or any other suitable cleavable linker known to those of average skill in the art. See, e.g., Leriche et al. (2012) Bioorg. Med. Chem. 20 571-582 and Bargh et al. (2019) Chem. Soc. Rev., 48, 4361. Accordingly, in some embodiments, the releasing step is performed using an agent that causes dissociation of any bond between the cleanly-releasable linker moiety and the capture support. In some embodiments, the releasing step includes cleavage of the cleanly-releasable linker moiety from the mRNA cap structure using, for instance, a mild acid solution.

[0138] The methods of purification thus generally follow the Covalent Capture & Release (CC& R) techniques described above, and as have also been applied inU. S. Patent No. 12,168,674 Bl and PCT International Publication No.WO2025 / 122986 Al for synthetic oligonucleotides.

[0139] It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the compounds, methods, and applications described herein may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following Examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.EXAMPLESExample 1. Exemplary Modified mRNA Cap Structures

[0140] FIGs. 1-3 illustrate various exemplary modified mRNA cap compounds that can be used in the synthesis and purification of capped mRNAs using the capture and release technologies described herein. The compounds are synthesized according to standard synthetic methods, including adaptation of the synthetic methods disclosed in U. S. Application No. 18 / 534,413, previously incorporated herein by reference.

[0141] FIG. 1 illustrates exemplary modified mRNA cap compounds of the disclosure, where the compounds include ribose rings modified with a cleanly-releasable linker moiety.

[0142] FIG. 2 illustrates exemplary modified mRNA cap compounds of the disclosure, where the compounds include nucleobases modified with a cleanly-releasable linker moiety.

[0143] FIG. 3 illustrates exemplary modified mRNA cap compounds of the disclosure, where the cleanly-releasable linker moiety comprises a residual carbonate or carbamate connecting group.Example 2, Synthesis of 2’-O-formyl DMT-acetal 3’-OMe-m7G-5’-ppp-5’-G and 2’-O-formyl DMT-acctal 3’-QMc-m7G-5’-ppp-5’-(2’-OMc A)-p-G

[0144] Reagents: 3’-0Me guanosine is available from Broadpharm, San Diego, CA, CAS RN: 10300-27-3, 2’-0Me guanosine is available from TCI Chemicals, Portland, OR, CAS RN: 2140-71-8. N2-isobutrylguanosine is available fromCayman Chemicals, Ann Arbor, MI, CAS RN: 64350-24-9. N2-Isobutyryl-2’-deoxyguanosine is available from TCI Chemicals, Portland, OR, CAS RN: 68892-42-2.Activation of formyl DMT-OH to form a cleanly-releasable linker moiety that comprises a selectively-reactive linker moiety

[0145] It is recognized that the 4-formyl-dimethoxytrityl (formyl DMT) moiety is a highly suitable cleanly-releasable linker moiety comprising a selectively-reactive linker moiety that can by appended to biomolecules via different linkers, thus allowing for traceless release. As illustrated in FIG. 4, formyl DMT can be coupled to target biomolecules via direct attachment, acetal, carbonate and carbamate linkages. Described below are methods to activate formyl DMT-OH for direct, acetal, carbonate and carbamate linkages, each of which serve as cleanly-releasable linker moieties.

[0146] Formyl DMT-OH 1 is prepared as described previously in U. S. Patent No. 12,168,674-B1. For direct activation, under anhydrous conditions a solution of oxalyl chloride (1.25 mmole) in DCM is added to a solution of formyl DMT-OH 1 (1.0 mmol) in DCM and is incubated at room temperature for 20 min. This solution is used directly to form the formyl-DMT-modified form of a biomolecule (2). For acetal activation, to a solution of formyl DMT-OH (1) (1 mmol) in acetic acid (20 mmol) is added DMSO (20 mmol) and acetic anhydride (20 mmol).Progress of the reaction is monitored by analytic HPLC or TLC. On completion, the reaction mixture is added dropwise to 10 M KOH aq. (0.01 mol) in an ice bath. Following completion, the reaction mixture is diluted with ethyl acetate and washed with brine and dried over anhydrous Na2SO4. The product is purified by silica gel column chromatography to afford the formyl DMT-O-methylthioacetal 3.This intermediate is combined with a hydroxyl-bearing biomolecule to form the formyl DMT-acetal-modified form. For carbonate or carbamate activation, to a solution of formyl DMT-OH (1) (1 mmol) in DMF is added triethylamine (2 mmol) and carbonyl diimidazole (1.25 mmol) and incubated for 30 min to yield the formyl DMT carbonyl-imidazole that on reaction with an alcohol or amine yields biomolecules bearing formyl DMT carbonate 4 or carbamate 5 modifications respectively.Preparing 2’-Q-formyl DMT-acetal 3'-QMe m7G diphosphate intermediate

[0147] To a solution of formyl DMT-OH 1 (1 mmol) in acetic acid (20 mmol) is added DMSO (20 mmol) and acetic anhydride (20 mmol). Progress of the reaction is monitored by analytic HPLC or TLC. On completion, the reaction mixture is added dropwise to 10 M KOH aq. (0.01 mol) in an ice bath. Then, the reaction mixture is diluted with ethyl acetate and washed with brine and dried over anhydrous Na2SO4. The product is purified by silica gel column chromatography to afford the formyl DMT-O-methylthioacetal 3. To a solution of 2N-isobutyryl-3’-OMe guanosine (6) (1.0 mmol) in anhydrous pyridine is added dropwise trimethylsilyl chloride (2.0 mmol) in pyridine in an ice bath. The mixture is stirred at 4°C until complete as monitored by analytical HPLC or TLC. The reaction mixture is quenched by adding ice-cold water and extracted 3 times with dichloromethane. The organic layer is dried over anhydrous Na2SC>4, filtered, and evaporated. The product is isolated by silica gel column chromatography to yield the desired product 2N-isobutyryl-5’-O-TMS-3’-OMe-guanosine 7. Following from the methods of Inagaki (Inagaki et al. (2023) Nature Communications 14:2657 (doi:10.1038 / s41467-023-38244-8)), to a mixture of 2N-isobutyryl-5’-O-TMS-3’-OMe-guanosine 7 (1.0 mmol), formyl DMT-O-methylthioacetal derivative (3) (1.1 mmol), and molecular sieves 3A (0.5 g), in tetrahydrofuran, is added A-iodosuccinimide (1.5 mmol) and then cooled to -40 °C. To the cooled suspension is added trifluoromethanesulfonic acid (1.5 mmol) and stirred at -40 °C until complete as monitored by TLC. The reaction mixture is quenched by adding triethylamine and then diluted with ethyl acetate. The mixture is washed with saturated aq. NaHCO3, saturated aq. sodium thiosulfate, and brine successively. The organic layer is dried over anhydrous Na2SC>4 and concentrated. The residue is purified by silica gel column chromatography afford compound 2N-isobutyryl-5’-O-TMS 2’-O-formyl DMT-acetal 3’-OMe-guanosine 8. A solution of 8 (1.0 mmol) in tetrahydrofuran is cooled in an ice bath and 1 M TBAF / THF (10.0 mmol) is added to the solution. After stirring at 0 °C until complete as monitored by analytical HPLC or TLC, the reaction mixture is diluted with dichloromethane and washed with water. The organic layer is dried over anhydrous Na2SC>4 and concentrated. The residue is purified by silica gel column chromatography to yield2N-isobutyryl-2’-O-formyl DMT-acetal 3’-OMe-guanosine. To a solution of the 2N-isobutyryl-2’-O-formyl DMT-acetal 3’-OMe-guanosine (1.0 mmol) in acetonitrile is added 28% ammonium hydroxide solution and the mixture is heated at 55 °C. After stirring at 55 °C for 6 hours, the reaction mixture is concentrated and azeotroped with benzene. The residue is suspended in a 1:3 mixture of dichloromethane and diethyl ether. The resulting precipitate is collected by filtration and washed with a 1:3 mixture of dichloromethane and diethyl ether. The obtained solid is dried in a desiccator over P2O5 under a vacuum to afford -2’-O-formyl DMT-acetal 3’-OMe-guanosine 9. To a solution of 9 (1.0 mmol) in anhydrous pyridine is added dropwise p-loluenesulfonyl chloride (2 mmol) in pyridine in an ice bath. The mixture is stirred at 4 °C until complete as monitored by HPLC. The reaction mixture is quenched by adding ice-cold water and extracted 3 times with dichloromethane. The organic layer dried over anhydrous Na2SO4 and evaporated. The product is isolated by silica gel column chromatography to yield the desired product 2’-O-formyl DMT-acetal 3’-0Me 5’-O-tosyl-guanosinc. To a suspension of 2’-O-formyl DMT-acetal 3'-0Mc 5’-O-tosyl-guanosine (1.0 mmol) and powdered 3A molecular sieves in CH3CN (1.27 mL), is added 725 mM tris-tetrabutylammonium pyrophosphate (1.5 mmol) in CH3CN. After stirring at 40 °C until complete as monitored by HPLC, the reaction mixture is diluted with water. The resulting suspension is centrifuged for 30 minutes at 4,500 rpm. The supernatant is collected, and the precipitate is resuspended in water. The resulting suspension is centrifuged for 30 minutes at 4,500 rpm. The supernatant is collected and purified by ion-exchange. The fractions containing the product are collected and concentrated to afford 2"-O-formyl DMT-acetal 3’-OMe-guanosine diphosphate. The resulting 2’-O-formyl DMT-acetal 3’-OMe-guanosine diphosphate (1.0 mmol) is dissolved in anhydrous DMF containing triethylamine (3.0 mmol) and reacted with methyl iodide (3.0 mmol). Ion-exchange chromatography affords the final product 2’-O-formyl DMT-acetal 3’-OMe-m7 guanosine diphosphate (10), abbreviated 2’-O-formyl DMT-acetal 3’-OMe-m7GDP. These reactions are illustrated in FIG. 5.Solution phase synthesis of 2’-Q-formyl DMT-acetal 3,-OMe-m7G-5’-ppp-5’-G and 2’-O-formyl DMT-acetal 3'-OMe-m7G-5’-ppp-5’-(2’-OMe A)-p-G

[0148] Following from the work of Thillier (Thillier et al. (2012) RNA 18, 856 (doi:10.1261 / ma.030932.111)) and Noel (Noel et al. (2023) ChemBioChem 24, 544, doi: 10.1002 / cbic.202300544), here the commercially available GMP is converted to its 5’-phospho-imidazolide 11 and reacted with 2'-O-formyl DMT-acetal 3’-OMe-GDP 10 in the presence of zinc chloride to yield 2’-O-formyl DMT-acetal 3’-OMe-m7G-5’-ppp-5’-G 12, which serves as a formyl-DMT-modified form of the anti-reverse cap analog (ARCA) where mild acid cleavage of the formyl-DMT releases the acetal and thereby cleanly releases 3’-OMe-m7G-5’-ppp-5'-G (ARCA). Similarly, the dimer 5’ phospho-(2' OMe A)-p-G is prepared in solution according to Senthilvelan (Senthilvelan et al. (2022) Org. Process Res. Dev. 26, 2771 (doi:10.1002 / cpzl.583)) and then converted to its 5’-phospho-imidazolide 13 and reacted with 2’-O-formyl DMT-acetal 3’-OMe-GDP 10 to yield 2’-O-formyl DMT-acetal 3’-OMe-m7G-5’-ppp-5’-(2’ OMe A)-p-G 14, which serves as a formyl-DMT-modified form of a standard commercial trinucleotide cap analog where mild acid cleavage of the formyl-DMT releases the acetal and thereby cleanly releases 3’-OMe-m7G-5’-ppp-5’-(2’ OMe A)-p-G. These reactions are illustrated in FIG. 6.Synthesis of 21-O-formyl DMT-acetal 3’-OMe-m7G-51-ppp-5’-(2’ OMe A)-p-G by solid phase oligonucleotide synthesis

[0149] In some embodiments, it may be preferable to form the formyl DMT- or other formyl-modified cap analogs via solid phase oligonucleotide synthesis (SPOS). Methods to form capped mRNA analogs including trinucleotide, tetranucleotide or longer cap analogs that can be applied as tools for cotranscriptional capping via in vitro transcription (IVT) reactions are well known from the literature. Briefly, an RNA dimer, trimer or longer oligonucleotide bearing appropriate modifications on the 2’ OH and / or the bases is formed by SPOS, 5’ phosphorylated and then modified with m7G to produce an m7G-5’-ppp-5’ cap structure, which can be cleaved from the solid support. As an example for forming the trinucleotide cap analog 14, following from the work of Thillier (Thillier et al. (2012) RNA 18, 856 (doi:10.1261 / rna.030932.111)), (2’-0Me N6AcA)-p-2’-OAc N2AcG 15 is prepared on solid support via couplingcommercially available 2’-OMe-A-CE phosphoramidite to commercially available Ac-G-RNA-CPG to form 15 which is chemically phosphorylated and then converted to its 5’-phospho-imidazolide, (2’-0Me N6AcA)-p-2’-OAc N2AcG 5’-phospho-imidazole 16. To 16 is added 2’-O-formyl DMT-acetal 3’-OMe-m7GDP 10 and ZnCb in anhydrous DMF and mixed to yield 17, the formyl DMT-modified trinucleotide cap analog protected and tethered to the solid support. Subsequently the solid support is treated with DBU to deprotect the acetyl protecting groups followed by incubation with ammonia in MeOH to cleave the product from the support. Following desalting, the desired 2’-O-formyl DMT-acetal 3’-OMe-m7G-5’-ppp-5’-(2’ OMe A)-p-G 14 is isolated. These reactions are illustrated in FIG. 7.Preparing 2'-O-formyl DMT-acetal GTP and N6-fomiyl DMT-carbamate GTP for enzymatic capping

[0150] In some embodiments, it may be preferable to perform enzymatic capping on RNA formed by in vitro transcription rather than to apply cotranscriptional capping with a cap analog. Here, taking advantage of the ability of Vaccinia capping enzyme or other capping enzymes to incorporate nucleotides other than GTP, it may be favorable to form a formyl-modified GTP that can be incorporated and modified to form a formyl m7G-5’-ppp-5’ cap. To a solution of N2-isobutrylguanosine (18; 1.0 mmole) in anhydrous pyridine is added tetraisopropyldisilyl dichloride (TiPDSiCh; 1.2 mmole) and incubated at room temperature. Progress of the reaction is followed by analytical HPLC or TLC. Following completion of the reaction, the reaction mixture is diluted with ethyl acetate and washed sequentially with 0.5 M IIC1, water and brine. The organic phase is dried over anhydrous sodium sulphate, filtered and concentrated to yield the desired product 19.

[0151] To form V2-Isobutyryl-3’,5’-O-TIPDS-2’-O-formyl DMT-acetal-guanosine (20), to a suspension of 2V2-isobutyryl-3’,5’-O-TIPDS-guanosine (19) (1.0 mmol), methylthioacetal derivative (6) (1.1 mmol), and molecular sieves 3A, in tetrahydrofuran, is added N-iodosuccinimide (1.5 mmol) and then cooled to -40 °C. To the cooled suspension is added trifluoromethanesulfonic acid (1.5 mmol) and stirred at -40 °C until complete as monitored by TLC. The reaction mixture is quenched by adding triethylamine and then diluted with ethyl acetate. The mixtureis washed with saturated aq. NaHC03, saturated aq. sodium thiosulfate, and brine successively. The organic layer is dried over anhydrous Na2SC>4 and concentrated. The residue is purified by silica gel column chromatography to afford compound 20. Then, a solution of 20 (1.0 mmol) in tetrahydrofuran is cooled in an ice bath and 1 M TBAF / THF (10.0 mmol) is added to the solution. After stirring at 0 °C until complete as monitored by analytical HPLC or TLC, the reaction mixture is diluted with dichloromethane and washed with water. The organic layer is dried over anhydrous Na2SC>4 and concentrated. The residue is purified by silica gel column chromatography to yield N2-isobutyryl-2’-O-formyl DMT-guanosine. To a solution of the N2-isobutyryl-2’-O-formyl DMT-acetal-G (2.0 mmol) in acetonitrile is added 28% ammonium hydroxide solution and the mixture is heated at 55 °C. After stirring at 55 °C for 6 hours, the reaction mixture is concentrated and azeotroped with benzene. The residue is suspended in a 1:3 mixture of dichloromethane and diethyl ether. The resulting precipitate is collected by filtration and washed with a 1:3 mixture of dichloromethane and diethyl ether. The obtained solid is dried in a desiccator over P2O5 under a vacuum to afford 2’ -O-formyl DMT-acetal-G 21. To form 2’-O-formyl DMT-acetal GDP (22), to a solution of 21 (1.0 mmol) in anhydrous pyridine, p-toluenesulfonyl chloride (2 mmol) in pyridine is added dropwise to in an ice bath. The mixture is stirred at 4 °C until complete as monitored by HPLC. The reaction mixture is quenched by adding ice-cold water and extracted with dichloromethane. The organic layer is dried over anhydrous Na2SO4 and evaporated. The product is isolated by silica gel column chromatography to yield the desired product 2’-O-formyl DMT-acetal 5’-O-tosyl-G. To a suspension of 2’-O-formyl DMT-acetal 5’-O-tosyl-G (1.0 mmol) and powdered 3 A molecular sieves in CH3CN (1.27 mL), is added 725 mM tris-tetrabutylammonium pyrophosphate (1.5 mmol) in CH3CN. After stirring at 40 °C until complete as monitored by HPLC, the reaction mixture is diluted with water. The resulting suspension is centrifuged for 30 minutes at 4,500 rpm. The supernatant is collected, and the precipitate is resuspended in water. The resulting suspension is centrifuged for 30 minutes at 4,500 rpm. The supernatant is collected and purified by ion-exchange. The fractions containing the product are collected and concentrated to afford 2’-O-formyl DMT-acetal GDP 22. Finally, to form thedesired 2’-O-formyl DMT-acetal GTP (23). (22) is dissolved in minimal water, passed through a Dowex 50WX8 (H+) column to yield the H+form, then is neutralized with tetrabutylammonium hydroxide (B NOH) to give theNDP-(Bu4N)2 salt followed by lyophilization. In a separate vial, (Bu4N)4P2O7 (3-10 equivalents) is dissolved in dry DMF (1-2 mL) and added to the NDP-(Bu4N)2 and ZnCl2 (1.0 equivalent). Reaction is monitored by anion exchange HPLC. After completion the reaction mixture is diluted with minimal water and the volatiles are evaporated under reduced pressure to remove DMF. The product 23 is purified by anion-exchange chromatography. These reactions are illustrated in FIG. 8.

[0152] Alternatively, it may be preferable to modify the guanosine base rather than the ribose sugar. As an example, to form N2-formyl DMT-carbamate-GTP (26), to a solution of guanosine (1.0 mmol) in DMF is added GDI-activated formyl DMT-OH (1.5 mmol) and dimethylaminopyridine (DMAP; 0.10 mmol) and stirred at 40-50 °C until formation of N2-formyl DMT-carbamate guanosine (24) is complete as monitored by analytical HPLC or TLC. Following completion the reaction mixture is diluted with ethyl acetate, washed with 1.0 N HCl, saturated Na2SO4 and brine. The organic phase is dried over anhydrous Na2SO4, filtered, and concentrated. The product 24 is purified by silica gel chromatography. To form N2-formyl DMT-carbamate guanosine diphosphate (25), to a solution of 24 (1.0 mmol) in anhydrous pyridine, p-toluenesulfonyl chloride (2 mmol) in pyridine is added dropwise in an ice bath. The mixture is stirred at 4 °C until complete as monitored by analytical HPLC or TLC. The reaction mixture is quenched by adding ice-cold water and extracted 3 times with dichloromethane. The organic layer is dried over anhydrous Na2SC>4 and evaporated. The product is isolated by silica gel column chromatography to yield N2-formyl DMT-carbamate 5’-O-tosyl-guanosine. To a suspension of N2-formyl DMT-carbamate 5’-O-tosyl-guanosine (1.0 mmol) and powdered 3A molecular sieves in CH3CN (1.27 mL) is added 725 mM tris-tetrabutylammonium pyrophosphate (1.5 mmol) in CH3CN. After stirring at 40 °C until complete as monitored by analytical HPLC followed by quenching by dilution with water. The resulting suspension is centrifuged for 30 minutes at 4,500 rpm. The supernatant is collected, and the precipitate is resuspended in water. The resulting suspension is centrifuged for 30 minutes at 4,500 rpm. Thesupernatant is collected and purified by ion-exchange. The fractions containing the product are collected and concentrated to afford N2-formyl DMT-carbamate GDP (25). Then, 25 is dissolved in minimal water, passed through a Dowex 50WX8 (H+) column to give the H+form, neutralized with tetrabutylammonium hydroxide (Bu4NOH) to give the NDP-(Bu4N)2 salt followed by lyophilization and dried further by azeotropic drying with anhydrous DMF. In a separate vial, (Bu4N)4H2P2O7 (3-10 equivalents) is dissolved in dry DMF (1-2 mL) and added to the NDP-(Bu4N)2 and ZnCl2 (1.0 equivalent). Reaction is monitored by anion exchange HPLC. After completion the reaction mixture is diluted with minimal water and the volatiles are evaporated under reduced pressure to remove DMF. The product N2-formyl DMT-carbamate GTP 26 is purified by anion-exchange chromatography. These reactions are illustrated in FIG. 9.Solution-phase synthesis of formyl-modified dinucleotide and trinucleotide cap analogs using formylbenzyl acetal (4FB-acetal) as the cleanly-releasable linker moiety that comprises a selectively-reactive linker moiety

[0153] There are many cleanly releasable linkers described in the literature that enable traceless release after cleavage. A favorable group of linkers are those that cleave in mild acid, making them highly compatible with release of intact 5’ capped mRNA via the Covalent Capture & Release (CC& R) purification process. Of these, one example is the 4FB-acetal linker, with the general structure R’OCH[R”]OR”’. Here, R’ is 4-formylbenzyl (4FB), serving as the selectively reactive linker moiety. R” may be a hydrogen, a methyl group or an alkyl or phenyl or other group that may alter pH cleavage efficiency. Here, R’” is m7G, which may be linked via the 2’ hydroxyl. As representative examples, we describe formation of dinucleotide and trinucleotide cap analogs bearing 2’-O-4FB-acetal as a cleanly-releasable linker moiety that comprises a selectively-reactive linker moiety. To form 4-(l-(methylthio)alkoxy)benzaldehyde (27), to a solution of 4-hydroxybenzaldehyde (1 mmol) in acetic acid (20 mmol) is added DMSO (20 mmol) and acetic anhydride (20 mmol). Progress of the reaction is monitored by analytic HPLC or TLC. On completion, the reaction mixture is added dropwise to 10 M KOH aq. (0.01 mol) in an ice bath. Following completion, the reaction mixture is diluted with ethyl acetate and washed with brine and dried over anhydrous Na2SO4. The product is purified by silica gel column chromatography toafford the 4-0-methylthioacetal-benzaldehyde 27. Then, the scheme shown in FIG.5 is followed, where 4-(l-(methylthio)alkoxy)benzaldehyde 27 is used in place of methylthioacetal formyl DMT 3, forming 2’ -O-formylbenzyl-acetal 3’-OMe-m7G (28) and then 2’ -O-formylbenzyl-acetal 3’-OMe-m7GDP (29) rather than 2’-O-formyl DMT-acetal 3’-OMe-m7G and 2’-O-formyl DMT-acetal 3’-OMe-m7GDP (10). Then, following the scheme in FIG. 6, GMP is converted to its 5’-phospho-imidazolide 11 and reacted with 2’ -O-formylbenzyl-acetal 3’-OMe-m7GDP (29) in the presence of zinc chloride to yield 2’ -O-formylbenzyl-acetal 3’-OMe-m7G-5'-ppp-5’-G 30. Here, 30 serves as a 4FB-acetal-modified form of the anti-reverse cap analog (ARCA) where mild acid cleavage of the 4FB-acetal cleanly releases 3‘-OMe-m7G-5’ -ppp-5 '-G (ARCA). Similarly, 5’ phospho-(2’ OMe A)-p-G prepared in solution according to Senthilvelan5and converted to its 5’-phospho-imidazolide 13 is reacted with 2’ -O-formylbenzyl-acetal 3’-OMe-m7GDP (29) to yield 2’-O-formylbenzyl-acetal 3’-OMe-m7G-5’-ppp-5’-(2’-O-Me A)-p-G 31. Mild acid cleavage of the 4FB-acetal cleanly releases the commercially available trinucleotide cap analog. These reactions arc illustrated in FIG. 10.Solid phase oligonucleotide synthesis to form 4FB-acetal-modified trinucleotide cap analog 2’ -O-formylbcnzyl-acctal 3'-OMe-m7G-5’-ppp-5’-(2’-O-Mc A)-p-G

[0154] As in FIG. 7, (2'-0Me N6AcA)-p-2’-OAc N2AcG 5 '-phospho-imidazole 16 is prepared on solid support. To 16 is added 2’ -O-formylbenzyl-acetal 3’-0Me-m7GDP 29 and ZnCl₂ in anhydrous DMF and mixed to yield 34, the 4FB-acetal-modified trinucleotide cap analog protected and tethered to the solid support. Subsequently the solid support is treated with DBU to deprotect the base acetyl protecting groups followed by incubation with ammonia in MeOH to cleave the product from the support. Following desalting, the desired 4FB-acetal-modified trinucleotide cap analog 2’ -O-formylbenzyl-acetal 3’-OMe-m7G-5’-ppp-5’-(2’-O-Me A)-p-G 31 is isolated. These reactions are illustrated in FIG. 11.Example 3. IVT Protocol Using Covalent Capture-and- Release mRNA Capping Reagents and Traditional Capping Reagents.

[0155] To demonstrate the utility of Covalent Capture-and-Release (CC& R) cap analogs versus traditional capping agents, each reagent is employed in an in vitrotranscription (IVT) reaction to synthesize a model mRNA encoding C.ypridiiifi luciferase (CLuc). For trinucleotide caps employing an “AG” initiation sequence, the HiScribe™ T7 mRNA Kit with CleanCap® Reagent AG (NEB catalog #: E2080S / L) is used. This kit contains a CLuc AG control DNA template, which is a linearized plasmid containing the CLuc gene under the transcriptional control of the T7 promoter. The initiating sequence has been changed to AG by site-directed mutagenesis to be compatible with AG initiating caps.

[0156] For caps employing a “G” or “GG” initiation sequence, such as the dinucleotide Anti-Reverse Cap Analog (ARCA) or GG trinucleotide caps, as well as post-transcriptional capping, the HiScribe® T7 ARCA mRNA Kit (NEB catalog #: E2065S) is used. This kit contains a CLuc GG control DNA template, which is a linearized plasmid containing the CLuc gene under the transcriptional control of the T7 promoter. The initiating sequence has not been changed from its native GG initiating sequence, and is therefore compatible with G or GG initiation. It is also compatible with post-transcriptional capping using enzymes such as vaccinia capping enzyme to install a native Cap 0 or CC& R Cap 0 structure. A separate Vaccinia Capping System kit (NEB catalog # M2080S) is used after IVT to incorporate m7G-PPP-N at the 5’ terminus of the IVT mRNA, which may be optionally further treated with 2 '-O- Methyl transferase to convert the Cap 0 to a Cap 1 structure.

[0157] Both kits produce CLuc as a run-off transcript with a length of -1.76 kb. It is prepared by first constructing a plasmid incorporating the T7 promoter sequence, followed immediately by the initiation dinucleotide configuration (AG or GG) appropriate for efficient incorporation of either trinucleotide cap, ARCA, or for post-transcriptional capping. The poly(A) tail is encoded directly into the plasmid to avoid the need for enzymatic tailing, and the DNA plasmid is fully linearized to allow efficient run-off transcription.

[0158] Each reaction is purified after IVT (and post-transcriptional capping, where required) to isolate pure mRNA using either kit recommendations (for traditional capping agents) or a hydrazide functionalized resin (for CC& R capping agents) to remove contaminating species such as NTPs, proteins, DNA template, uncapped mRNA, double- stranded RNA (dsRNA), etc. Protocols for removal ofthese contaminants necessarily vary for traditional capping agents vs. CC& R caps, and are detailed herein.IVT Reactions - AG Initiation Sequence

[0159] In vitro transcription reactions are typically assembled in a total volume of twenty microliters using certified nuclease-free water and buffer stocks contained within the kit. The reactions are prepared at room temperature using certified nuclease-free tubes and tips, with all reagents gently mixed and pulse- spun before use. Transcription buffer is added at a IX final concentration, ATP is supplied at a final concentration of 6 mM, and GTP, CTP and UTP are all supplied at a final concentration of 5 mM. CleanCap® Reagent AG (included) is supplied at 4 mM final concentration as recommended by the manufacturer. CleanCap® Reagent AG (3’ OMe) (TriLink Cat. #: N-7413) is purchased separately and used at the same final concentration. CC& R Trinucleotide Cap AG analogs (structures 14 and 31) are similarly supplied at 4 mM final concentration each. 1 pg of linear template DNA (1 pL) is added, followed by DTT (5 mM final concentration). Finally, 2 pL of T7 RNA Polymerase Mix is added and the tube contents are gently mixed to start the IVT reaction. Reactions are incubated for 2 hours in a thermocycler at a constant temperature of 37 °C.Components i 20 uL Reaction i Final Cone.Xue lease- free Water 4 pL1 OX T7 CleanCap® Reagent AG Reaction Buffer 2 pL IX 6O 111M ATP 2 pl. 6 mM 50 mM GTP 2 pL 5 mM 50 m. M (TP 2 pl. 5 m. M 50 m. VH TP 2 pl. 5 m. M 40 mM CC& R Trinucleotide Cap AG analogi-or- 2 pL 4 mM 40 mM CleanCap® Reagent AG (or CleanCap®Reagent AG 3’ OMe)Linear Template DNA 1 pL 1 pg 1) 1 1 (0. IM) 1 pl. 5 m. MT7 RNA Polymerase Mix 2pLTable 1. Components of “AG” initiation IVT reactions.IVT Reactions - GG Initiation Sequence

[0160] In vitro transcription reactions using “G” or “GG” initiation are generally performed in a similar manner to AG initiation, with some important considerations. For dinucleotide caps such as ARCA, the nucleoside triphosphate GTP competes for initiation with the ARCA G nucleotide. Therefore, to achieve high capping efficiency, GTP “starvation” is typically employed to favor initiation with ARCA over GTP. In this case, all nucleoside triphosphates are generally reduced while the ARCA concentration is kept relatively high (for instance: 1 mM GTP, 4 mM ARCA, 1.25 mM CTP, UTP, and >1.25 mM ATP final concentration). This is necessary to achieve relatively high capping yields in the final product for traditional ARCA, but is wasteful of that molecule and simultaneously provides low mRNA yields. With the CC& R dinucleotide caps, GTP (and other NTP) starvation is generally not necessary, as the CC& R purification process is specific only for capped product, allowing lower amounts of cap (or higher NTP concentrations) to be used over the relatively inexpensive NTPs, followed by efficient covalent separation of capped vs. uncapped species which is typically not possible with non-covalent purification strategies.

[0161] However, in order to standardize conditions between ARCA and CC& R dinucleotide caps in these studies, and to maintain a high capping efficiency for the legacy capping agent, the GTP “starvation” conditions illustrated above are used in the IVT reactions. Conventional ARCA from the HiScribe® T7 ARCA mRNA Kit is compared to CC& R dinucleotide caps (structures 12 and 30) under identical IVT reaction conditions.

[0162] Post-transcriptional capping does not require a cap analog during IVT, and therefore no GTP “starvation” or significant differences in NTP concentrations are generally required. Due to this, higher NTP concentrations (10 mM each) may be used for high-yield IVT reactions with post-transcriptional capping. In this instance, the CLuc GG control DNA template is used with kit components from the AG kit, with the omission of any capping agent. NTPs are used at 10 mM, andany difference in final volume is made up by reduction of nuclease free water to maintain proper buffer and enzyme concentrations. IVT is performed using otherwise identical conditions to the AG kit to produce uncapped RNA.

[0163] After IVT RNA synthesis, the reaction is purified using the Monarch® Spin RNA Cleanup Kit (NEB Cat # T2050S) to remove unincorporated NTPs and proteins, and eluted in nuclease-free water. RNA is heated at 65 °C for 5 minutes to denature RNA, followed by incubation on ice for 5 minutes. 10X Capping Buffer from the NEB Vaccinia Capping System Kit is added to a final concentration of IX, then split in half. To half of the reaction is added 1 mM (final concentration) GTP to serve as the conventional control. To the other half is added the same final concentration of either CC& R GTP (structure 23 or 26). SAM is added to each reaction at 1 mM. 2 uL Vaccinia Capping Enzyme is added and reactions are incubated at 37°C for 30 minutes to form the Cap-0 structure. Optionally, each Cap-0 sequence can be converted to Cap 1 with 2'-O-Methyltransferase using commercially available kits.Purification of Standard IVT Reactions (Commercially Available Trinucleotide and Dinucleotide Caps, and Post-Transcriptional Capping)

[0164] Following IVT using standard capping reagents or post-transcriptional capping with Vaccinia and GTP, DNA template must be removed prior to use in in vitro or in vivo assays, and especially for therapeutic use. This is accomplished by adding two microliters of RNase-free DNase I and incubating the mixture for fifteen minutes at 37°C (dinucleotide cap) or room temperature (trinucleotide caps). Note that heating the product mixture above room temperature may adversely affect yield and transcript quality of the trinucleotide cap mRNA. After DNase digestion, the mRNA is purified, for example, by lithium chloride precipitation or by silica membrane adsorption (e.g., Quiagen RNeasy Cleanup Kit) and resuspended in nuclease-free water. This purification step removes degraded DNA, unincorporated nucleoside triphosphates, and proteins.

[0165] Next, 5’ triphosphate is removed from uncapped mRNA using Antarctic Phosphatase (AP). AP treatment is required prior to in vivo (especially therapeutic) use to remove the very highly immunogenic 5’ triphosphate group of uncapped mRNA, which would otherwise activate RIG-I, IFIT5, and other intracellularinnate immune sensors. Antarctic Phosphatase Kit (NEB Catalog #: M0289L) is used for removal of 5’-PPP according to kit instructions. Briefly: 10X Antarctic Phosphatase Reaction Buffer is added to a final concentration of IX in 20 uL of mRNA solution. Next, 2 uL of AP at 5,000 units / mL is added and incubated at 30 minutes at 37°C. AP, Pi, and contaminants are removed using, for example, lithium chloride precipitation or silica membrane adsorption (e.g., Quiagen RNeasy Cleanup Kit) and resuspended in nuclease-free water containing 1 mM Sodium Citrate, pH 6.4.

[0166] Purified mRNA concentration for each sample is quantified via UV-Vis spectroscopy at 260 nm, and / or fluorometrically using an RNA-selective quantitation reagent, such as the Qubit™ RNA High-Sensitivity Assay Kit or the Quant-iT™ RiboGreen RNA Reagent, performed according to the manufacturer’s recommendations, prior to analysis.Purification of CC& R IVT Reactions (CC& R Trinucleotide Cap AG analogs, CC& R Dinucleotide Caps, and CC& R Post-Transcriptional Capping)

[0167] Following in vitro transcription, each reaction mixture is diluted two-fold using 200 mM MES Buffered Saline (MBS), pH 4.7 (200 mM MES, 150 mM NaCl, pH 4.7), to reduce the viscosity of the reaction and to equilibrate the mRNA into a buffer compatible with CC& R on hydrazide resin. Total mRNA concentration is then estimated fluorometrically using a highly RNA-selective quantitation reagent, such as the Qubit™ RNA High-Sensitivity Assay Kit or the Quant-iT™ RiboGreen RNA Reagent, each performed according to the manufacturer’s recommendations. These fluorometric assays permit selective quantification of single-stranded RNA in the presence of template DNA, unincorporated NTPs, proteins, and other reaction components, thereby enabling accurate determination of the total mass of CC& R mRNA available for capture.

[0168] Hydrazide functionalized resin (Thermo Scientific Pierce UltraLink™ Hydrazide Resin, Cat #: 53149) is equilibrated in 100 mM MES, 150 mM NaCl, pH 4.7 by washing with 3 - 5 bed volumes of buffer. The equilibrated resin is then combined with each CC& R capped mRNA at a loading density exceeding one milligram of mRNA per milliliter of resin to allow aldehyde cap-bearing transcripts to encounter reactive hydrazide groups on the resin. The suspension isgently mixed via end-over-end mixing, or, alternatively, by slow introduction of the solution to a stationary phase column to promote uniform contact between the aldehyde moiety on the CC& R cap and the hydrazide groups on the resin. Aniline is included during the capture step at a concentration of > 10 mM to catalyze hydrazone formation. Covalent hydrazone formation is allowed to proceed for a period of at least ten minutes at ambient temperature. During this interval, fully capped CC& R mRNA becomes immobilized on the resin through formation of a stable hydrazone linkage, while uncapped RNA, template DNA, proteins, NTPs, and other transcription reaction components remain unbound in either the supernatant or chromatography column flow-through.

[0169] Optionally, prior to application of the IVT reaction to the capture resin, the mRNA may first be desalted using a size-exclusion or silica resin (e.g., Sephadex G-25 or RNeasy) to remove unincorporated CC& R cap, although this step is generally unnecessary as the resin has a sufficient capacity to capture both the unreacted aldehyde-containing cap molecules as well as capped mRNA.

[0170] After completion of the capture step, the resin is washed extensively in a neutral pH buffer (such as PBS) to remove template DNA, nucleoside triphosphates, uncapped mRNA, and proteins. A series of washes is subsequently performed using a low ionic strength buffer at near-neutral pH, such as 1 - 5 mM MOPS, Bis-Tris, Phosphate, HEPES, or another biological buffer possessing a pKa near seven. Each wash is performed using three to five bed volumes of buffer, or with gentle agitation (in batch mode) to ensure complete removal of contaminants. These low ionic strength washes efficiently remove small to medium length double-stranded RNA species, short abortive transcripts, template DNA, unreacted NTPs, and protein components of the IVT mixture.

[0171] Longer double-stranded RNA contaminants which are resistant to separation may require additional washing with lower ionic strength buffers, nuclease-free water, or hybrid-destabilizing solutions (such as urea, guanidinium salts, or even formamide), optionally at elevated temperatures (e.g., 37°C - 70°C) to disrupt the intermolecular interactions between these contaminants and the desired mRNA transcript. These optional elevated temperature washes facilitateremoval of even the most refractory dsRNA contaminants while leaving the desired intact mRNA construct covalently bound to the solid support.

[0172] After complete removal of non-covalently bound impurities, the resinbound mRNA is equilibrated in an appropriate solution (e.g., low ionic strength neutral pH buffer), then is subjected to controlled acidic cleavage to tracelessly remove the cleanly-releasable linker moiety and release the native mRNA molecule into solution. For applications requiring a volatile buffer system, cleavage may be performed using acidic (e.g., pH 4 - 5) aqueous solutions of ammonium acetate, ammonium formate / formic acid, ammonium acetate / acetic acid, ammonium propionate / propionic acid, or other volatile buffer systems known to those skilled in the art. These volatile buffers are particularly advantageous when the resulting mRNA is to be lyophilized and reformulated in a defined buffer or nuclease free water without residual non-volatile salts. Addition of a bulking agent such as tert-butanol may be used to enhance lyophilization characteristics when desired.

[0173] Alternatively, after complete removal of non-covalently bound impurities, the resin-bound mRNA is equilibrated in an appropriate solution (e.g., low ionic strength neutral pH buffer), then is subjected to controlled acidic cleavage using a non-volatile acidic buffer to cleave the cleanly-releasable linker moiety and to tracelessly generate the native capped mRNA directly in the final formulation buffer. Suitable non-volatile buffers include Tris-HCl, sodium acetate, phosphate buffer, MES-buffers, or other suitable buffers with or without saline. Stabilizing excipients may be added at this stage to produce a formulation directly compatible with downstream lipid nanoparticle (LNP) encapsulation.

[0174] Following cleavage, the released native mRNA is collected and the resin is rinsed with an additional volume of the same cleavage buffer to maximize recovery. The combined eluates are clarified by 0.22 pm filtration, as required for sterile handling, after which the mRNA may be further polished by ultrafiltration / diafiltration, size exclusion chromatography, or other methods, if desired, or directly formulated into lipid nanoparticles.

[0175] Based on the underlying chemistry and process design, CC& R purification is expected to obviate the need for an additional poly-dT affinitypurification step. In CC& R workflows, in vitro transcription is conducted under non-limiting nucleotide conditions, thereby avoiding GTP starvation and preserving RNA polymerase processivity through the 3' poly (A) region. mRNA molecules are selectively retained via covalent capture and subjected to stringent washing conditions that remove uncapped transcripts, dsRNA, DNA, proteins, truncated products, short RNA species, and residual reaction components while retaining chemically intact, full-length mRNA. As a result, the recovered mRNA population exhibits high 3 '-end fidelity and poly (A) tail completeness, reducing or eliminating enrichment for partially polyadenylated transcripts. Accordingly, poly-dT purification, which is commonly employed in traditional capping and purification workflows to compensate for poly(A) heterogeneity and truncation, is likely not required for CC& R-purified mRNA, thereby simplifying processing and reducing mRNA loss.

[0176] In certain situations, or in applications requiring extremely high mRNA purity — such as regulated manufacturing workflows for therapeutics — any 3'-end heterogeneity may be addressed through selective capture of polyadenylated transcripts using poly(dT) affinity resin. This step enriches for transcripts containing the full-length encoded poly(A) tail and removes truncated RNAs, prematurely terminated transcripts, and products arising from polymerase fall-off within the template. When such “Ultrapure mRNA” is required, poly(dT) capture may be performed prior to CC& R to ensure that only fully polyadenylated transcripts proceed through that process.

[0177] To perform poly(dT) affinity capture, the IVT reaction is first diluted two- to four-fold in a high-salt hybridization buffer, typically consisting of 0.5-1.0 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA. This buffer promotes stable hybridization of the encoded poly(A) tract to the immobilized oligo(dT) groups on the affinity resin. Poly(dT) resin, supplied either as oligo(dT)-cellulose or as synthetic dT-functionalized magnetic or agarose beads, is equilibrated in the same buffer by sequential washes to remove storage preservatives and ensure optimal binding.

[0178] The diluted mRNA mixture is then applied to the equilibrated poly(dT) resin and incubated for fifteen to thirty minutes at ambient temperature with gentleagitation. During this binding interval, transcripts containing a complete or nearcomplete poly (A) tail hybridize selectively to the oligo(dT) strands, whereas truncated RNA lacking sufficient poly(A) length remains unbound in the supernatant. After hybridization the resin washed, or is separated from the supernatant by centrifugation or magnetic capture, and the unbound fraction is removed. The resin is then washed with ten to twenty bed volumes of high-salt buffer to eliminate non-specifically associated RNA species, partially polyadenylated transcripts, residual template DNA, and short abortive RNAs.

[0179] Elution of fully polyadenylated mRNA is achieved by lowering the ionic strength and raising the temperature to disrupt base-pairing between the poly(A) tail and the oligo(dT) groups. Elution is performed using nuclease-free water or a low-salt buffer such as 10 mM Tris-HCl, pH 7.0, prewarmed to 65-70°C. The resin is incubated for several minutes under these conditions to promote efficient release, after which the eluate is collected. A second elution may be performed to maximize recovery. The combined eluates are rapidly cooled on ice to prevent RNA secondary-structure formation and to protect mRNA integrity. A suitable buffer, such as MES Buffered Saline, pH 4.7, is then added in preparation for CC& R purification as described previously.

[0180] Purified mRNA concentration for each sample is quantified via UV-Vis spectroscopy at 260 nm, and / or fluorometrically using an RNA-selective quantitation reagent, such as the Qubit™ RNA High-Sensitivity Assay Kit or the Quant-iT™ RiboGreen RNA Reagent, performed according to the manufacturer’s recommendations, prior to purity analysis.Analysis of mRNA Purity

[0181] After purification, mRNA is optionally dissolved in an appropriate buffer (if previously lyophilized), especially one suitable for LNP formulation.Quantification of mRNA is performed either by UV absorbance at 260 nm, or by fluorometric RNA quantitation, as described earlier. A number of assays are performed to confirm 5’ cap integrity, determine double- stranded RNA content, residual impurity content (DNA, protein, NTPs and other small molecules), assess mRNA length distribution and transcript heterogeneity, innate immune activation, and mRNA potency and functional quality. These assays are described herein.

[0182] 5' Cap Analysis is performed via LC-MS of digested oligonucleotides to determine the incorporation efficiency of traditional vs. CC& R cap analogs. In this assay, each mRNA preparation is subjected to controlled enzymatic digestion to generate a defined mixture of capped and uncapped 5 '-terminal oligonucleotides, followed by chromatographic separation and mass spectrometric detection of the resulting cap-containing vs. uncapped fragments (Galloway et al. (2020) Open Biology 10, 190306 (doi:10.1098 / rsob.190306); Wang et al. (2019) Nucleic Acids Research 47 (20), el30 (doi:10.1093 / nar / gkz751)). This approach enables quantitative comparison of capping efficiency and cap structure distribution between traditional cap analogs and CC& R caps.

[0183] For cap-end mapping, purified mRNA samples are first exchanged into a volatile, MS-compatible buffer such as 10 mM ammonium acetate, pH 6.8-7.0, using ultrafiltration or desalting columns. The samples are then digested with an endonuclease or combination of nucleases selected to preserve the integrity of the 5' cap structure while cleaving the RNA at internal phosphodiester bonds.Commonly, nuclease Pl or RNase T2 is employed to hydrolyze the RNA into 5'-monophosphate nucleotides and short oligonucleotides, leaving the 5' cap attached to a short RNA segment. Alternatively, a sequence-specific endoribonuclease may be used to generate a defined 5'-terminal fragment of known length. Digestion reactions are incubated at 37 °C for 30-60 minutes under conditions recommended by the enzyme manufacturer, after which the reactions are terminated by heat inactivation of the enzyme or by addition of organic solvent.

[0184] The resulting mixture of nucleotides and capped oligonucleotides is clarified by centrifugation and, if necessary, filtered through a 0.22 pm membrane to remove particulates prior to LC-MS analysis. Chromatographic separation is performed by reverse-phase or hydrophilic interaction chromatography using a column compatible with polar, highly charged analytes. Mobile phases typically consist of aqueous ammonium acetate or ammonium formate (5-20 mM) and an organic phase of acetonitrile or methanol, formulated to be fully volatile for electrospray ionization. A shallow gradient is applied to resolve distinct capcontaining species (e.g., nFGpppN-, nFGpppNN, and the like) from background nucleotides and small oligoribonucleotides.

[0185] Mass spectrometric detection is carried out in negative-ion or positive-ion electrospray mode, depending on the ionization characteristics of the cap structures being studied. Extracted ion chromatograms are generated for each expected cap species based on their exact mass-to-charge ratios (m / z), including diagnostic fragment ions corresponding to methylated guanosine, triphosphate linkages, and proprietary structural features unique to each standard or CC& R cap analog.Calibration curves are prepared using synthetic standards of known cap structures where available, or by using internal standards spiked into the digestion mixtures at defined concentrations.

[0186] Peak areas for each cap-containing oligonucleotide are integrated and normalized either to total RNA input or to a non-capped internal fragment to calculate capping efficiency. The percentage of capped transcripts is determined by comparing the summed signal of all capped 5 '-termini to the combined signal of capped and uncapped 5 '-terminal species. Comparative analyses are performed on mRNA produced with traditional cap analogs and on mRNA produced with CC& R caps under identical IVT and digestion conditions. This LC-MS-based cap analysis thereby provides a sensitive and structurally specific measure of cap incorporation efficiency, cap homogeneity, and the relative distribution of cap species.

[0187] Impurity analysis consists of dsRNA, DNA, residual NTP, and protein quantitation assays. dsRNA ELISA using, e.g., J2 antibody (Schönborn et al. (1991) Nucleic Acids Res. 19 (11), 2993-3000 (doi:10.1093 / nar / 19.11.2993); Holland et al. (2024) Vaccines 12 (8), 899 (doi: 10.3390 / vaccines 12080899)) is considered the industry standard for dsRNA detection and quantification. An ELISA is performed using each traditional mRNA capped and purified mRNA and each CC& R capped and purified mRNA in an ELISA assay using anti-dsRNA antibody J2 conjugated to HRP. The assay is conducted in 96-well high-binding polystyrene plates, which are first equilibrated to ambient temperature. Each well is coated with mRNA sample diluted into carbonate-bicarbonate coating buffer (typically 50 mM, pH 9.6), with sample amounts ranging from 50 to 200 ng per well, depending on assay sensitivity requirements and the relative amount of contaminating dsRNA in each sample. The plates are sealed to prevent evaporationand are incubated for two hours at ambient temperature or overnight at 4°C to ensure efficient adsorption of RNA to the well surface.

[0188] Following adsorption, the wells are washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBS-T) to remove unbound material. Non-specific binding sites are then blocked using 3% bovine serum albumin (BSA) in PBS or an equivalent blocking buffer, with the plates incubated for one hour at room temperature. This blocking step substantially reduces background signal and improves assay linearity across the dynamic range.

[0189] After blocking, the wells are washed again with PBS-T and incubated with the J2 monoclonal anti-dsRNA antibody conjugated to horseradish peroxidase (HRP). The antibody is diluted to the manufacturer-recommended working concentration (typically in the range of 1:2,000 to 1:10,000 in 1% BSA / PBS-T). The plates are incubated for one hour at ambient temperature with gentle agitation to facilitate uniform binding of the antibody to dsRNA structures present in each mRNA preparation. Unbound antibody is subsequently removed by five washes with PBS-T.

[0190] Colorimetric detection is performed by addition of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate to each well. The HRP-mediated oxidation of TMB generates a blue chromophore, the intensity of which is proportional to the amount of dsRNA bound in each well. The reaction is allowed to proceed for a defined period (typically 10-20 minutes, depending on dsRNA content) under subdued light until sufficient color development is observed. The reaction is then quenched by addition of an acidic stop solution, such as 1 N sulfuric acid, converting the chromophore to a yellow end-product. The absorbance at 450 nm is measured immediately using a calibrated microplate reader.

[0191] Quantification is performed using a standard curve prepared from a known dsRNA reference material, such as synthetic polyinosinic-polycytidylic acid [poly(I: C)], serially diluted across the working range of the assay. The absorbance values of the mRNA samples are interpolated against this standard curve to determine the absolute amount of dsRNA contamination in each preparation. Both traditional capped and purified mRNA and CC& R capped and purified mRNA are analyzed in parallel using identical assay conditions. Theresulting dsRNA levels are expressed as nanograms dsRNA per microgram of mRNA or as a percentage of total RNA content, depending on the levels of contaminating dsRNA.

[0192] All samples are assayed in triplicate to ensure statistical robustness, and each experimental run includes negative controls (coating buffer only), positive controls (defined dsRNA standards), and internal process controls (spiked RNA complementary to the mRNA sequence and pre-annealed). The assay provides a sensitive and quantitative measure of dsRNA content across differently capped mRNA preparations and enables direct comparison of dsRNA removal efficiency achieved by the CC& R workflow relative to traditional capping and purification processes.

[0193] ELISA results are further confirmed via Dot Blot with dsRNA-specific antibody and a dsRNA standard as described above. Dot blot analysis is performed as an orthogonal confirmation of dsRNA content in each mRNA preparation. Nitrocellulose membranes (0.45 pm pore size) are pre- wetted briefly in nuclease-free water and subsequently equilibrated in lx SSC buffer (150 mM NaCl, 15 mM sodium citrate, pH 7.0). Serial dilutions of each mRNA sample of known concentration are prepared in nuclease-free water to generate a quantitative loading series. Defined dsRNA standards, such as high-molecular-weight polyinosinic-polycytidylic acid [poly(LC)], are diluted in parallel across a concentration range suitable for calibration, typically from 1 ng / pL down to 10 pg / pL.

[0194] Aliquots (1-2 pL) of each mRNA sample dilution and each poly(I: C) standard are applied directly onto the equilibrated membrane using a micropipette, ensuring even spacing between spots. The membrane is allowed to air dry for ten to fifteen minutes, during which the RNA becomes immobilized through hydrophobic and electrostatic interactions with the nitrocellulose matrix. After drying, the membrane is immersed in blocking buffer consisting of 5% (w / v) nonfat dry milk or 3% (w / v) BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST), and incubated for one hour at ambient temperature with gentle agitation. This blocking step is necessary to minimize non-specific binding of the antibody and reduce background noise during detection.

[0195] Following blocking, the membrane is washed briefly in TBST and then incubated with the dsRNA-specific monoclonal antibody J2. The antibody is diluted into TBST containing 1% BSA at the manufacturer’s recommended working concentration (typically 1:1,000 to 1:5,000). The membrane is incubated with the primary antibody for one hour at ambient temperature or overnight at 4°C to permit selective binding of the antibody to dsRNA structures present in each spotted sample. Unbound antibody is removed by three to five sequential washes in TBST.

[0196] A secondary antibody conjugated to horseradish peroxidase (HRP), typically rabbit or goat anti-mouse IgG-HRP, is applied following dilution into TBST containing 1% BSA. The membrane is incubated for thirty to sixty minutes at room temperature, after which it is washed extensively with TBST to remove unbound secondary antibody-HRP conjugate.

[0197] Following incubation with the secondary antibody-HRP conjugate, and extensive washing to remove unbound reagent, the membrane is developed using a chromogenic peroxidase substrate. A solution of 3,3'-diaminobenzidine (DAB) or 4-chloronaphthol (4CN) is freshly prepared according to the manufacturer’s instructions and applied to the membrane until visible signal develops. The HRP-conjugated secondary antibody catalyzes local deposition of an insoluble colored precipitate at the site of dsRNA-J2 antibody binding, yielding discrete and sharply defined spots corresponding to each RNA sample and poly(I: C) standard. Color development is monitored continuously and is stopped by rinsing the membrane in deionized water once optimal contrast is achieved. The dried membrane is then imaged using standard gel or blot documentation equipment, and spot intensities are quantified by densitometry. The chromogenic approach provides a stable, nonfading signal that permits archiving and reanalysis of dsRNA dot blot results while maintaining high sensitivity and compatibility with quantitative comparison against the poly(I: C) calibration series.

[0198] This dot blot assay provides a qualitative and semi-quantitative confirmation of the ELISA-derived dsRNA measurements. Because the J2 antibody recognizes double- stranded RNA structures independent of nucleotide sequence, the dot blot serves as a robust orthogonal method for validating dsRNAremoval during mRNA purification and for comparing dsRNA levels between traditional capped and purified mRNA samples and CC& R capped and purified mRNA.

[0199] Residual DNA in each sample is quantified using qPCR and / or digital droplet PCR. This assay can detect pg-fg levels of residual DNA in the final mRNA product. Residual template DNA is quantified using quantitative PCR (qPCR) and, when enhanced sensitivity is required, digital droplet PCR (ddPCR) (Sanderson et al. (2022) Vaccine 40 (12), 1813-1821 (doi:10.1016 / j.vaccine.2022.01.040); Hindson et al. (2011) Analytical Chemistry 83 (22), 8604—8610 (doi:10.1021 / ac202028g)). These assays are performed to determine the presence of trace DNA impurities remaining after IVT and subsequent purification steps. Both qPCR and ddPCR enable detection of DNA in the picogram to femtogram range, thereby providing a sensitive measure of DNA contamination in the final mRNA product.

[0200] For qPCR analysis, primers are designed to amplify a defined region of the template DNA, typically a 100-200 bp amplicon positioned within the coding region or 5' UTR to ensure detection of any residual full-length or fragmented template. A standard curve is generated using serial ten-fold dilutions of the linearized DNA template, spanning at least six orders of magnitude and including concentrations down to 1 fg / pL. Each mRNA sample is optionally treated with RNase A to remove potential interference from RNA secondary structures, followed by heat denaturation at 95 °C for two minutes to disrupt RNA-DNA hybrids. Aliquots of each sample are added to a qPCR master mix containing DNA polymerase, dNTPs, MgCl₂. primers, and a double-stranded DNA-intercalating fluorescent dye such as SYBR Green I. Reactions are assembled in triplicate in optical qPCR plates and sealed to prevent evaporation.

[0201] qPCR is performed using a thermocycler equipped with real-time fluorescence detection. The cycling protocol typically consists of initial denaturation at 95°C, followed by 35-40 cycles of denaturation, annealing, and extension using cycling times appropriate for the selected amplicon. Fluorescence is measured at the end of each extension step, and quantification cycle (Cq / Ct) values are calculated automatically by the instrument software. Ct values for eachmRNA sample are interpolated against the DNA standard curve to determine the absolute amount of DNA present. The detection limit routinely reaches the low femtogram range per reaction, permitting sensitive quantification of residual template DNA in the purified mRNA preparations.

[0202] Alternatively, when maximum sensitivity is required, residual DNA is quantified using digital droplet PCR. For ddPCR, the same primer set and amplicon are used, but reactions are partitioned into thousands of nanoliter droplets using a microfluidic droplet generator. Each droplet acts as an individual PCR microreactor, increasing quantification precision and enabling absolute measurement without reliance on a standard curve. The droplet-partitioned reactions are thermocycled under conditions similar to qPCR. Following amplification, the droplets are passed through a droplet reader that records the fluorescent signal from each droplet. Positive droplets contain amplified DNA, whereas negative droplets do not. The concentration of template DNA is calculated using Poisson statistical correction to account for the probability of more than one DNA molecule being present within a single droplet.

[0203] The resulting DNA concentration is expressed either as picograms of DNA per microgram of mRNA or as copies of template DNA per microgram of RNA. Internal negative controls (no-template controls) and positive controls (known template concentrations) are included in every run to monitor assay performance and ensure specificity. These assays confirm the extent to which each workflow removes template DNA and validate that CC& R purification effectively eliminates DNA, likely without the need for DNase treatment.

[0204] Protein impurities are assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining to enable sensitive visualization of protein contaminants derived from the IVT reaction mixture, such as T7 RNA polymerase, RNase inhibitors (if used), and other enzymatic components (Pardi et al. (2018) Nature Reviews Drug Discovery 17 (4), 261-279 (doi:10.1038 / nrd.2017.243)). The analysis is performed using precast polyacrylamide gels (typically 4-12% or 10-20% gradient gels), which are equilibrated to ambient temperature prior to sample loading. Each mRNA preparation is diluted into SDS sample buffer containing 2% SDS, 50 mM Tris-HC1 (pH 6.8), 10% glycerol, and 0.01% bromophenol blue, with 50-100 mM dithiothreitol (DTT) or 0-mercaptoethanol added as a reducing agent. The samples are heated at 70-95 °C for five minutes to ensure complete denaturation of proteins and disruption of quaternary structures.

[0205] Following denaturation, equal volumes corresponding to 100-300 ng of total protein (estimated by micro-BCA assay vs. appropriate protein controls) are loaded into individual wells, alongside a prestained molecular weight ladder. Electrophoresis is performed in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at a constant voltage, typically 120-150 V, until the dye front reaches the lower edge of the gel.

[0206] Upon completion of electrophoresis, the gel is fixed immediately in a solution containing 50% methanol and 10% acetic acid for thirty minutes with gentle agitation. The gel is then rinsed sequentially with methanol and deionized water to remove fixative and prepare the gel for sensitization. A sensitizing solution, typically 0.02% sodium thiosulfate, is applied for one minute and the gel is washed thoroughly with deionized water.

[0207] Silver staining is performed using a standardized silver nitrate-based kit (such as the SilverQuest™ Silver Staining Kit; ThermoFisher Scientific Cat #: LC6070) or manually prepared reagents. The gel is incubated in 0.1% silver nitrate solution for twenty minutes at ambient temperature, followed by brief rinsing to remove unbound silver ions. Development is initiated by immersion in a solution containing sodium carbonate (e.g., 2%) with a trace amount of formaldehyde (0.05-0.1 %), which reduces silver ions bound to protein sites and generates visible darkened bands. The gel is monitored continuously during development; once signal intensity reaches the desired level, the reaction is quenched using 5% acetic acid to halt further silver reduction and to stabilize the image.

[0208] Bands corresponding to protein impurities present in each mRNA sample are visualized and documented using a gel imaging system. Band intensities may be quantified using densitometry software to compare the relative abundance of protein contaminants remaining after purification as compared to positive control bands consisting of known amounts of protein. Traditional capped and purified mRNA samples and CC& R capped and purified mRNA samples are analyzed sideby side under identical electrophoretic and staining conditions, along with positive controls to allow quantification. This assay provides a highly sensitive measure of protein impurity levels, with detection sensitivities down to the low-ng range.

[0209] NTPs and other small molecule impurities are assayed by HPLC (anion-exchange or reverse-phase) and / or LC-MS small-molecule panel to detect trace NTPs, cap analogs, pyrophosphate, and abortive RNAs. These contaminants are analyzed using high-performance liquid chromatography (HPLC) and, when higher specificity is required, liquid chromatography-mass spectrometry (LC-MS). These analyses confirm removal of non-volatile or reactive small molecules and provide an orthogonal measure of the purification efficiency achieved by traditional workflows versus CC& R purification (Basila et al. (2021) Mol. Ther. Nucleic Acids 24, 1013-1025 (doi:10.1016 / j.omtn.2021.04.015); Guimaraes et al. (2024) Mass Spectrom. Rev. 43 (5), 1066-1090 (doi:10.1002 / mas.21856)).

[0210] For anion-exchange HPLC analysis, purified mRNA samples are diluted into a low-ionic-strength buffer compatible with the stationary phase, typically 20 mM Tris-HCl, pH 8.0. A strong anion-exchange column (e.g., quaternary amine functionalized column) is equilibrated in buffer A (20 mM Tris-HCl, pH 8.0), and elution is performed using a linear gradient of buffer B (20 mM Tris-HCl containing 1 M NaCl). The chromatographic method is optimized to achieve baseline separation of ATP, GTP, CTP, UTP, their corresponding diphosphates and monophosphates, inorganic pyrophosphate, and any remaining cap analogs. Detection is performed by UV absorbance at 254 nm, which selectively monitors aromatic nucleobases and phosphorylated nucleotides. Retention times for each species are confirmed using authentic standards prepared at known concentrations. Integration of the chromatographic peaks permits quantification of residual NTPs and cap analogs in each sample relative to an external calibration curve.

[0211] Reverse-phase HPLC is similarly performed using a C8 or polymer-based reversed-phase column capable of resolving small polar molecules under ion-pairing conditions. Typical eluents include aqueous ammonium acetate or ammonium formate (pH 6.5-7.0) combined with increasing concentrations of acetonitrile. Ion-pairing reagents such as triethylammonium acetate (TEAA) may be included to improve retention of highly polar nucleotides. UV detection at 254nm is employed to distinguish residual NTPs, abortive RNAs of <10 nucleotides, and cap analogs, each of which displays characteristic retention behavior under these conditions. This method provides a complementary measure of smallmolecule removal that is directly comparable across mRNA preparations.

[0212] For LC-MS analysis, samples are diluted into a volatile buffer system compatible with electrospray ionization, such as 5-10 mM ammonium acetate or ammonium formate. A reversed-phase column or hydrophilic interaction chromatography (HILIC) column is used depending on the polarity of the analytes. The chromatographic gradient is optimized to separate NTPs, cap analogs, pyrophosphate, nucleotide degradation products, and short abortive transcripts. Mass spectrometric detection is performed in negative-ion mode for nucleotide phosphates and in positive-ion mode for cap analogs when appropriate. Extracted ion chromatograms corresponding to exact m / z values of the analytes are used for quantification, with calibration performed using serial dilutions of authentic standards. This method provides structural confirmation of the identity of each impurity and affords sensitivity into the low-femtomole range.

[0213] The results from HPLC and LC-MS small-molecule analyses are used to identify and quantify the level of unreacted nucleotides, residual cap structures, and degradative byproducts. Traditional capped and purified mRNA samples and CC& R capped and purified mRNA samples are analyzed in parallel. The extent of removal of small-molecule contaminants is expressed either as molar concentration of impurity per microgram of mRNA or as percent impurity relative to total smallmolecule content.

[0214] Quantitative assessment of mRNA length distribution and transcript heterogeneity is performed using ion-pair reverse-phase high-performance liquid chromatography (IP-RP-HPLC). This method permits high-resolution separation of full-length mRNA from truncated species, short abortive transcripts generated during promoter escape, read-through transcripts produced when transcription terminates beyond the encoded poly(A) tail, double-stranded RNA species, and capped versus uncapped variants. The assay provides a chromatographic fingerprint of transcript integrity and is used to compare the quality of mRNAgenerated using traditional capping and purification workflows with that produced using CC& R.

[0215] Samples are first diluted into a volatile, ion-pair reagent-containing mobile phase to ensure compatibility with chromatographic separation. A typical mobile phase system consists of mobile phase A (100 mM triethylammonium acetate [TEAA], pH adjusted to 7.0) and mobile phase B (TEAA in 50%-60% acetonitrile), although alternative ion-pair reagents such as hexylammonium acetate or dibutylammonium acetate may be employed depending on column chemistry. The use of TEAA as an ion-pairing agent enhances retention of the polyanionic RNA on the hydrophobic stationary phase and enables resolution of RNA species differing in length by as little as 10-20 nucleotides.

[0216] Chromatography is performed on a C18 reversed-phase column with wide-pore geometry (typically 300 A) to accommodate the large hydrodynamic volume of long mRNA molecules. The column is equilibrated in mobile phase A at ambient or slightly elevated temperature. Elevation of column temperature to 50-60°C is often employed to reduce RNA secondary structure and improve peak shape, while maintaining RNA stability.

[0217] Each mRNA sample is injected at a defined mass load, typically 0.5-2.0 pg per injection, and eluted using a shallow linear gradient of mobile phase B over 20-40 minutes. The gradient slope is optimized to achieve baseline separation between full-length mRNA and shorter or longer RNA species. Elution is monitored at 260 nm, allowing detection of all nucleic-acid-containing species. Full-length mRNA elutes as the dominant peak, whereas tmneated RNAs, abortive transcripts, dsRNA species, and read-through transcripts appear as earlier or later eluting minor peaks depending on their hydrophobicity and degree of secondary structure.

[0218] Peak identification is facilitated by comparison with control transcripts of known lengths or by fraction collection followed by denaturing agarose gel electrophoresis or LC-MS analysis for confirmation. Peaks corresponding to truncated transcripts typically appear earlier in the gradient, whereas read-through transcripts, which possess extended 3' sequences, elute later. dsRNA contaminantsoften display broadened or asymmetric peaks due to increased rigidity and altered interaction with the stationary phase.

[0219] Chromatographic peaks are integrated to quantify the relative abundance of each transcript species. The percentage of full-length mRNA is calculated as the ratio of the full-length peak area to total peak area. The abundance of truncated species, abortive transcripts, and dsRNA contaminants is similarly quantified. These values provide a direct measure of the heterogeneity present in each mRNA preparation, enabling comparison across purification workflows. CC& R-purified mRNA typically exhibits reduced levels of dsRNA and fewer truncated transcripts relative to traditional capping and purification workflows, consistent with selective covalent capture and extensive washing steps.

[0220] This IP-RP-HPLC method thereby provides a robust and quantitative assay of transcript integrity, capturing subtle variations in mRNA length, capping heterogeneity, and dsRNA contamination that are not readily observed by electrophoretic methods alone.

[0221] Innate immune activation potential of each mRNA preparation may be evaluated using a cell-based interferon response assay calibrated to a defined polyinosinic-polycytidylic acid |poly(l: C) ] standard. This assay measures induction of type I interferon-stimulated signaling pathways upon exposure of mammalian cells to the purified mRNA samples (Alexopoulou et al. (2001) Nature 413, 732-738 (doi: 10.1038 / 35099560); Kariko et al. (2005) Immunity 23 (2), 165-175 (doi:10.1016 / j.immuni.2005.06.008)). The method serves as a functional readout of dsRNA contamination and innate immune stimulatory motifs within the RNA population and allows direct comparison of immune activation triggered by traditionally capped and purified mRNA versus CC& R capped and purified mRNA.

[0222] Human embryonic kidney (HEK293), THP-1 monocyte-derived, or other interferon-responsive reporter cell lines are seeded into 96- well tissue culture plates at a density of 5 x 104to 1 x 10scells per well and allowed to adhere overnight in complete growth medium (e.g., DMEM supplemented with 10% fetal bovine serum and antibiotics). For reporter-based assays, cells stably expressing luciferase, secreted alkaline phosphatase (SEAP), or similar interferon-stimulatedresponse element (ISRE) reporters are used to enable quantifiable measurement of pathway activation.

[0223] On the day of the assay, mRNA samples are diluted into serum-free medium or Opti-MEM® reduced-serum medium and formulated with a transfection reagent that facilitates cell uptake at a defined charge ratio. Typical formulations use lipid-based transfection reagents at final mRNA loads of 5-100 ng per well, depending on the dynamic range of the reporter system. Each mRNA preparation is evaluated across a titration series to determine dose-dependent changes in interferon activation. In parallel, a poly(I: C) standard curve is prepared using serial dilutions spanning three to four orders of magnitude, providing a calibration series for quantitative comparison of immune activation potency.

[0224] Cells are incubated with the mRNA-lipid complexes or poly(EC) controls for a defined exposure period, typically six to twenty-four hours, under standard culture conditions (37°C, 5% CO2). During this interval, dsRNA-associated molecular patterns present within the mRNA preparations engage pattern recognition receptors (e.g., MDA5, RIG-I, or TLR3), initiating downstream signaling cascades that culminate in interferon-stimulated transcriptional activation and reporter output.

[0225] Following incubation, reporter activity is measured according to the manufacturer’s instructions. For luciferase reporters, cells are lysed, and luminescence is quantified using a luminometer with appropriate integration times. For SEAP-based reporters, conditioned medium is transferred to a secondary plate containing a chromogenic or fluorogenic phosphatase substrate, and signal development is quantified spectrophotometrically or fluorometrically. For cytokine assays, such as IFN-f> ELISA, conditioned supernatant is collected and analyzed using a commercial sandwich ELISA kit.

[0226] Interferon response values for each mRNA sample are normalized to background signal from untreated cells and plotted against the poly(EC) calibration curve. The magnitude of activation induced by each mRNA preparation is expressed as “poly(EC) equivalent potency,” providing a quantitative measure of innate immune stimulation attributable to dsRNA contaminants or structural features within the mRNA itself. Assays are performed in biological duplicates ortriplicates, with appropriate negative controls (transfection reagent only, mock RNA) included in each experiment.

[0227] This cell-based assay provides a functional orthogonal assessment of innate immune activation and complements quantitative dsRNA measurements obtained by ELISA and dot blot. Reduced interferon stimulation confirms the removal of immunostimulatory RNA species during covalent capture and release, thereby demonstrating improved product quality and reduced innate immune activation potential.

[0228] Finally, mRNA potency and functional quality are assessed using in vitro translation systems based on the Cypridina luciferase (CLuc) bioluminescent reporter mRNA, providing a highly sensitive, quantitative functional readout of translational performance (Thompson et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94 (17), 862-867 (doi: 10.1073 / pnas.94.17.862); Kariko et r? / . (2011) Molecular Therapy 19 (9), 1624-1631 (doi:10.1038 / mt.2011.150)). These assays directly evaluate the ability of each mRNA preparation to direct protein synthesis and thereby report on critical transcript attributes including 5' cap integrity, 5'-UTR structure, poly(A) tail functionality, nucleotide composition, and overall structural quality.

[0229] In vitro translation systems such as rabbit reticulocyte lysate, HeLa cell extract, and other mammalian cell-free translation platforms are suitable for this purpose and enable direct comparison of mRNA produced using conventional capping and purification workflows versus CC& R purified mRNA. The use of CLuc, a naturally secreted bioluminescent enzyme, affords exceptional sensitivity and a broad dynamic range without the need for external excitation, minimizing background signal and assay interference.

[0230] For rabbit reticulocyte lysate assays, translation reactions are assembled according to manufacturer specifications, typically in 10-20 pL reaction volumes containing nuclease-treated lysate, a complete amino acid mixture, supplemental potassium and magnesium salts, and RNase inhibitors as appropriate. mRNA samples are diluted in nuclease-free water and added to the translation mixture at defined concentrations (commonly 25-250 ng per reaction), with each preparation evaluated across a concentration series to enable dose-response analysis. Reactionsare incubated at 30 °C for 60-120 minutes, during which CLuc protein is synthesized in proportion to the translational competence of the input mRNA.

[0231] For HeLa cell extract or alternative mammalian translation systems, reactions are assembled in an analogous manner, with buffer composition and energy-regenerating components adjusted to meet the requirements of the specific lysate. These systems provide a more physiologically relevant translational environment and are particularly sensitive to deficiencies in mRNA capping efficiency, 5'-end heterogeneity, internal secondary structure, and poly(A)-dependent stability.

[0232] Following completion of the translation reaction, CLuc activity is quantified by addition of Cypridina luciferin substrate and measurement of emitted light using a luminometer or luminescence-capable microplate reader. Because CLuc catalyzes a bioluminescent reaction without the need for excitation light, background signal is minimal, enabling detection of subtle differences in translational efficiency between mRNA preparations. Luminescence values are recorded as relative light units (RLU) and arc directly proportional to the amount of functional CLuc protein produced.

[0233] For each translation system, negative control reactions lacking mRNA and positive controls comprising a well-characterized reference CLuc mRNA are included to verify assay performance and ensure inter-assay comparability.Quantitative potency values are calculated by normalizing luminescent signal to the amount of input mRNA, yielding a measure of translational efficiency expressed as RLU per nanogram of mRNA. These normalized values permit direct comparison of functional potency across mRNA preparations.

[0234] Increased CLuc activity observed for CC& R-purified mRNA typically correlates with enhanced capping of purified mRNA, reduced double- stranded RNA contamination, and improved transcript integrity, thereby providing a sensitive functional confirmation of the biochemical and structural quality attributes measured by complementary analytical assays. Collectively, this CLuc-based in vitro translation assay provides a robust and highly sensitive functional endpoint for mRNA performance and serves as an essential component of a comprehensive analytical framework for evaluating mRNA quality and potency.

[0235] All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein.

[0236] While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined by reference to the appended claims, along with their full scope of equivalents.

Claims

What is Claimed is:

1. A compound having a structure:, or a chemically acceptable salt thereof;wherein R1, R2, R4, R7, and R8is each independently -H, -alkyl, or a cleanly-releasable linker moiety;R3is -H, a nucleotide residue, or an oligonucleotide residue; andB1is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue;wherein the compound comprises at least one cleanly-releasable linker moiety; and wherein the at least one cleanly-releasable linker moiety comprises a selectively-reactive linker moiety.

2. The compound of claim 1, wherein R2is -CH.

3. The compound of claim 1, wherein B1is an adenine or guanine residue.

4. The compound of claim 1, wherein R1, R2, R4, R7, and R8is each independently -H, -CH3, or the cleanly-releasable linker moiety.

5. The compound of claim 1 having a structure:OOO7N PH3R! J N+H F / \\ O O O TNN I-O-I1j’1-O-I1j’1-O-I1j’1-O B1h~N o o oOR1OR2OR3OR4, or a chemically acceptable salt thereof;wherein T’ is the cleanly-releasable linker moiety.

6. The compound of claim 5, wherein R2is -CH3.

7. The compound of claim 5, wherein B1is an adenine or guanine residue.

8. The compound of claim 5, wherein R1, R2, R4, R7, and R8is each independently -H or -CH3.

9. The compound of claim 1 having a slruclure:salt thereof;wherein R5is -H, a nucleotide residue, or an oligonucleotide residue;R6and R9is each independently -H, -alkyl, or a cleanly-releasable linker moiety; andB2is a nucleobase residue, a protected nucleobase residue, or a modified nuclcobasc residue.

10. The compound of claim 9, wherein R2is -CH3.

11. The compound of claim 9, wherein B1is an adenine or guanine residue.

12. The compound of claim 9, wherein R1, R2, R4, R6, R7, R8, and R9is each independently -H, -CH3, or the cleanly-releasable linker moiety.

13. The compound of claim 1 having a structure:acceptable salt thereof;wherein R5is -H, a nucleotide residue, or an oligonucleotide residue;R6and R9is each independently -H, -alkyl, or a cleanly-releasable linker moiety; andB2is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue.

14. The compound of claim 13, wherein R1, R2, R4, R6, R7, R8, and R9is each independently -H, -CH3, or the cleanly-releasable linker moiety.

15. The compound of claim 13 having a structure:R8‘.10 gMOacceptable salt thereof;wherein T’ is the cleanly-releasable linker moiety.

16. The compound of claim 13 having a structure:OR5or, or a chemically acceptable salt thereof;wherein T’ is the cleanly-releasable linker moiety.

17. The compound of claim 15, wherein R2is -CH3.

18. The compound of claim 15, wherein R1, R2, R4, R7, and R8is each independently -H or -CH3.

19. The compound of any one of claims 1-18, wherein the cleanly-releasable linker moiety comprises a stimulus-cleavable linker moiety, a self-immolative spacer moiety, or a modified trityl group.

20. The compound of claim 19, wherein the cleanly-releasable linker moiety comprises a photo-cleavable linker, a pH-cleavable linker, a linker cleavable by a reactive oxygen species (ROS), or a redox-cleavable linker.

21. The compound of claim 19, wherein the cleanly-releasable linker moiety comprises the stimulus-cleavable linker moiety and the modified trityl group.

22. 1’he compound of claim 19, wherein the cleanly-releasable linker moiety comprises the stimulus-cleavable linker moiety, the self-immolative spacer moiety, and the modified trityl group.

23. The compound of claim 19, wherein the cleanly-releasable linker moiety has a structure:Ph P ih" 'Ph,>xC',wherein L’ comprises the selectively-reactive linker moiety, Ph is an optionally substituted phenylene moiety, each Ph’ is independently an optionally substituted phenyl group, and C’ is a connecting group or a bond.

24. The compound of claim 19, wherein each Ph’ is independently substituted with one or more C1-C4 alkoxy groups, one or more C1-C4 alkyl groups, or a combination of C1-C4 alkoxy groups and C1-C4 alkyl groups.

25. The compound of claim 19, wherein C’ comprises a carbonate residue, a carbamate residue, an optionally substituted straight-chain or branched C1-C10 alkylene, wherein each carbon atom is optionally substituted with a heteroatom, or is a bond.oO26. The compound of claim 19, wherein C’ comprisesH27. The compound of claim 24, wherein the cleanly-releasable linker moiety has a structure:wherein each M’ is independently a C1-C4 alkoxy group, a C1-C4 alkyl group, -H, or a combination thereof.

28. The compound of claim 27, wherein at least one M’ is a methoxy group.

29. The compound of claim 28, wherein the cleanly-releasable linker moiety has a structure:

30. The compound of claim 29, wherein the cleanly-releasable linker moiety has a structure:H3CO31. The compound of any one of claims 1-30, wherein the selectively-reactive linker moiety comprises a reactive carbonyl group, a reactive oxyamino group, a reactive hydrazino group, a component of a click reaction, or a derivative of any thereof.

32. The compound of claim 31, wherein the selectively-reactive linker moietyOor a derivative of any thereof.

33. A method of preparing a capped mRNA comprising the steps of:a) providing the modified mRNA cap compound of any one of claims 1-32; andb) generating an mRNA comprising the modified mRNA cap compound.

34. The method of claim 33, wherein the generating step comprises a co- transcriptional capping step.

35. The method of claim 33, wherein the generating step comprises a post- transcriptional capping step.

36. The method of claim 33, further comprising the step of:reacting the mRNA comprising the modified mRNA cap compound with a solid support comprising a reactive oxyamino group, a reactive hydrazino group, a reactive carbonyl group, or a component of a click reaction to selectively bind the mRNA comprising the modified mRNA cap compound to the solid support.

37. The method of claim 36, further comprising the step of washing the solid support.

38. The method of claim 37, further comprising the step of releasing a selectively-bound mRNA comprising the modified mRNA cap compound from the solid support.

39. The method of claim 38, wherein the releasing step cleanly releases the cleanly-releasable linker moiety from the modified mRNA cap.

40. The method of claim 36, wherein the selectively-reactive linker moiety comprises a reactive carbonyl group, a reactive oxyamino group, a reactive hydrazino group, a component of a click reaction, or a derivative of any thereof.

41. The method of claim 40, wherein the selectively-reactive linker moiety, or a derivative of any thereof.

42. A compound having a structure:o o o R8Ho-ij’-o-ij’-o-ij’-o — i B1Oo o o1OR3OR4,or achemically acceptable salt thereof;wherein R3, R4, and R8is each independently -H, -alkyl, or a cleanly-releasable linker moiety; andB1is a nucleobase residue, a protected nucleobase residue, or a modified nucleobase residue;wherein the compound comprises at least one cleanly-releasable linker moiety; and wherein the at least one cleanly-releasable linker moiety comprises a selectively -reactive linker moiety.

43. The compound of claim 42, wherein B1is an adenine or guanine residue.

44. The compound of claim 42, wherein R3, R4, and R8is each independently - H, -CH3, or the cleanly-releasable linker moiety.

45. The compound of claim 42 having a structure:O O O HO-fj’-O-lj’-O-fj’-O B1O- O OOR3OT'O O O T'Ho-ij’-o-ij’-o-ij’-oOo o oOR3OR4, or a chemically acceptable salt thereof;wherein T’ is the cleanly-releasable linker moiety.

46. The compound of claim 45, wherein B1is an adenine or guanine residue.

47. The compound of claim 45, wherein R3, R4, and R8is each independently -H or -CH3.

48. The compound of claim 42 having a structure:or a chemically acceptable salt thereof.

49. The compound of claim 48, wherein R3, R4, and R8is each independently -H, -CH3, or the cleanly-releasable linker moiety.

50. The compound of claim 42 having a structure:, or a chemically acceptable salt thereof;wherein T’ is the cleanly-releasable linker moiety.

51. The compound of claim 50, wherein R3, R4, and R8is each independently -H or -CH3.

52. The compound of any one of claims 42-51, wherein the cleanly-releasable linker moiety comprises a stimulus-cleavable linker moiety, a self-immolative spacer moiety, or a modified trityl group.

53. The compound of claim 51, wherein the cleanly-releasable linker moiety comprises a photo-cleavable linker, a pH-cleavable linker, a linker cleavable by a reactive oxygen species (ROS), or a redox-cleavable linker.

54. The compound of claim 52, wherein the cleanly-releasable linker moiety comprises the stimulus-cleavable linker moiety and the modified trityl group.

55. The compound of claim 52, wherein the cleanly-releasable linker moiety comprises the stimulus-cleavable linker moiety, the self-immolative spacer moiety, and the modified trityl group.

56. The compound of claim 52, wherein the cleanly-releasable linker moiety has a structure:Ph'Ph iPh’> XC'x^wherein L’ comprises the selectively-reactive linker moiety, Ph is an optionally substituted phenylene moiety, each Ph’ is independently an optionally substituted phenyl group, and C’ is a connecting group or a bond.

57. The compound of claim 52, wherein each Ph’ is independently substituted with one or more C1-C4 alkoxy groups, one or more C1-C4 alkyl groups, or a combination of C1-C4 alkoxy groups and C1-C4 alkyl groups.

58. The compound of claim 52, wherein C' comprises a carbonate residue, a carbamate residue, an optionally substituted straight-chain or branched C1-C10 alkylene, wherein each carbon atom is optionally substituted with a heteroatom, or is a bond.oNAO59. The compound of claim 52, wherein C’ comprisesH60. The compound of claim 57, wherein the cleanly-releasable linker moiety has a structure:wherein each M’ is independently a C1-C4 alkoxy group, a C1-C4 alkyl group, -H, or a combination thereof.

61. The compound of claim 60, wherein at least one M’ is a methoxy group.

62. The compound of claim 61, wherein the cleanly-releasable linker moiety has a structure:

63. The compound of claim 62, wherein the cleanly-releasable linker moiety has a structure:

64. The compound of any one of claims 42-63, wherein the selectively-reactive linker moiety comprises a reactive carbonyl group, a reactive oxyamino group, a reactive hydrazino group, a component of a click reaction, or a derivative of any thereof.

65. The compound of claim 64, wherein the selectively-reactive linker moiety H O S AN'NHa ZYN'NH2ANAN, NH2ANAN, NH2comprises:H, O H H H HOoH or a derivative of any thereof.

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