Separation and purification of short-, medium-, and long-stranded RNAs by RP-HPLC using different mobile phases and C18 columns with various pore sizes.

Nucleic acids, which have been employed in medicines for various diseases, are attracting attention as a new pharmaceutical model. Depending on the target substances, nucleic acid medicines with various nucleic acid chain lengths (several tens of nucleotides [nt] to several thousands of nt) exist. The purification of synthesized nucleic acids is crucial as various impurities remain in the crude product after synthesis. Presently, reversed-phase high-performance liquid chromatography (RP-HPLC) represents an effective purification method for nucleic acids. However, the information regarding the HPLC conditions for separating and purifying nucleic acids of various chain lengths is insufficient. Thus, this technical note describes the separation and purification of short-, medium-, and long-stranded nucleic acids (several tens of nt to thousands of nt) by RP-HPLC with various mobile phases and octadecyl-based columns with various pore sizes, such as normal (9-12 nm), wide (30 nm), and super wide (>30 nm) pores.

Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores.

Messenger ribonucleic acids (mRNAs) have been used in vaccines for various diseases and are attracting attention as a new pharmaceutical paradigm. The purification of mRNAs is necessary because various impurities, such as template DNAs and transcription enzymes, remain in the crude product after mRNA synthesis. Among the various purification methods, reversed-phase high-performance liquid chromatography (RP-HPLC) is currently attracting attention. Herein, we optimized the pore size of the packing materials, the mobile phase composition, and the temperature of the process; we also evaluated changes in the separation patterns of RNA strands of various lengths via RP-HPLC. Additionally, single-stranded (50-1000 nucleotides in length) and double-stranded (80-500 base pairs in length) RNAs were separated while their non-denatured states were maintained by performing the analysis at 60 °C using triethylammonium acetate as the mobile phase and octadecyl-based RNA-RP1 with super-wide pores (> 30 nm) as the column. Furthermore, impurities in a long-stranded RNA of several thousand nucleotides synthesized by in vitro transcription were successfully separated using an RNA-RP1 column. The columns used in this study are expected to separate various RNA strands and the impurities contained in them.

Separation and identification of the DL-forms of short-chain peptides using a new chiral resolution labeling reagent.

There are several reports of D-amino acids being the causative molecules of serious diseases, resulting in the formation of, for example, prion protein and amyloid ß. D-Amino acids in peptides and proteins are typically identified by sequencing each residue by Edman degradation or by hydrolysis with hydrochloric acid for amino acid analysis. However, these approaches can result in racemization of the L-form to the D-form by hydrolysis and long pre-treatment for hydrolysis. To address these problems, we aimed to identify the DL-forms of amino acids in peptides without hydrolysis. Here, we showed that the DL-forms in peptides which are difficult to separate on a chiral column can be precisely separated by labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamine-amide (D-FDLDA). Additionally, the peptides could be quantitatively analyzed using the same labeling method as for amino acids. Furthermore, the detection sensitivity of a sample labeled with D-FDLDA was higher than that of the conventional reagents Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (L-FDAA) and Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (L-FDLA) used in Marfey's method. The proposed method for identifying DL-forms of amino acids in peptides is a powerful tool for use in organic chemistry, biochemistry, and medical science.