COSMOSIL RNA Series

RNA-based therapeutics represent a powerful new tool for gene therapy. In RNA production, impurities such as nucleotide triphosphates, abortive transcripts, and DNA templates can affect downstream applications by triggering immune responses. Gel-based methods of purification, such as preparative denaturing polyacrylamide gel electrophoresis (PAGE) and agarose gels, are limited by poor resolution of RNA size and potential chemical modifications from reagents such as formaldehyde. To date, high performance liquid chromatography (HPLC) remains a staple method for purification of messenger RNA (mRNA) and RNA oligonucleotides. Presented here are two HPLC methods: a size exclusion-based method for the purification of long mRNA, and a reversed-phase method for the purification of oligonucleotides.

Detects impurities not observable with electrophoresis.
Scale up to preparative separations.
Separate long RNA strands from 100 to 5,000 bases.
Two modes available: reversed-phase and size-exclusion.

References

>> Read our paper in Analytical Sciences on HPLC separation of RNA using RNA-RP1

>> See our poster for TIDES 2019

>> COSMOSIL Application Search

Preparative separation of long-stranded ssRNA

Reversed-phase column: COSMOSIL RNA-RP1

chromatograms showing purification of mRNA sample

For this analysis, in vitro transcribed long-chain ssRNA (4.8 knt) precipitated using lithium chloride was used as the sample. When the long-chain ssRNA was analyzed using electrophoresis, the impurity was not detected (see page 6); however, using COSMOSIL RNA-RP1, a peak of an impurity thought to be of different length was detected (see chromatogram ① above). Using preparative separation by HPLC, the impurity peak was successfully removed (see chromatogram ③ above).

Two modes to choose from

The COSMOSIL RNA series is comprised of RNA-RP1, a reversed-phase column, and RNA-SEC-1000 and RNA-SEC-2000, size-exclusion columns. Chromatograms of these columns using the same ssRNA ladder are compared below.

Reversed-phase: COSMOSIL RNA-RP1

As the hydrophobicity of RNA strands generally increases with length, long strands are typically eluted after short strands. However, the hydrophobicity also differs by sequence, so it is not necessarily the case that strands are eluted in order of length. By optimizing the analysis conditions, it is possible to improve separation. For example, it is possible to adjust the mobile phase, temperature, and gradient slope. See the references on page 8 for details.

Size-exclusion: COSMOSIL RNA-SEC-1000, RNA-SEC-2000

Compared to COSMOSIL RNA-SEC-1000, which has a pore size of 100 nm, RNA-SEC-2000, with a pore size of 200 nm, is suitable for analysis of large strands. Use different pore sizes depending on the strand sizes you need to separate.

Properties of packing material

Pore size

Long-strand nucleic acids have a very large molecular size, so they do not enter the pores of normal packing material, so the individual peaks do not separate. The super-wide pores allow the nucleic acids to enter, enabling much better separation.

HPLC columns for general use typically have silica gel with an average pore size of 8 to 12 nm, whereas COSMOSIL RNA-RP1 uses silica gel with super-wide pores.

RNA-SEC-1000 and RNA-SEC-2000 use silica gel with 100 and 200 nm pores, respectively, so long-chain nucleic acids can enter the pores and be separated.

Target molecular sizes

Our recommendations for target molecular sizes for reversed-phase and size-exclusion columns are noted below.

* These figures only represent the range of general suitability; it does not indicate that any nucleic acids in the range can be separated.

Suitable range for reversed-phase chromatography
For mid-length to long-stranded nucleic acids above 100 nt, the super-wide pore silica gel in COSMOSIL RNA-RP1 is recommended. For oligonucleotides, columns with smaller pores, such as 3C18-EB (average pore size 12 nm) or COSMOCORE 2.6C18 (average pore size 9 nm) are recommended.
Suitable range for size-exclusion chromatography
Using COSMOSIL RNA-SEC-1000, it is possible to separate smaller strands of less than 100 nt, up to long strands of thousands of nucleotides. Using RNA-SEC-2000, even longer strands of greater than 5,000 nt can be separated.

Analysis conditions

Analysis temperature

⬤ Single-strand nucleic acids

Reversed-phase: COSMOSIL RNA-RP1 temperature optimization for reversed-phase

Conditions
Column:       COSMOSIL RNA-RP1 2.0 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine Acetate（pH 7.0）
              B: Acetonitrile
              B conc. 7.5% → 17.5%（0 → 20 min）, 17.5%（20 → 24 min）
Flow rate:    0.2 mL/min
Temperature:  **°C
Detection:    UV 260 nm
Sample:       ssRNA Ladder（New England Biolabs #N0362S）
              2.5 times dilution × 2 µL

Size-exclusion: COSMOSIL RNA-SEC-2000 temperature optimization for size exclusion

Conditions
Column:       COSMOSIL RNA-SEC-2000 4.6 mm I.D. × 250 mm
Mobile phase: 20 mmol/L Phosphate Buffer (pH 7.0)
Flow rate:    0.3 mL/min
Temperature:  **°C
Detection:    UV 260 nm
Sample:       ssRNA Ladder（New England Biolabs #N0362S）
              2.5 times dilution × 2 µL

At low temperatures, single-strand nucleic acids tend to form higher-order structures, resulting in broad peaks. This maybe improved by analyzing at higher temperature.

⬤ Double-strand nucleic acids

Reversed-phase: COSMOSIL RNA-RP1 temperature optimization for reversed-phase

Conditions
Column:       COSMOSIL RNA-RP1 2.0 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine Acetate（pH7.0）
              B: Acetonitrile
              B conc. 5% → 20%（0 → 20 min）, 20%（20 → 24 min）
Flow rate:    0.2 mL/min
Temperature:  **°C
Detection:    UV 260 nm
Sample:       dsRNA Ladder（New England Biolabs #N0363S）
              2.5 times dilution × 2 µL

For analyzing double-strand nucleic acids in their nondenatured state, care is required when setting analysis temperature, as the strands tend to dissociate at high temperatures. Around the dissociation temperature, small differences in temperature can lead to very different separations. See our paper for details.

⬤ Mix of single- and double-strand nucleic acids

Reversed-phase: COSMOSIL RNA-RP1 temperature optimization for reversed-phase

Conditions
Column:       COSMOSIL RNA-RP1 2.0 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine Acetate（pH7.0）
              B: Acetonitrile
              B conc. 5% → 20%（0 → 20 min）, 20%（20 → 24 min）
Flow rate:    0.2 mL/min
Temperature:  60°C
Detection:    UV 260 nm
Sample:       dsRNA Ladder（New England Biolabs #N0363S）
              2.5 times dilution × 2 µL
              + Low Range ssRNA Ladder（New England Biolabs
              #N0364S）2.5 times dilution × 2 µL

By analyzing at a temperature at which single strands do not form higher-order structures and double strands do not dissociate, it is possible to simultaneously analyze both.

Considering loading capacity

Size-exclusion: COSMOSIL RNA-SEC-1000

testing loading capacity for size exclusion

Conditions
Column:       COSMOSIL RNA-SEC-1000 7.5 mm I.D. × 300 mm
Mobile phase: 20 mmol/L Phosphate Buffer (pH7.0) 
Flow rate:    1.0 mL/min
Temperature:  65°C
Detection:    UV 260 nm
Sample:       Low Range ssRNA Ladder
              (New England Biolabs #N0364S)

Even injecting 40 uL of sample, a similar chromatogram to a 2 uL injection was obtained (vertical axis scale differs). Loading capacity differs by sample, so determine it experimentally using an analysis column before scaling to preparative size.

Application data

COSMOSIL RNA-RP1 (RNA ladder)

Low Range ssRNA Ladder application for Low Range ssRNA Ladder

Conditions
Column:       COSMOSIL RNA-RP1 4.6 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine Acetate（pH 7.0）
              B: Acetonitrile
              B conc. 5% → 20%（0 → 20 min）, 20%（20 → 24 min）
Flow rate:    1.0 mL/min
Temperature:  65°C
Detection:    UV 260 nm
Sample:       Low range ssRNA Ladder（New England Biolabs #N0364S）
              2.5 times dilution × 5 µL

ssRNA Ladder application for ssRNA Ladder

Conditions
Column:       COSMOSIL RNA-RP1 2.0 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine Acetate（pH 7.0）
              B: Acetonitrile
              B conc. 7% → 10%（0 → 20 min）, 10%（20 → 24 min）
Flow rate:    0.2 mL/min
Temperature:  65°C
Detection:    UV 260 nm
Sample:       ssRNA Ladder（New England Biolabs #N0362S）
              2.5 times dilution × 2 µL

COSMOSIL RNA-RP1（DNA ladder）

pBR322 / MspI digest application for pBR322 / MspI digest

Conditions
Column:       COSMOSIL RNA-RP1 4.6 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine Acetate（pH 7.0）
              B: Acetonitrile
              B conc. 10% → 17%（0 → 20 min）, 17%（20 → 25 min）
Flow rate:    1.0 mL/min
Temperature:  40°C
Detection:    UV 260 nm
Sample:       pBR322 / MspI digest（Nippongene #317-01501）
              200 ng/µL × 5 µL

100 bp DNA Ladder application for 100 bp DNA Ladder

Conditions
Column:       COSMOSIL RNA-RP1 4.6 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine acetate（pH 7.0）
              B: Acetonitrile
              B conc. 12% → 18%（0 → 20 min）, 18%（20 → 25 min）
Flow rate:    1.0 mL/min
Temperature:  40°C
Detection:    UV 260 nm
Sample:       100bp DNA Ladder（Takara Bio #3407A）
              130 ng/µL × 5 µL

Fragments that are difficult to separate using agarose gel electrophoresis can be separated using COSMOSIL RNA-RP1.

COSMOSIL RNA-RP1 (separation of impurities)

ssRNA synthesized by in vitro transcription purification of RNA impurity

Conditions
Column:       COSMOSIL RNA-RP1 2.0 mm I.D. × 100 mm
Mobile phase: A: 100 mmol/L Triethylamine Acetate + 20 mmol/L Phosphate Buffer (pH 7.0)
              B: Methanol / Acetonitrile = 50 / 50
              B conc. 11% → 13%（0 → 20 min）, 13%（20 → 24 min）
Flow rate:   0.2 mL/min
Temperature: 60°C
Detection:   UV 260 nm
Sample:      pQBI T7-GFP RNA, digested with BglII and EagI (3.0 knt), 100 ng/µL × 5 µL
             pQBI T7-GFP RNA, digested with HindIII and BamHI （4.8 knt）, 100 ng/µL × 5 µL

electophoresis result not detecting RNA impurity

Upon analyzing long-chain ssRNA purified using lithium chloride precipitation, impurity peaks not detected with agarose gel electrophoresis were confirmed. This shows that it is possible to separate impurities in vitro transcribed ssRNA using reversed-phase HPLC.

COSMOSIL RNA-SEC-1000・RNA-SEC-2000（RNA ladder）

Low Range ssRNA Ladder（65°C） application

Conditions
Column:       COSMOSIL RNA-SEC-1000 4.6 mm I.D. × 250 mm
Mobile phase: 20 mmol/L Phosphate Buffer（pH 7.0）
Flow rate:    0.3 mL/min
Temperature:  65°C
Detection:    UV 260 nm
Sample:       Low range ssRNA Ladder（New England Biolabs #N0364S）
              2.5 times dilution × 2 µL

dsRNA Ladder（40°C） application

Conditions
Column:       COSMOSIL RNA-SEC-1000 4.6 mm I.D. × 250 mm
Mobile phase: 20 mmol/L Phosphate Buffer（pH 7.0）
Flow rate:    0.3 mL/min
Temperature:  40℃
Detection:    UV 260 nm
Sample:       dsRNA Ladder（New England Biolabs #N0363S）
              2.5 times dilution × 2 µL

ΦX174 / HincII digest application

Conditions
Column:       COSMOSIL RNA-SEC-1000 4.6 mm I.D. × 250 mm
Mobile phase: 20 mmol/L Phosphate Buffer（pH 7.0）
Flow rate:    0.3 mL/min
Temperature:  40°C
Detection:    UV 260 nm
Sample:       ΦX174 / HincII digest（Takara Bio #3406A）
              200 ng/µL × 1.25 µL

20 bp DNA Ladder application

Conditions
Column:       COSMOSIL RNA-SEC-1000 4.6 mm I.D. × 250 mm
Mobile phase: 20 mmol/L Phosphate Buffer（pH 7.0）
Flow rate:    0.3 mL/min
Temperature:  40°C
Detection:    UV 260 nm
Sample:       20bp DNA Ladder（Takara Bio #3409A）
              200 ng/µL × 2.5 µL

ΦX174 / HaeIII digest application

Conditions
Column:       COSMOSIL RNA-SEC-2000 4.6 mm I.D. × 250 mm
Mobile phase: 20 mmol/L Phosphate Buffer（pH 7.0）
Flow rate:    0.3 mL/min
Temperature:  40°C
Detection:    UV 260 nm
Sample:       ΦX174 / HaeIII digest（Takara Bio #3405A）
              200 ng/µL × 1.25 µL

100 bp DNA Ladder application

Conditions
Column:       COSMOSIL RNA-SEC-2000 4.6 mm I.D. × 250 mm
Mobile phase: 20 mmol/L Phosphate Buffer（pH 7.0）
Flow rate:    0.3 mL/min
Temperature:  40°C
Detection:    UV 260 nm
Sample:       100bp DNA Ladder（Takara Bio #3407A）
              130 ng/µL × 4 µL

Product specifications

Packing Material	RNA-RP1	RNA-SEC-1000	RNA-SEC-2000
Separation mode	Reversed-phase	Size-exclusion
Silica gel	Fully-porous high-purity spherical silica gel
Average particle size (µm)	5
Average pore size (nm, approx.)	—	100 (1,000 Å)	200 (2,000 Å)
Bonded phase	Octadecyl group	Hydrophilic group
Suitable pH range	2 – 7.5
Maximum pressure (MPa)	15

Ordering information

Product Name	Catalog Number	PKG Size
COSMOSIL RNA-SEC-1000 Packed Column (7.5 mm I.D. x 300 mm)	19380-21	1 pkg
COSMOSIL RNA-SEC-1000 Packed Column (4.6 mm I.D. x 250 mm)	21088-01	1 pkg
COSMOSIL RNA-SEC-1000 Guard Column (7.5 mm I.D. x 50 mm)	20785-91	1 pkg
COSMOSIL RNA-SEC-2000 Packed Column (7.5 mm I.D. x 300 mm)	19381-11	1 pkg
COSMOSIL RNA-SEC-2000 Packed Column (4.6 mm I.D. x 250 mm)	21095-01	1 pkg
COSMOSIL RNA-SEC-2000 Guard Column (7.5 mm I.D. x 50 mm)	21096-91	1 pkg
COSMOSIL RNA-RP1 Packed Column (2.0 mm I.D. x 100 mm)	21078-31	1 pkg
COSMOSIL RNA-RP1 Packed Column (4.6 mm I.D. x 100 mm)	21079-21	1 pkg
COSMOSIL RNA-RP1 Packed Column (10.0 mm I.D. x 100 mm)	21080-81	1 pkg