Semi-preparative scale single-stranded RNA purification

Sean M. McCarthy and Martin Gilar

Waters Corporation, Milford, MA, US

introduction

Oligonucleotide synthesis is a very efficient and high yield process. A typical yield of oligonucleotide reaction on a solid support is 98 to 99.5% per coupling step. In a typical multi-stage oligonucleotide synthesis, impurities are clustered together, even for a general-sized 21-base oligonucleotide with a total yield of 67-90%, the yield of longer-chain oligonucleotides. Correspondingly lower. Researchers often need to use materials that are higher in purity than the initial synthesis mixture. Therefore, oligonucleotides for gene knockout, genotyping, and diagnostic purposes typically require purification after synthesis. Economically viable solutions for laboratory-scale oligonucleotide purification are rare, and existing methods such as ion exchange chromatography and polyacrylamide gel electrophoresis are often cumbersome and time consuming. In the present application specification, we describe a method for purifying a low-cost, fast medium amount of material, single injection loading up 140 nmoles, using Waters ACQUITY UPLC ^® system and oligonucleotide separation techniques ( OST) column chemistry can achieve a final purity of over 95%. The proposed purification scale matches well with standard oligonucleotide synthesis scales (50 to 250 nmol). The following method can complete oligonucleotide purification in 15 to 30 minutes to obtain a high purity product.

Results and discussion

sample

RNA Oligonucleotide 5'-CCU UGU AAU CGC UUG ACG ATT -3' was purchased from the supplier and reconstituted in 110 μL of 0.1 M triethylamine acetate (TEAA) to give approximately 2.8 nmol/μL Solution. To prevent degradation, the samples were prepared shortly before use.

HPLC conditions

RNA oligonucleotides were purified by a Waters Alliance ^® HPLC Bioseparation System using a Waters XBridgeTM BEH OST C18 4.6 x 50mm 2.5 μm column separated by ion-pair reversed phase chromatography. ¹

Liquid Chromatography System: Waters Alliance HPLC Bioseparation System

Column: XBridge OST BEH C18 4.6 x 50mm, 2.5 μm

Column temperature: 60 ÌŠC

Flow rate: 1.0mL/min

Mobile phase A: 0.1M TEAA, pH 7.5

Mobile phase B: 80:20 0.1MTEAA/ACN

Gradient: 30 – 52.5% B elution 10.0 minutes (0.15% ACN/min)

Detection: PDA, 260nm

The isolated product was detected at 260 nm using a Waters PDA detector. Mobile phase A consisted of 0.1% M acetic acid triethylamine (TEAA) and mobile phase B was 80:200.1 MTEAA/acetonitrile. The column temperature was maintained at 60 °C.

As shown in Figure 1, despite the high efficiency of oligonucleotide synthesis, there are many failed sequences in the 21-matrix.

Although the column is overloaded with a larger mass load, the resolution remains N-1, N-2... The impurities elute before the main peak. Appropriate cleavage of the main peak of the 21-matrix oligonucleotide from the apex yields a product of extremely high purity.

The selected fraction collection window shown in Figure 2 represents a different mass load. After peak collection, samples can be aliquoted and dried as needed for long-term storage. The volatility of TEAA makes ion removal of the buffer component very easy. The oligonucleotides purified after evaporation of the solvent are virtually salt-free.

UPLC condition

The purity of the purified RNA oligonucleotide was verified by the ACQUITY UPLC System. As shown in Figure 3, our purification method effectively reduces the failure sequence impurities, resulting in oligonucleotides that are much higher in purity than commercially available unpurified oligonucleotides.

LC System: Waters ACQUITY UPLC System

Column: ACQUITY UPLC OST C18 column,

2.1 x 50mm, 1.7 μm

Column temperature: 60 ÌŠC

Flow rate: 0.2 mL/min

Mobile phase A: 0.1M TEAA, pH 7.5

Mobile phase B: 80:20 0.1MTEAA/ACN

Gradient: 35 – 85% B elution 10.0 minutes

(1% ACN/min)

Detection: PDA, 260 nm

in conclusion

The purification strategy of single-stranded RNA oligonucleotides described herein is fast and low cost, and can produce high purity materials. Using OST column chemistry and the Alliance HPLC system, a large number of crude single-stranded RNA products can be successfully purified in a short period of time, resulting in high purity (about 95%) of the material; based on the ratio of the peak area collected to the total peak area of ​​the sample Estimated, the yield can reach 55%.

This method is extremely useful for the purification of single-stranded RNA for RNAi experiments, which are critical for ensuring purity and target specificity. In addition, due to the volatility of T EAA, this strategy can store purified oligonucleotides, and in the absence of an unnecessary salt of T EAA, unnecessary impurities generated in other purification means can be avoided. Overall, the strategy proposes a comprehensive purification method that is superior to currently available methods. In addition, this purification method is very economical when considering the time cost of sample purification, the reagents, and the lifetime of the Waters XBridge OST chromatography.

references

[1] UPLC Isolation of Oligonucleotides: Method Development. Waters Application Note. 2007: 720002383EN.

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