PMID- 30705318
OWN - NLM
STAT- MEDLINE
DCOM- 20200817
LR  - 20240104
IS  - 2045-2322 (Electronic)
IS  - 2045-2322 (Linking)
VI  - 9
IP  - 1
DP  - 2019 Jan 31
TI  - HPLC methods for purity evaluation of man-made single-stranded RNAs.
PG  - 1019
LID - 10.1038/s41598-018-37642-z [doi]
LID - 1019
AB  - Synthetic RNA oligos exhibit purity decreasing as a function of length, because 
      the efficiency of the total synthesis is the numerical product of the individual 
      step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 
      nucleotides (nt) have been reported, but they fall short for purity evaluation of 
      100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted 
      as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both 
      identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at 
      high temperature in the presence of an organic cosolvent is the current 
      analytical strategy. Here we report that IP-RP is less suitable compared to the 
      conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the 
      relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a 
      protocol to determine the on-the-column stability of any RNA, and establish the 
      applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless 
      well resolving HPLC methods are used for batch-to-batch evaluation of man-made 
      RNAs, process development will remain shortsighted, and observed off-target 
      effects in-vitro or in-vivo may be partially related to low purity and the 
      presence of shorter sequences.
FAU - Kanavarioti, Anastassia
AU  - Kanavarioti A
AD  - Yenos Analytical LLC, 4659 Golden Foothill Pkwy, Suite 101, El Dorado Hills, CA, 
      95762, USA. tessi.kanavarioti@gmail.com.
LA  - eng
GR  - R43 HG010051/HG/NHGRI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20190131
PL  - England
TA  - Sci Rep
JT  - Scientific reports
JID - 101563288
RN  - 0 (RNA, Guide, CRISPR-Cas Systems)
RN  - 0 (RNA, Messenger)
RN  - 63231-63-0 (RNA)
RN  - 9014-25-9 (RNA, Transfer)
SB  - IM
MH  - Chromatography, High Pressure Liquid/*methods
MH  - Nucleic Acid Conformation
MH  - Nucleic Acid Denaturation
MH  - RNA/*analysis/chemistry
MH  - RNA Stability
MH  - RNA, Guide, CRISPR-Cas Systems/analysis/chemistry
MH  - RNA, Messenger/analysis/chemistry
MH  - RNA, Transfer/analysis/chemistry
PMC - PMC6356003
COIS- Anastassia Kanavarioti is the founder and director of Yenos Analytical LLC, a 
      for-profit company delivering custom analytical solutions for synthetic and 
      transcribed nucleic acids, and engaged in the development and manufacturing of 
      osmylated nucleic acids. There is no competing interest or relationship, 
      commercial or otherwise, between the two companies, namely Thermo Fisher 
      Scientific and Yenos Analytical and their employees, besides the fact that A.K. 
      both in her recent capacity as well as for earlier work has purchased and used 
      Thermo Fisher Scientific (Dionex) products, among which are the two DNAPac HPLC 
      columns used in this study.
EDAT- 2019/02/02 06:00
MHDA- 2020/08/18 06:00
PMCR- 2019/01/31
CRDT- 2019/02/02 06:00
PHST- 2018/08/09 00:00 [received]
PHST- 2018/12/11 00:00 [accepted]
PHST- 2019/02/02 06:00 [entrez]
PHST- 2019/02/02 06:00 [pubmed]
PHST- 2020/08/18 06:00 [medline]
PHST- 2019/01/31 00:00 [pmc-release]
AID - 10.1038/s41598-018-37642-z [pii]
AID - 37642 [pii]
AID - 10.1038/s41598-018-37642-z [doi]
PST - epublish
SO  - Sci Rep. 2019 Jan 31;9(1):1019. doi: 10.1038/s41598-018-37642-z.