Electrophoresis - 2022 - Minkner - Oligonucleotide Separation Techniques For Purification and Analysis What Can We Learn

Received: 30 March 2022 Revised: 9 September 2022 Accepted: 23 September 2022
DOI: 10.1002/elps.202200079
REVIEW
Oligonucleotide separation techniques for purification and

analysis: What can we learn for today’s tasks?
Robert Minkner1 Jirayu Boonyakida2,3 Enoch Y. Park2,3 Hermann Wätzig1
1 Institute
of Medicinal and
Pharmaceutical Chemistry, Technische Abstract
Universität Braunschweig, Braunschweig, Nucleic acids are the blueprint of life. They are not only the construction plan
Germany
2 Department
of the single cell or higher associations of them, but also necessary for function,
of Bioscience, Graduate
School of Science and Technology, communication and regulation. Due to the pandemic, the attention shifted in
Shizuoka University, Shizuoka, Japan particular to their therapeutic potential as a vaccine. As pharmaceutical oligonu-
3 Laboratory of Biotechnology, Green cleotides are unique in terms of their stability and application, special delivery
Chemistry Research Division, Research
systems were also considered. Oligonucleotide production systems can vary and
Institute of Green Science and
Technology, Shizuoka University, depend on the feasibility, availability, price and intended application. To achieve
Shizuoka, Japan good purity, reliable results and match the strict specifications in the pharma-
Correspondence
ceutical industry, the separation of oligonucleotides is always essential. Besides
Hermann Wätzig and Robert Minkner, the separation required for production, additional and specifically different sep-
Institute of Medicinal and Pharmaceutical aration techniques are needed for analysis to determine if the product complies
Chemistry, Technische Universität
Braunschweig, Beethovenstr. 55, 38106 with the designated specifications. After a short introduction to ribonucleic acids
Braunschweig, Germany. (RNAs), messenger RNA vaccines, and their production and delivery systems, an
Email: h.waetzig@tu-bs.de and
overview regarding separation techniques will be provided. This not only empha-
robert.minkner@tu-bs.de
sises electrophoretic separations but also includes spin columns, extractions,
Color online: See article online to view precipitations, magnetic nanoparticles and several chromatographic separation
Figure 1 in color.
principles, such as ion exchange chromatography, ion-pair reversed-phase, size
exclusion and affinity.
KEYWORDS
capillary electrophoresis, chromatography, oligonucleotides, RNA/DNA, separation
Abbreviations: AEC, anion exchange chromatography; DEAE, diethylaminoethyl (e.g., cellulose or sepharose); DOTMA,
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; FDA, United States Food and
Drug Administration; HEC, hydroxyethyl cellulose; HIC, hydrophobic interaction chromatography; iCIEF, imaged capillary isoelectric focusing; IP
RP, ion-pair reversed-phase; IVT, in vitro transcribed; knt, kilo nucleotides; LNPs, lipid nanoparticles; MNPs, magnetic nanoparticles; nt, nucleotides;
PF, pulsed field; SXC, steric exclusion chromatography.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium,
provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2022 The Authors. Electrophoresis published by Wiley-VCH GmbH.
2402 www.electrophoresis-journal.com Electrophoresis 2022;43:2402–2427.

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MINKNER et al. 2403
1 INTRODUCTION Engineered RNAs include ribozymes, riboswitches and

riboregulators [16, 17]. Besides regulation of gene expres-
1.1 Multifaceted species of ribonucleic sion, RNA is also a crucial component of all CRISPR/Cas
acid (RNA) systems. It can be designed to specifically bind to ligands
like small molecules, proteins or nucleic acids [18, 19].
Nucleic acids carry genetic information of each form of
life, from prokaryotic cells to eukaryotic cells. Though
viruses are neither cells nor living beings but organic 1.2 RNA in medical and pharmaceutical
particles, sometimes defined as ‘organisms at the edge science
of life’, they still carry nucleic acid as the genetic mate-
rial for their existence. There are two main classes of The development of RNA-based therapeutic modalities
nucleic acids (1) deoxyribonucleic acid (DNA) and (2) is primarily based on two approaches: engineering of
ribonucleic acid (RNA). In general, DNA in nature is a (1) antisense RNA or RNA interference (RNAi) and (2)
double-helix molecule of deoxyribonucleotides that stores mRNA.
the hereditary information of living beings in biologi- RNAi is a normal cellular process for controlling gene
cal systems, whereas RNA is usually a single-stranded expression by promoting mRNA degradation after recog-
polymeric molecule made up of ribonucleotides and par- nising by a sequence-specific siRNA or by miRNA. Human
ticipates in the coding, decoding, regulation and expres- diseases can be caused by the expression of mutated genes
sion of genes. Due to the physicochemical properties of or the abnormal expression of normal genes; hence, tar-
DNA, the molecule has been used in many applications in geting pathologically involved genes/mRNA could be a
the nanotechnology aspect, including data storage, DNA practical approach for diseases that conventional drugs
microscopy, protein crystallisation, and DNA origami, cannot treat. The efficacy of RNAi in animal models has
rather than in vaccinology [1–5]. However, this informa- been proven for several diseases, including viral infections,
tion has been extensively reviewed elsewhere. In this nervous system disorders, tumours and genetic diseases
review, we are focusing on RNA and separation techniques [20–22]. As of 2021, there are two RNAi-based therapies
for RNA purification. approved by United States Food and Drug Administra-
With the advances in science and technology over the tion (FDA); both are siRNA-based drugs. Although no
past decades, RNA biology has been extensively and deeply miRNA-based drug is available on the market yet, three
studied through the development of high-throughput RNA miRNA-based drugs are currently undergoing clinical
sequencing (RNA-seq) for their roles [6, 7]. RNAs can be trials [15].
roughly categorised into two main groups: coding RNA
and non-coding RNA (ncRNA). Coding RNA generally
refers to messenger RNA (mRNA), which serves as a tem- 1.2.1 RNA as vaccine
plate for protein synthesis. ncRNA can be further divided
into two groups based on the length; small non-coding The concept of mRNA for a vaccine started in 1978
RNA (<200 bases) and long non-coding RNA (lncRNA, when protein expression was induced in mouse lym-
>200 bases) [8], or by functionality: housekeeping ncR- phocytes and a human cell line after delivering mRNA-
NAs or regulatory ncRNAs. Housekeeping ncRNAs are carrying liposomes [23, 24]. In 1989, the first synthetic
ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small cationic lipid nanoparticle (LNP) made up from N-[1-
nuclear RNAs (snRNAs) and small nucleolar RNA. Regu- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chlo-
latory ncRNAs are microRNAs (miRNAs), small interfer- ride (DOTMA) was reported for in vitro transcribed (IVT)
ing RNA (siRNA), circular RNAs, piwi-interacting RNA, mRNA delivery [25]. Since then, various cationic and ion-
tRNA-derived small RNAs (tsRNAs) and lncRNAs [9, isable lipid types have been explored for mRNA delivery.
10]. Housekeeping ncRNAs are abundantly expressed in In the following year, the IVT-mRNA encapsulated inside
cells and regulate fundamental cellular functions, whereas a liposome was successfully delivered into mouse mus-
regulatory ncRNAs regulate gene expression at epige- cle, and protein expression was detected [26]. This was
netic, transcriptional and post-translational levels [11–13]. a ground-breaking demonstration of the mRNA delivery
Among the ncRNAs, miRNAs are the most extensively system in an animal model.
studied as biomarkers in human diseases, particularly neu- The mRNA technology has been authorised for massive
rodegenerative diseases, cancers and therapeutic targets use in humans for the first time during the SARS-CoV-2
[14, 15]. pandemic as a vaccine. The first-generation coronavirus
RNAs have been engineered for new biological func- disease 2019 (COVID-19) vaccines approved by the FDA
tions to engineer biological systems in synthetic biology. for use in an emergency were both mRNA-based vaccines,
2404 MINKNER et al.
BNT162b2 [27] and mRNA-1273 [28]. This system has sev- and endosomal escape [38]. DSPC increases LNP stability
eral advantages over conventional vaccine platforms as it by facilitating a formation of a stable bilayer structure
is more rapid, simpler and can be synthesised in vitro. The beneath the PEG surface layer [39].
mRNA platform is more tolerable than the other vaccine
platforms. It can avoid using noxious chemicals for virus
attenuation and cell cultures that may be contaminated 1.3.2 Other delivery systems
with adventitious viruses or the contamination of microor-
ganisms and their metabolites from the protein production Apart from the lipid-based nanoparticle, several alter-
process [29–31]. The theoretical risks of mutagenesis and native systems have been experimented with polymer-
viral tropism exist in the viral vector platform but not in based, peptide-based and viral vector systems [40]. The
the mRNA platform [32, 33]. first polymer-based mRNA delivery system was diethy-
laminoethyl (DEAE)-dextran; however, the efficiency was
lower than for lipid-based systems [25]. Later, sev-
1.3 Special delivery systems for RNA eral cationic polymers and biodegradable cationic poly-
vaccines mers were developed to improve the delivery efficacy.
Poly(amidoamine) and polyethyleneimine (PEI) are exam-
1.3.1 Lipid nanoparticles (LNPs) ples of cationic polymers [41, 42]. However, they are not
clinically advanced due to their toxicity. Furthermore,
This section is of particular relevance because of the cur- they tend to have a lower cellular clearance due to their
rent pandemic, not only for the RNA analysis, separation high molecular weight [36]. Therefore, low-molecular-
and purification but also for the vaccine. The property of weight PEI modified and biodegradable polymers (e.g.,
mRNA as a template for protein synthesis has empowered poly(beta-amino) esters, poly (CBA-co-4-amino-1-butanol)
and transformed vaccinology. The major hurdle of this sys- and poly(lactic-co-glycolic acid)) were developed to reduce
tem is the rapid degradation of mRNA molecules by RNase toxicity [43–45].
in physiological fluids. So, for cellular delivery, lipid-based The versatility of peptides has attracted attention for
encapsulation is the top choice for successful deliveries of the development of peptide-based mRNA delivery sys-
mRNA through liposomes. The synthetic LNP made up of tems. The first peptide for mRNA delivery was protamine,
DOTMA, a cationic lipid was the first of its kind [34]. The a cationic peptide that forms a complex with mRNA
two mRNA vaccine candidates against SARS-CoV-2 use through electrostatic interaction [46]. Later, cationic cell-
cationic LNP for delivering S-protein encoding mRNA; penetrating peptides such as RALA [47], KALA [48] and
ALC-0315 (((4-hydroxybutyl)azanediyl)bis(hexane-6,1- PepFect14 [49] were developed to improve the delivery
diyl)bis(2-hexyldecanoate)) in BNT162b2 formulation efficiency. However, there are several downsides to the
and SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(8- peptide-based delivery system, including poor chemical
(nonyloxy)-8-oxooctyl)amino)octanoate) in mRNA-1273 and physical stability in serum, which results in a short cir-
formulation [35]. These cationic lipids form complexes culatory half-life [50]. Viral vectors from genetically engi-
with nucleic acids, which increases their resistance against neered viruses have been used as a vehicle for nucleic acid
nuclease activity and enables successful mRNA transport delivery, particularly adeno-associated viruses [51]. Given
into target cells. the advantages of positive-strand RNA viruses, in which
The LNP should facilitate organ specificity, enhance genomes can be released into the cytoplasm for replication
cellular uptake and provide efficient endosomal escape and directly used for protein translation by the host ribo-
[36, 37]. These features can be improved by the addition somal machinery [52], several viruses have been employed
of helper lipids, for example, cholesterol, 1,2-distearoyl- for mRNA delivery, for example, alphaviruses [53], fla-
sn-glycero-3-phosphocholine (DSPC) or polyethylene viviruses [54], picornaviruses [55] and rhabdoviruses [56].
glycol (PEG)-lipid. Examples for PEG-lipids are 2-
[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide
(PEG2000-DMA) in BNT162b2 formulation or 1.4 Short overview of oligonucleotide
1,2-dimyristoyl-rac-glycero3-methoxypolyethylene glycol- production methods and general
2000 (PEG 2000-DMG) in mRNA-1273 formulation. The considerations
PEG-lipid helps to stabilise the mRNA-LNP structure
against aggregation during manufacturing and storage RNAs can be generated by three main approaches: (1)
[35]. Cholesterol stabilises the LNP by modulating the chemical synthesis, (2) enzymatic synthesis (IVT) and (3)
fluidity of the membrane, promoting membrane fusion in vivo expression from siRNA expression vector in cells.
MINKNER et al. 2405
1.4.1 Chemical synthesis of RNAs merase promoter and a DNA template of the desired
RNA sequence [57]. To date, several platforms have been
The chemical synthesis of RNAs requires the least applied for RNA molecules expression and production,
effort among the three methods but is most expen- for example, Escherichia coli, Saccharomyces cerevisiae and
sive. The RNAs are produced on a solid-phase with mammalian cells [70–72]. However, host cellular com-
2′-hydroxyl protecting groups (PGs) that provide ribonu- ponents, RNases and non-target RNAs are the major
cleoside phosphoramidites [57]. The generally used 2′- concerns of in vivo RNA production [73]. Chromatographic
hydroxyl PGs are fluoride-labile tert-butyldimethylsilyl RNA separations using the Ni-NTA (affinity) or AEC
(TBDMS) ether, 2′-O-[t-butyldimethylsilyl], and 2′-O- columns have been applied to purify the RNAs from cell
[(triisopropylsilyl)oxy]methyl, which has to be removed by lysate [73, 74]. Additionally, ion-exchange chromatography
a fluoride ion after the RNA synthesis [58, 59]. Therefore, (IEC) separation at two different pH values and hydropho-
to obtain the high purity deprotected RNA, the fluoride bic interaction chromatography (HIC) can be applied to
reagent and remaining chemicals must be removed by an separate RNAs with overlapping sizes [74].
extra purification step with an anion exchange chromatog-
raphy (AEC) or a reversed-phase (RP) chromatography
[60]. This extra step has significantly increased the cost of 2 NON-CHROMATOGRAPHIC
RNA production by this method. SEPARATION TECHNIQUES
1.4.2 Enzymatic synthesis (IVT) of RNAs 2.1 Extraction, precipitation and

commercial solid-phase extraction
RNA can be made by IVT of a DNA template with a bacte-
riophage DNA-dependent RNA polymerase (e.g., T7, SP6 Extraction and precipitation are often used and needed
or T3) to catalyse the synthesis of the target RNA [61, 62]. to purify oligonucleotides from the sample matrix. This
This is done in a cell-free environment. Thus, cell-derived is also true for pre-treatment steps prior to further sepa-
impurities are absent, which makes the product much ration steps, such as chromatography, in order to achieve
safer for clinical use [63]. The DNA template must be lin- higher purity and prevent various detrimental effects, for
earised before IVT either by restriction digestion or ampli- example, fouling of the column. For mRNA, the same
fying the target via PCR. The IVT reaction component gen- purification methods can be applied as for RNA. In addi-
erally contains four types of ribonucleoside triphosphates, tion, Rosa et al. advised compliance to the current good
RNA polymerase, the cofactor for RNA polymerase MgCl2 manufacturing practise [66]. Typical steps are DNAse treat-
and a Tris buffer. After the IVT, a DNA template has to be ment, precipitation or extractions, but truncated or dsRNA
removed by DNase before purifying the transcribed RNA cannot be effectively removed by these methods [66]. The
[64–66]. To achieve the clinical standard of RNA therapeu- chemical extraction methods, such as phenol/chloroform,
tics, transcribed RNA must be purified to remove reaction solid-phase and LiCl precipitation, were already reviewed
components and aberrant RNA transcripts, as they may in detail elsewhere [75–77]. As these methods have existed
induce an undesired response in a host [67–69]. As we later for a long time, optimised protocols for specific sample
describe in Section 2.1, the IVT-RNA can be purified using matrices are already at hand, such as for E. coli by extrac-
a commercial RNA purification kit, isopropanol- or LiCl tion [78], Helicobacter pylori by kit-based approach [79],
precipitation for a laboratory-scale preparation. For large- from grapevine [80], miRNAs and mRNAs from milk by
scale purification (see Section 2), chromatographic-based a mix of kit, and extraction [81] or spermatozoal RNA
techniques (e.g., ion-pair reversed-phase [IP RP] chro- from bulls with an extraction method and/or commer-
matography, AEC and affinity chromatography), tangen- cial kit [82]. This technical literature for the isolation of
tial flow filtration (TFF) or TFF-coupled LC are required RNA and their quality control is manifold and still reg-
for a sufficient removal of impurities [66]. These tech- ularly updated, reflecting the protocols’ continuous use
niques can often be used for purification and, with some and improvements [83–88]. This shows that precipitation
exceptions such as TFF, for analysis. is a standard in the literature, and LiCl precipitation is
the method of choice for long RNAs with at least 100
nucleotides (nt), as it is also the case for DNA. Shorter
1.4.3 In vivo expression of RNAs RNA, tRNA and proteins are not much affected by it. How-
ever, higher concentrations are required (>400 mg/ml) for
RNA molecules can also be produced in vivo using a LiCl, and if not applicable, one can rely on salt and ethanol
cell with an expression cassette containing an RNA poly- precipitation [89].
2406 MINKNER et al.
Silica-based spin columns for purification purposes and purified from the gel matrix [99]. If the RNA is kept
regarding their regeneration and reuse are discussed in the in a denatured state for further analysis after agarose gel
Supporting Information section [90–92]. electrophoresis, glyoxal [100] or formaldehyde can be used
Precipitation, extractions and kits are helpful and widely as a denaturing reagent [101]. Recently, a protocol for size-
used, even though spin-column-based chromatographic dependent (denaturing) separation of RNA via PAGE was
separations are standard for oligonucleotide separation. also updated [102]. Preparative PAGE is an option if a large
This is also because they are often more cost-effective for sample quantity is not needed, only moderate purity is
small scales or if high purity is not required. These meth- required, and the funds for other equipment are not avail-
ods are often part of pre-purification strategies to decrease able. For example, approximately 15 µl with 500 ng/µl can
impurities prior to further purification steps. Reproducibil- be obtained, with a recovery in the range of 50% [103, 104].
ity of the kits is usually high but can vary as it depends on However, problems with acrylamide leftovers can arise
the sample matrix, loading amount and different providers using PAGE, as acrylamide cannot be completely removed
[75, 85, 91, 93]. Unfortunately, commercial kits often do and can bind to the RNA. This may affect or compromise
not disclose the specific details of their function or compo- the data or interfere with further processes, such as NMR
nents [93]. In at least one study, it has been investigated, in analysis [105]. For toxicological consequences, please refer
this case, with five different kits for DNA extraction from to Section 5 and Table 3.
faeces. It was shown that the reproducibility is sufficient To sum it up, gel electrophoresis is a valid and frequently
for each kit itself, but there may be differences in absolute used method for separation, but mainly for analytical
DNA yield and purity between these kits [94]. purposes. However, everybody should consider updating
one’s laboratory protocol for gel electrophoresis from time
to time. Some methods may have been used for many
2.2 Gel electrophoresis decades, and improvement can often be applied in one’s
own research.
Gel electrophoresis is a long-established method for the
separation of DNA or RNA, which is able to separate
DNA and RNA effectively, and the respective relative elec- 3 CHROMATOGRAPHY
trophoretic mobility is sufficiently reproducible [95]. RNA INTERACTION-BASED TECHNIQUES
analysis on gel often requires hazardous reagents such as
ethidium bromide and can lack sensitivity. Both could be 3.1 General considerations
improved, and the LOD was increased to 12.5 ng RNA
using a commercially available kit [96]. Further improve- Chromatography is a widespread separation technique
ments are still possible as replacing buffer compounds can for (bio-)molecules in the pharmaceutical industry and
improve the speed and separation of DNA and RNA in is selective, versatile and scalable. Size exclusion chro-
gel electrophoresis. For DNA separation of 100 bp → 5 kb, matography (SEC), IP RP, IEC, HIC, affinity and core
10 mM sodium boric acid and for >3 kb 5 mM lithium bead are typical separation principles. The latter principle
acetate were recommended, whereas for RNA separa- implements a nonfunctionalised outer layer with a func-
tion, it was 5 mM sodium boric acid or 5 mM lithium tionalised core to trap the target structure. On the other
acetate. This differs from the commonly used Tris-based hand, packed beads and monolithic columns are different
buffers. This allowed rapid separations on agarose gels at types of stationary phase material. All these are mentioned
a high voltage of 300 V, without significant Joule heat- in a recent review concerning mRNA vaccines [66]. Mono-
ing or even melting the gel, as the current was reduced lithic columns are a newer type of stationary phase for the
due to lower conductivity. The gel melting happened column. Compared to the classical packed beads, they con-
with Tris-boric acid EDTA gels because the increas- sist of one porous block with several pores and channels,
ing current increased the temperature [97]. Switching which provides better flow rates and increased surface
to a Hepes/triethanolamine or Tricine/triethanolamine area, leading to improved separation performance. Mono-
buffer system could not only improve the resolution and liths can be used for all separation principles, which have
separation of high molecular RNA species on agarose– their advantages and disadvantages, unrelated to the used
formaldehyde gels but also decrease the analysis time column material [66, 106]. Often several chromatographic
and the amount of (toxic) formaldehyde. However, an steps are required for the desired high purity of oligonu-
improved resolution and separation effect on DNA was not cleotides [66]. RNA phosphorothioate oligonucleotides are
observed [98]. usually produced as diastereomers. For their purification,
Separation by non- or denaturing preparative PAGE is HPLC is commonly applied, whereby often strong AEC
common, whereby the target RNA will be cut out afterward or RP is used [107]. AEC can also be used after par-
MINKNER et al. 2407
tial purification via extraction and/or SEC. For example, due to the high lipid content, it can be assumed that the
the purification of RNA constructs from E. coli by this purity may still be optimised. However, the quantifica-
principle led to approximately 90% purity. Another possi- tion was performed with AEC, but the separation before
bility is to insert a tag into the RNA and purify the RNA LC–MS was performed with an RP18 column so a direct
via affinity chromatography [78]. Many reviews focused comparison is difficult.
on RNA chromatography, especially for AEC and IP RP Investigations showed that a DEAE CIM monolithic col-
chromatography, as they are the most used ones. Some- umn had a slightly increased retention time for dsRNA
times the following mass spectrometry (MS) applications than a Gen-Pak FAX column (also DEAE), indicating
are included [76] or the focus is on native/denaturing other factors influencing binding. On a quaternary amine
ncRNA purification [108]. Many consider affinity purifica- (QA) CIM monolithic column, the retention time was
tion, LiCl precipitation, denaturing PAGE, SEC, (HPLC), shorter. In general, the binding capacities of the mono-
IP RP and AEC as (gold-) standards [109], but there is lithic columns were higher, with the DEAE variant being
consensus that (si)RNA purification with these techniques the best. However, separation efficiency, lower necessary
still needs improvement. For example, probably Asura- salt concentration and recovery were more favourable on
gen in the United States and CureVac GmbH in Europe the QA column. The QA material could achieve a clear
have some additional know-how, which, however, remains separation of dsRNA and ssRNA. Separation of dsRNA
proprietary knowledge [110]. with different sizes was possible up to 1 kb. However, base-
Chromatography purification is not necessarily easy, line separation was not possible when the size difference
and the method and the analyte of interest should be well was too small, but clear, distinguishable peaks were still
known. This includes many considerations such as mate- achieved. This approach was superior compared to the
rials, methods, interactions, hybridisations, the target of LiCl precipitation [113].
interest and aim [111]. AEC is a work horse for purifications, as it usually
provides high binding capacity accompanied by high load-
ing capacity. Of course, its performance depends on the
3.2 Anion exchange chromatography specific case. However, it is the first choice to sepa-
(AEC) rate different oligonucleotides with N − x (‘N minus x’)
deletion.
AEC is considered an easy, inexpensive and mild sep-
aration principle. This is also true for oligonucleotides.
Sometimes denaturing conditions are necessary, but these 3.3 Ion-pair reversed-phase (IP RP)
can cause aggregation, misfolding or degradation itself. chromatography
Improvements in non-denaturing methods and column
material, such as monolithic, are steadily done, as RP and IP RP chromatography use the same stationary
reviewed in 2014 [75]. AEC also proved to be suitable for phase, but IP RP LC employs additional reagents for the
N − x (‘N minus x’) deletion separation because different ion pairing. Hence, the toxicity of the mobile phase and
construct lengths have a different charge [106]. reagents may be an issue [75]. Therefore, it is a good ana-
A non-denaturing AEC–HPLC with a step gradient was lytical tool but not useful for preparative steps, as toxic or
used to quantify siRNA (up to 21 base pairs). Interesting denaturing reagents require additional purification steps.
is the one-step sample preparation with proteinase K and The ion-pairing reagent masks the charged molecule so
a lysis buffer, including 60 mM Tris, 100 mM EDTA to that the separation is mainly based on hydrophobicity. IP
inhibit Rnase, 20 mM DTT especially for Rnase A, 400 mM RP LC is very beneficial for the separation of oligonu-
guanidine hydrochloride as a denaturant and 0.1% Triton cleotide impurities with base sequence change because the
X-100 as a surfactant at pH 9, which proved to be very overall retention is based on the base composition, as they
effective. Moreover, different siRNAs of the same length have a different hydrophobicity (C < G < A < T) [106] (see
with different base pair composition were separated on an also Ref. [112]). IP RP LC on HPLC showed that proteins
AEC column, which shows that hydrophobic interactions such as Rnase are removed, and therefore, the RNA stor-
also affect the separation. The siRNA and their chain- age stability increases. One early study resolved different
shorted metabolites were separated with a lower limit of lengths and types of RNAs, even if not all could be bound
quantification of 6 ng/ml by an overall recovery of >95%. or perfectly separated [114].
Investigations regarding mass were done with ion-pairing RP IP HPLC is usable as a purification and analysis
chromatography on an RP18 column [112]. No information method (see Table 1). In the following study, the mobile
on purity is available, but as the siRNA extraction proto- phases contained triethylammonium acetate (TEAA) as an
col for MS-analysis of the metabolites had to be adapted IP reagent and acetonitrile. Elution was done via linear gra-
2408
TA B L E 1 Example ion-pair reversed-phase (IP RP) chromatography methods

Mobile Mobile
Purposea Column phase A phase B Gradient Temperature Detection Separation results Reference
Analysis and WAVE DNASep 0.1 M TEAA, 25% (v/v) phase For example, flow rate RNA: 75◦ C; Absorption at Resolving RNA ladder [114]
purification Cartridge pH 7.0 A/acetonitrile 0.9 ml/min; dsDNA: 50◦ C 260 nm ranging from 155 to
(ACN) 38%–40% B in 1 min, 1770 nt at 75◦ C (not
to 60% B in 15 min, completely baseline
to 66% B in 6 min, to separated); RNA
70% B in 0.5 min, to quantification; purity
100% B in 0.5 min, analysis of RNA
held at 100% B for degradation/translation
1 min, to 38% B in failures
1 min, held at 38% B
for 2 min
Purification XBridge C18 OST 0.1 M TEAA, 80/20% (v/v) Flow rate 1 ml/min ssRNA: 60◦ C DAD; Yield of ssRNA: 55%–70% [115]
column, pH 7.0 phase A/ACN ssRNA: 30%–52.5% B in Duplex: 20◦ C absorption with purity >95%
4.6 mm × 50 mm 30 min, at 60◦ C from 220 to The purity of prior
with 2.5 µm Duplex: 25%–75% B in 350 nm annealed siRNA was
particles 30 min >98%
On column annealing:
similar as ssRNA result,
but strong secondary
structure significantly
reduced yield
(Continues)
MINKNER et al.
TA B L E 1 (Continued)
Mobile Mobile
MINKNER et al.
Purposea Column phase A phase B Gradient Temperature Detection Separation results Reference
Analysis Waters ACQUITY 0.1 M TEAA, 80/20% (v/v) Flow rate 0.2 ml/min; Between 20 and DAD; Separation of [115]
OST C18 column pH 7.0; phase A/ 35%–65% B in 10 min 70◦ C in 5◦ C absorption at single-stranded and
with 1.7 µm In LC – MS ACN LC – MS steps 260 nm duplex RNA species was
particles also In LC – MS Variable, for example, LC – MS LC – MS possible with good
(UHPLC 25 mM hexyl- also flow rate: 20◦ C Single- resolution. MS analysis
method) ammonium ACN 0.2 ml/min; 35%–85% quadrupole improved with
acetate, B in 10 min MS with ESI ammonium acetate by
pH 7.0 reversed elution order
Analysis DNAsep column 0.1 M TEAA, 75% phase Flow rate 1 ml/min 50 and 75◦ C UV detector at Different elution [116]
50 mm × 4.6 mm pH 7.0 A/25% ACN Different gradients 260 nm behaviour of dsRNA
I.D. and dsDNA because of
structural differences
Analysis (best BEH C18 UPLC 0.1 M TEAA, 20% A and 80% Flow rate 0.2 ml/min; 80◦ C DAD at 260 nm A good stereoisomer [118]
conditions column pH 7 ACN 2 µl injection separation but no
only) volume; 6%–12% B in complete baseline
19.5 min, 12%–14% B separation. LOD was
in 5.5 min, 14%–30% 4.2 ng. Detection of
B in 7 min degradation products;
ACN is a key
component for
diastereomer
separation; choosing the
right column
temperature, column, IP
reagent and the organic
modifier is important
Analysis (best BEH phenyl 0.1 M dibuty- ACN Flow rate 0.4 ml/min; 65 DAD, with UV Good separation of [119]
conditions) column lammonium injection volume at 260 nm for siRNAs and
(150 mm × 2.1 mm) acetate 5 µl; 30%–35% B in siRNA; phospholipids; data
(DBAA) 5 min, 35%–100% B corona CAD regarding temperature,
in 5 min and held for IP reagents and column
100% B for 5 min phospholipids type
Note: If not mentioned otherwise, all buffers are aqueous.
Abbreviations: siRNA, small interfering RNA; TEAA, triethylammonium acetate.
a
According to the publication, other intended use is possible.
2409
2410 MINKNER et al.
dient and analysis with a slightly modified mobile phase reagent improved the results; the separation principle
by a UPLC–UV–MS method. The RP IP LC purification was mainly the RP IP. Interestingly, enhancing the IEC
yielded >95% pure ssRNA (21 nt) and >98% for their cor- principle by using NaCl or NaBr as a salt gradient led to an
responding duplexes if the crude complementary oligonu- improvement in peak shape and a good separation even
cleotides were annealed before the purification. This study for the ‘base-flip’ isomers. Only the strong IEC column
is interesting because the duplex stability regarding melt- did not achieve a result due to the strong RNA binding to
ing temperature was investigated. Short oligonucleotides the column [117].
normally have low melting points. Nevertheless, in this
study, 20◦ C was chosen because duplexes with a melting
point of 37.5◦ C already started to degrade at 25◦ C in the 3.3.1 IP RP LC applications
experiments. The authors attempted purification with on-
column annealing of the complementary siRNA to shorten Synthetic, stereoisomeric siRNAs are often modified with
the purification process. This scalable approach resulted in a phosphorothioate moiety instead of the phosphodiester
similar results, in which a small amount of siRNA did not linkage, introducing stereocenter(s). Incidentally, even if
form a duplex. However, this approach is not generalisable this modification indeed stabilises the siRNA, it is still
as oligonucleotides with strong secondary structures had a possible that the introduced sulphur atom is replaced by
significantly reduced yield [115]. Regarding analysis, RP IP oxygen through desulfurisation. Li et al. [118] investigated
UPLC–MS was able to separate the siRNA duplexes from several parameters, such as columns, organic modifier,
their impurities; ‘mismatched’ truncated duplexes, which reagent type and concentrations, to establish an IP RP
contain at least one shorter/failed sequence. The duplexes UHPLC method to separate stereoisomeric siRNAs. A
were stable during the UPLC and had a good resolution in sample mixture consisted of two stereoisomers from the
hexyl ammonium acetate but melted during the ionisation sense strand and four stereoisomers from the anti-sense
process. Interestingly, ammonium acetate improved the strand, which led to eight different stereoisomeric siRNA
MS analysis as ionisation of the intact duplexes was possi- duplexes. The loaded siRNA sample concentration was
ble, but the elution order of the analytes was reversed, and approximately 0.1 mg/ml prepared in a 20 mM phos-
the separation efficiency suffered. Moreover, incomplete phate buffer. The best separation conditions on a BEH
duplex formation and on-column melting were observable C18 UPLC column (see Table 1 and Ref. [118]) led to a
with shorter strand length (from 21 to 16 nt). Both meth- good stereoisomer separation, but not a complete base-
ods, purification and analysis, were non-denaturing for the line separation as three compounds were still partially
siRNA [115]. overlapping. The limit of detection was 4.2 ng (with
In RP IP LC, DNA and RNA show differences; dsRNA 2 µl loaded sample). To detect oxidation (deterioration)
eluates earlier than their same-sized DNA counterpart. products, the siRNA duplex was treated with iodine for
One reason is the replacement of thymine to uracil with desulfurisation, and six different degradation products
a small effect. Another seems to be coincident with the could be detected and separated [118]. In a follow-up
structural–conformational differences, which results in a study, the simultaneous separation of several siRNAs and
less hydrophobicity of RNA and, therefore, less binding lipids was investigated, which are model components for
affinity to the alkylated stationary phase [116]. Moreover, (filled) LNPs. The investigation covered several parameters
at elevated temperatures of 50 and 75◦ C, dsRNA showed such as stationary phase, temperature, IP reagent, con-
higher resolution than dsDNA. The separation was not centration, and mobile phase and their respective impacts
only size-dependent, as different ssRNA fragments of the were well summarised. Applying the best separation con-
same size could be resolved. This method was successfully ditions (see Table 1 and Ref. [119]) and overlapping the
applied to analyse human telomerase RNA [116]. optimised single chromatograms showed that a good sep-
Mainly, strong IEC or RP IP LC is applied for oligonu- aration was achieved (Figure 1). Only one siRNA and the
cleotide separation. These methods achieve different lipid 1,2-dioleoyl-3-trimethylammonium-propane chloride
separation results, and their combination has now salt (DOTAP) showed a peak broadening, which needs to
been compared together on three different mixed-mode be further improved without negatively affecting the other
columns (IEC + RP IP; weak and strong IEC parts). The included compounds. In the end, the separation of siRNAs
mixed-modes can lead to a worse separation of RNA and lipids from an experimental LNP drug was successful,
N − x deletion variants (compared to IEC) but to a better even using their base or hydrogen peroxide stressed sam-
separation of ‘base-flip’ isomers (compared to IP RP). ples [119]. As shown, purification depends not only on the
However, peak broadening and tailing increased strongly type of separation but also on the used material of the sta-
when ammonium acetate was used as a buffer compound, tionary phase. This material effect on oligonucleotides was
as recommended by the vendor. Using TEAA as an IP investigated on a C18 monolithic column, and four packed
MINKNER et al. 2411
F I G U R E 1 UHPLC–UV–CAD separation and detection of small interfering RNA (siRNA) duplexes (1) and phospholipids (2) with
DBAA as the ion-pair reagent on a BEH phenyl column. Conditions: Mobile phase A was aqueous 0.1 mol/L DBAA, Mobile phase B: ACN.
BEH phenyl column (150 × 2.1 mm). Column temperature: 65◦ C. The mobile phase gradient profile was 30%–35% B from 0 to 5 min, 35%–100%
B from 5 to 10 min and 100% B from 10 to 15 min. The flow rate was 0.4 ml/min, and the injection volume was 5 µl. A diode array detector with
UV absorbance detection at 260 nm was used to monitor siRNA, and a corona CAD was used for phospholipids. The siRNA sample
concentrations were approximately 0.1 mg/ml in 20 mmol/L phosphate buffer. The lipid concentrations were about 0.3 mg/ml. Source:
Reprinted from Ref. [119], copyright (2019) Elsevier
C18 columns with different particle sizes. Also, the effect of the right ones. Using an optimised protocol on a
elevated temperature was investigated. On the monolithic monolithic poly(styrene-divinylbenzene) column, N-
column, oligonucleotides with hairpin structures always acetylaminofluorene-adducted oligonucleotides isomers
eluted earlier than their random coiled counterparts, and were successfully separated and identified. A complex
all retention times decreased with increasing temperature. mixture consisting of oligomers with different lengths and
On the other hand, no distinct trend was recognisable on modifications was also investigated for further evaluation.
the packed columns, as different and changing retention The components, mainly the unmodified and the modified
times were achieved depending on temperature, construct oligonucleotides, could be separated. Yet positional iso-
length and column. However, it seems that hairpin struc- mers could not be resolved well, especially closely situated
tures provided weaker interaction and that the retention adducts [122]. Sample sensitivity, signal intensity and
order among the columns with the different particle size complex sample preparation are major issues in analysing
approaches the behaviour of the monolithic column with oligonucleotides when performing LC–MS. Because
a particle size of 10 µm. Therefore, it can be assumed that ion-pairing reagents are needed for chromatographic
the tight and narrow pores and channels in the packed separation, the influence on MS sensitivity concerned
column influence the oligonucleotide structure, either sta- the authors. Among other things, different ion-pairing
bilising or destroying the secondary structure. This leads reagents resulted in up to an eightfold difference in the
to longer or shorter retention, depending on the target signal intensity, the type and rate of the organic solvent
oligonucleotide structure [120]. having a significant impact [123].
Predicting the outcome of a separation even before Interestingly, regarding purification, a high concen-
the actual separation is very intriguing. In one study, tration (1 µg/ml) of the internal standard, as ‘sacrifi-
this attempt was made, as we discuss in the Supporting cial oligonucleotide’, was used to saturate possible non-
Information section [121]. specific binding surfaces. The amount and type have to
be chosen appropriately as it may not be beneficial or
cause cross-talking between the different MS/MS channels
3.3.2 IP RP LC in combination with a mass [123]. An HPLC–MS strategy quantified monophosphate-
spectrometry (MS) detector substituted impurities from phosphorothioate oligonu-
cleotides. As the IP RP cannot separate these constructs
High-resolution RP IP LC electrospray ionisation and coelution happens, the MS provided the selectivity
tandem MS/MS was used to separate isomeric oligonu- to quantify the impurity while retaining the ability to
cleotides with or without adducts. Ion-pairing reagents quantify other synthesised impurities [124].
have to be chosen thoughtfully, as isomeric forms To sum it up, RP IP chromatography is not only a good
of oligonucleotides can only be separated with method to separate different sequences of oligonucleotides
2412 MINKNER et al.
but is also able to separate duplexes, RNA from DNA, 3.5 Affinity purification
stereoisomeric oligonucleotides and RNAs from lipids, and
it can be used for quantification. It can be expected that the 3.5.1 Affinity tags
range of applications will further increase, as several inves-
tigations for understanding the underlying mechanism, Affinity purification often provides a selective way to sep-
interactions and principles were performed and surely will arate the target from the impurities; thus, this technique
be performed in the near future. offers scaling-up for preparative or even industrial level
but can be costly. Affinity separation is based on selective
ligand–acceptor bindings and can be achieved, for exam-
3.4 Size exclusion chromatography ple, by Oligo(dT) as a catcher for the poly(A) tail, by amino
acid affinity, or by different tags (see also Table 2) [75].
SEC is a practical size-dependent separation, but it can be However, an additional tag can lead to an instability of
time-consuming. It is also known as gel chromatography. the construct, among other things, which can lead to mis-
The sample needs pre-treatment (e.g., prior to removing folding or aggregation, but it has to be preserved until
proteins as much as possible), and the eluate is diluted the purification [130]. In addition, the tag often needs to
[75]. However, it is a standard purification technique and is be removed from the construct, introducing further cleav-
also established for RNA purification [125]. To avoid PAGE ing and purification steps. There are different tags for
and acrylamide leftovers, agarose-based SEC can be used RNA purification, such as complementary-/aptamer affin-
after partial purification, which already removed many ity tags, which are bound to a fluorophore. This kind of
proteins with a phenol/chloroform extraction. The purity tag allows for tracking the target easily during the pro-
was over 99%, and the method is eligible for NMR anal- cess, and in the case of some fluorophores, single-molecule
ysis [105]. A 1,2-dihydroxypropane derivatised diol SEC detection can be possible [131]. Another possibility is fus-
column, followed by ICP–MS, can also be used for RNA ing RNA into ribozymes with an affinity sequence. After
affinity purification. This method was optimised for two binding, the RNA is released by adding small molecules
specific RNAs to quantify the phosphorus content. No salt to trigger a cleavage. However, more RNA than just the
was used besides the 0.1 mol/L, pH 8.1 Tris-buffer to avoid target sequence needs to be synthesised, and (depend-
unspecific RNA binding to the column. The column sepa- ing on the mechanism) additional proteins need to be
rated RNAs with sizes below 500 nt [126]. To investigate removed [132].
the structure–function relationship of S-Box RNA, SEC Besides standard tags, other useful structures can be
was used to baseline separate the RNA from the simple used, as long as they can associate with the target struc-
sample matrix E. coli in a denaturing manner with urea ture. This applies not only to oligonucleotides but also to
and obtained purity of >99%. However, this method was proteins or other substances. Therefore, an RNA aptamer
not able to baseline separate a previously crystalised S-Box also functioned as a purification tag because it had an
RNA (199 nt) from hepatitis delta virus ribozyme (67 nt). affinity to Sephadex, a widely used column material for
Also, it retained a shoulder containing precursor RNA SEC. The aptamer showed high affinity to Sephadex G-100
(277 nt) [127]. Although this still led to an improvement beads, but the recovery was relatively low at ∼30%, and
in yield, it also means that the method is at best suitable the elution was done under denaturing conditions [133].
for simple sample matrices with a large size difference Schizophyllan (SPG) may seem unusual but has an affin-
between the analytes. ity to poly(A) and poly(C) tails with a length of more than
More recent is the steric size exclusion chromatography 30 nt. An SPG-modified column was able to bind poly(A)
(SXC), in which the target interacts with the hydrophobic and poly(C) RNAs, but not poly(G) RNA, and to recover
stationary phase through mutual steric exclusion from up to 93% and 97% of the bound RNAs, using only salt-free
PEG. Originally, SXC was introduced for virus and IgG (distilled) water at 70◦ C. This system was also able to purify
purification [128]. A study investigated oligonucleotide mRNA from the yeast with a purity of approximately 87%,
purification using SXC, with interesting, informative and mRNA was not detectable in any fraction except the
results. A perfect separation of a phage genome was not elution fraction and could still be used for RT-PCR [134].
achieved for ssRNA, but for dsRNA [129]. We discuss this
in more detail in the supplement file. SEC is one possible
option for operators to purify oligonucleotides if other 3.5.2 Interaction affinity
approaches did not yield favourable results. It can be even
more beneficial in terms of recovery. Generally, the SXC Simple amino acids also show affinity to oligonucleotides,
approach may be the better choice than the traditional even with differences between DNA and RNA. This was
SEC. used to develop and validate an analytical RNA-arginine-
MINKNER et al. 2413
T A B L E 2 Publications using affinity tags for affinity separation of oligonucleotides, with binding capacity and recovery, if provided by
the respective authors
Affinity tag Binding part of sample Binding capacity Recovery Reference
Oligo(dT) Poly(A) tail [170] No detailed [170] >95% [75, 170]
numbers, but
>95%
RNA aptamer tags (w/o) Specific sequence [171]— [161] 20%–25% from crude [75, 131,
fluorophore [172]— extract 171–173]
[172] 87%
Amino acids based, arginine Nucleotide bases, [174]— [174] 96% ± 17% [75, 135, 174]
multi-contact with the
RNA backbone or RNA
bases
Amino acids based, histidine Nucleotide bases, [175]— [175] 90.0% (sRNA) and 51.5% [75, 175]
stacking/hydrophobic (rRNA)
interactions and
histidine–RNA direct
hydrogen-bonding
Aptamer D8 Dextran B512 (Sephadex) ∼50% (2.5–5 × 104 ∼30% [133, 171]
c.p.m. or
0.5–1 pmol)
Schizophyllan (SPG) Poly(A) and poly(C) ∼100% 93% (poly(C)), 97% (poly(A)) [134]
Nucleobase based, adenine Adenine–thymine (Uracil) 11.86 mg/g 47.5% [136]
(Cryogel) interactions
Complementary Specific sequence – 1–2 µg yield of ca. 200 µg RNA [137]
5ʹ-amino-modified DNA from ca. 4 mg total RNA
oligonucleotide
Abbreviations: rRNA, ribosomal RNA; sRNA, small RNA.
affinity separation on a column. The limit of detection was have their limitation. tsRNA was purified via AEC after
20 ng/µl, and linearity was confirmed up to 200 ng/µl as extraction and then enriched using affinity chromatogra-
the upper limit investigated. The coefficient of variation phy, in which 5ʹ amino-modified DNA oligonucleotides
(CV) for intra-day variability did not exceed 1.45% for dif- complementary to the target tsRNA were immobilised
ferent concentrations and 2.97% for inter-day variability. on a Sepharose column [137]. Preparative SEC, RNase
The CV for the RNA concentration accuracy was highest H-mediated RNA removal or urea-PAGE gel elution
at 5.55% for 30 ng/µl; for the higher concentrations, it was separated the tsRNA from the parental tRNA. Around 1–
at most 1.03%. This method showed a good separation of 2 µg-specific tsRNA could be purified from around 200 µg
RNA and DNA from HeLa cells or chemically synthesised of total RNA, and the final purity is approximately 83%.
RNAs. However, it was not shown what kind of separation However, the authors declared that an overestimation
from other impurities such as proteins was achieved [135]. of the purity is possible since sequencing-based RNA
Affinity separation can also be performed using rather identification can lead to bias, and therefore some RNA
uncommon materials such as cryogels. These are at low sequences are not detected [137].
temperature-produced gels, which are normally superma- Hydroxyapatite (HAP) gel-mediated fractionisation
croporous, have a spongelike-flexible structure and good proved useful for the separation of dsDNA, ssDNA,
flow dynamics. A cryogel based on adenine was produced, dsRNA and ssRNA. HAP is a complex mixed-mode
characterised and used as a chromatographic phase for separation, as it is based on metal (Ca2+ ) affinity and
the RNA purification from rotaviruses. The cryogel bound cation exchange chromatography with the negative PO4 3−
about twice as many RNA as a commercial kit. However, functional groups. Elution is done with increasing phos-
the release is with 47.5% only half as effective, so that phate concentration in the gradient. A good separation of
both approaches are similarly efficient [136]. tsRNAs ssDNA, dsDNA, dsRNA and ssRNA was achieved with
are currently in the focus of research, but as they are a model sample, but the published data of a ‘real-life’
often post-transcriptionally modified, synthetical tsRNAs sample does not allow any conclusion regarding the
2414 MINKNER et al.
purity of the different separated nucleic acids. The linker- field, the same as in gel electrophoresis. When CE is men-
amplified shotgun libraries used to investigate the viral tioned, in most cases, the capillary zone electrophoresis
composition showed DNA virus-related sequences in the (CZE) is meant. The European Pharmacopoeia (10th edi-
RNA fraction and no RNA-related sequences in the DNA tion, 4th supplement) defines CZE in chapter 2.2.47 as the
fractions. However, whether these associations are based simplest variant as for the separation the analyte is only
on DNA impurities in the RNA fraction or because of (still dissolved in the background electrolyte (BGE), and the sep-
unknown) sequence homologies is unclear. Nevertheless, aration is achieved by the different migration speed. This
HAP enabled the simultaneous investigation of RNA is based on the different charge-to-mass ratios of the ana-
and DNA viruses and allowed the metagenomic data lytes. Furthermore, it is a technique primarily for analysis,
investigation [138]. as only small amounts of sample are needed. Therefore,
only small amounts can be fractionated. Occasionally CZE
or variants can be used in a preparative manner if only
3.5.3 Indirect affinity separation, especially ng amounts of the target substances are required. One of
for special applications the CE variants is capillary gel electrophoresis (CGE). Like
PAGE, a gel (like) matrix is incorporated into the capillary,
These previously mentioned affinity separation strategies which functions as a sieving matrix to achieve a separation
were targeted approaches; however, separation can also be based on the size. CE is already established to sequence
achieved more indirectly. Double-tag purification strate- DNA to analyse miRNA as a microchip-based approach
gies are not only useful for very pure products but can, [141] and considerations have to be done for using CGE to
for example, also be used to investigate the interactions analyse oligonucleotides, such for, for example, gel matrix,
of mRNA and non-coding (nc)RNA or ribonucleoprotein sample introduction or extraction, separation conditions
complexes (RNP). In one study [139], the RNA of inter- or detection [142]. An excellent overview of CE applica-
est was fused with MS2 hairpin loops as the tag. After the tions for the analysis of oligonucleotides was given by Nai
association of ncRNA or RNP, the cells were lysed and the et al. [143].
complex bound to an added fusion protein of MS2 recogni-
tion site and GST-tag. Then, the purification was simply
done via GST-affinity magnetic beads [139]. Cross-link- 4.1.1 Aspects of modified CE methods
assisted mRNP purification is another option to purify viral
RNA, which is interacting with proteins [140]. Because it is Investigations of pulsed-field CE (PFCE) showed that this
a rather special application in this content, we discuss it in results in higher resolution of larger DNA of 1.5 kbp on
the Supporting Information section. costs of the resolution of smaller dsDNA compared to
We gave some insight into the many ways affinity purifi- standard CZE. In the case of RNA, the results are similar,
cation can be applied. Depending on the situation, it could whereby with PFCE, RNA > 1.0 kilo nucleotide (knt)
be helpful to try an ‘out of the box’ approach instead of the migrates a little bit slower. By increasing hydroxyethyl
‘standard tags’. This was, for example, done with the SPG, cellulose (HEC) polymer concentration, the resolution of
which is a polysaccharide from a fungus. Of course, the use RNA could be improved, especially for <1.0 knt, but even
of newer kinds of base material, such as cryogels, is attrac- at 1.2%, HEC subpeaks appeared, and peak widening was
tive, but these often require more fundamental research observed. Interestingly, the data suggests that RNA in com-
and are not usually considered to establish a separation bination with 4 mol/L urea has a linear mass relationship
process. It can still be useful to keep them in mind, espe- in PFCE, which would allow a correct determination of the
cially for challenging separations. It goes without saying molecular weight. On the contrary, the authors showed
that in most cases, a standard tag will provide good or even that RNA with 2 mol/L acetic acid does not offer this
excellent results and, therefore, should be used at first. linear behaviour, and therefore, the gel polymer could be
affected by this agent. The modulation depth of the pulse,
which is the ratio of the AC field to the DC field, was also
4 MISCELLANEOUS SEPARATION investigated under urea conditions. However, the correla-
PRINCIPLES tion coefficient of mobility and molecular mass decreased
with higher modulation depth, so the authors settled on
4.1 Capillary electrophoresis a relative 100% modulation depth, but depending on the
sample and settings, a higher modulation depth up to 200%
Capillary electrophoresis (CE) differs from the previously may also be beneficial. Moreover, adjustments to modu-
described chromatographic techniques as the separation is lation depth and pulse frequency were buffer dependent
mainly based on the charge, shape and size in an electric [144].
MINKNER et al. 2415
Another exciting work was performed to directly anal- temperature stress demonstrated this in the range of
yse a single-cell nucleic acid content, based on the idea of 8–60◦ C. This demonstrates the usefulness of iCIEF, espe-
a previous work [145]. An isotachophoresis (ITP) method cially for the stability analysis and sample characterisation
was used on a microfluid chip to measure rRNA from a of mRNA LNPs [148]. Using this publication, Krebs et al.
single cell. In ITP, the target analyte is focused on the inter- [149] developed the iCIEF method further to adjust it to
face by trailing electrolyte with low mobility and leading another mRNA vaccine for stability and quality investi-
electrolyte with high mobility. Spacer ions between them gations. They settled for two slightly different methods,
can lead to a sharp separation of the analytes, as the sam- in which method A is better for stability information and
ples accumulate in the interfacial area of their respective may possibly show different LNPs or LNPs with different
mobility. Single A20 cells (mouse lymphocyte cells) were mRNA loading. Method B, on the other hand, enables high
separated and lysed inside the chip in all experiments. The reproducibility pI measurement (<0.5%), and the peak
total RNA amount fluctuated, and had a mean of 14.1 pg, area also correlates well with the mRNA loading, even in
but the modal value was at 11.0 pg. It was reasoned that this stressed samples. In addition, the peak area here is less
is due to cell population heterogeneity and cell cycle dif- influenced by particle size than method A [149].
ferences. A positive relationship was confirmed between
the relative DNA and the RNA amount. The CZE data was
in good agreement with a fluorescence-activated cell sorter 4.1.2 Capillary gel electrophoresis (CGE)
(FACS) analysis. In contrast, FACS cannot provide the total
RNA amount, separation of nuclear DNA and RNA and CGE is slightly different from a standard CE, as it contains
has no fractions for further analysis. However, in the sup- gel-like structures or other matrices, which enable separa-
plement, the authors discussed that they could not be sure tion mainly by size through a sieving effect. The intrinsic
that all RNA was really separated from the nucleus DNA, charge is usually masked and a similar mass-to-charge
and therefore, the DNA measurement may be affected. ratio of the analytes is achieved. Originally, cross-linked
Lastly, even if this method is promising, further improve- polymers similar to classical gel electrophoresis were
ments have to be done for automation and (allowing for) used. However, these had several drawbacks, such as
follow-up analysis of the fractioned analytes [146]. ITP was degradation and carry-over effects. Therefore, low-
recently performed on a different microchip and used in viscous, non-cross-linking polymers were increasingly
combination with a sieving matrix to separate RNA with investigated and used. Already in 1995 [150], CGE was
sizes of 2–35 nt, >35 nt, or as single nucleotides from each investigated for the analysis of RNA using these entangled
other. The sample amount was 17 µl, and all fractions could polymers. Cross-linked polymers are not easily prepared
be recovered if desired, and 90% of single nucleotides and and handled, and their shelf-life is short. This early work
65% of RNA >35 nt were depleted from the 2–35 nt frac- transferred DNA analysis knowledge to RNA analysis
tion. In comparison, gel electrophoresis yielded better size using cross-linked and entangled polymers. Entangled
separation but lacked the recovery of the target RNA. The polymers, especially hydroxymethyl cellulose (HPMC)
usefulness of ITP was proven, as this method had a higher based, gave better results and separated low molecular
reproducibility, RNA-seq from the different fractions was weight RNA, tRNA and 5S-RNA, in a range of 30–160 bp
possible, and separation was achieved in approximately [150]. Already early on, the sieving matrix HPMC was opti-
10 min [147]. mised to separate RNA ranging from 100 to 6000 nt [151].
Imaged capillary isoelectric focusing (iCIEF) is another Molecular mass/mobility ratio, gel concentration, temper-
tool based on CE and enables analyte(s) separation via ature, electrical field and denaturants were systematically
charge and isoelectric point (pI) determination. Lough- investigated. This protocol mostly enabled the separation
ney et al. [148] characterised the surface charge of LNPs of small RNA species (tRNA, 5.8s rRNA, snRNA etc.; 60–
containing mRNA, where the chosen ampholyte mixture 300 nt) and the base line separation of 18s rRNA (1868 nt)
contained pH 5–8 and pH 3–10 in a 2:1 ratio with 10% and 28s rRNA (5025 nt) from a human glioma cell line [151].
glycerol as a stabilising agent. This method was repro- CGE was further improved to separate RNA up to 6.5 knt
ducible and showed linearity in the concentration range of by utilising semi-diluted HEC polymers as a sieving matrix
0.56–9.0 µg/ml mRNA (equivalent to 7.2–115 µg/ml lipids). with urea as a denaturant [152]. Polymer studies continued
Moreover, it was shown that the apparent pI depends as an HEC 250 polymer was used to separate rotavirus
on the ratio of cationic (charged) lipids to total lipids or RNA, after linear polyacrylamide and polyethylene oxide
the kind of cationic lipid and that the detection signal were investigated as a sieving matrix. HEC concentration
at 280 nm is generated from mRNA and lipid particles. of 1.3% (w/v), electrical field between 200 and 260 V/cm
Furthermore, the stability was also assessable, as stressed and a YOPRO-1 dye concentration of 500 nmol/L were
samples showed different peak shapes and profiles. A 24-h considered most appropriate. This method also allowed
2416 MINKNER et al.
quantification based on known concentrations of RNA via RNAs, it could be argued that 1.0% may be the better option
a calibration curve [153]. HEC developed into one of the regarding resolution. Increasing the modulation depth led
most used polymers for RNA oligonucleotide separation, to increased RNA mobility, most likely due to Joule heat-
but optimisations are still sought. The different molecular ing, which in turn led to a lower viscosity of the sieving
weight of HEC with 90k, 250k, 720k and 1300k were inves- matrix, which then decreased the resolution. Regarding
tigated to separate long ncRNA. The resolution of the RNA the resonance frequency of the oligonucleotide reorienta-
construct improved with increasing molecular weight and tion time at which mobility is lowest, the group pointed out
concentration (up to 1.4, 1.2, 1.2 and 0.6%, respectively). that it is 12.5 Hz for RNA and 31.3 Hz for DNA. From the
However, the resolution dropped for larger RNA of >4000 presented data, it seems that for RNA separation, a pulse
or >5000 nt respectively, when the concentration was frequency of ≥25 or ≤8 Hz is best for mobility, with ≥25 Hz
>0.6% by HEC 250k and 720k. By analysing the RNA appearing to be the better choice in terms of resolution
mobility in different environments, it was concluded that [157].
higher concentrations of low molecular weight HEC 90k As shown, CGE is one of the most efficient tools for
are beneficial for the separation resolution of small RNAs RNA analysis, but for large RNA, it can be insufficient.
(<1000 nt) and lower concentrations of high molecular A recently published study [158] overcame this problem
weight HEC for large RNA (>1000 nt). Optimal was 1.2% by using a denaturing, non-aqueous approach with for-
HEC 250k, which allowed a wide range of 100–10000 nt mamide instead of water in the BGE for CGE. Afterwards,
RNA to be resolved well. As expected, the running time gel optimisation resulted in a much higher resolution for
increased with higher molecular weight and higher the 1500/2000 nt peaks from an RNA ladder and was used
concentration due to the increased viscosity [154]. Poly- to analyse a putative mRNA vaccine construct. It is ben-
acrylamide is still used, despite its toxicity and possible eficial that no heating or cooling steps for the sample
non-specific interaction due to the amid group [155]. preparation or additional denaturants are needed, but as
PEG dimethacrylate (PEGDMA), which also provides a trade-off, the analysis run time increased approximately
cross linking, was used as a sieving matrix to circumvent threefold [158].
this. Using glucose and a DNA ladder, it was shown RNA integrity can also be checked in a CGE set-up with
that PEGDMA-23G-9.4% gives similar results as a 10% a 490 nm cyan light-emitting diode inducing fluorescence
polyacrylamide. Unfortunately, ammonium persulfate in a polyvinylpyrrolidone (PVP)-self-coated capillary with
and tetramethylethylenediamine are still needed for the 0.5% poly(ethylene) oxide as a separation matrix [159].
polymerisation process, which are harmful and hazardous With high separation voltage (20 kV), the separation time
substances. Moreover, the final separation was only done was decreased, but with lower voltages, the separation
with sugars cut out of mAb and not with an oligonucleotide efficiency was better as the peak height and the resolu-
sample. So, the efficiency regarding oligonucleotides still tion increased. Most likely, the fluorescence dye dissociates
needs to be evaluated appropriately [155]. from the DNA at higher voltages, which lead to a decreased
Apart from the sieving matrix, optimisation can also peak intensity. It was also shown that heat treatment is nec-
be achieved for the BGE or the denaturing agents. Differ- essary to undo RNA hybridisation [159]. PVP as a sieving
ent carboxylic acids were investigated for the BGE, and matrix and glycerol were also investigated using a design-
2 mol/L acetic acid showed the best results for RNA sep- of-experiment approach to optimise several factors of the
aration. Unfortunately, the fluorescence dye had only 20% CGE. The optimal compositions for those two were in the
intensity left and no intensity with DNA, compared to range of 1%–2% (m/m) PVP and 10%–20% (m/m) glycerol.
without acetic acid in the BGE. However, this technique The latter one increased the resolution without decreas-
is faster as this method allows in-gel denaturation without ing the separation time because the mixture’s viscosity
the need for prior in vitro denaturation [150]. In a follow-up was lower than a higher concentration of PVP only. One
investigation, the method was further improved by switch- optimised gel composition (1.32% PVP, 10% glycerol and
ing back to formaldehyde as a denaturant, but with 5% 112.5 mM HEPES) led to a better resolution with decreased
1,2,5-thiadiazole as an additive. With this, the detection separation time than a commercially available gel [160].
limit was further 10-fold lowered while suppressing the Another sieving matrix option is 1% polyethylene oxide
Joule heating with a lower conductivity at neutral pH [156]. with ethidium bromide as fluorescence dye, in which 28S
PF is also an option for CGE and was investigated to sep- rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA were sep-
arate RNA. In the following study, alkaline separation with arated with an LoD of 100 pg/µl. However, sample injection
PFCGE was faster, but the resolution decreased, which was time needed to be increased to 6 min, and the separation
compensated by increasing HEC concentration. The ini- voltage had to be decreased to 5 kV, as the dissociation of
tial concentration of 0.8% HEC was considered the best the dye increased at a higher voltage. Therefore, the signal
choice in terms of separation time. In the case of smaller intensity decreased, but the resolution also increased at a
MINKNER et al. 2417
lower voltage, similar to a previously mentioned study (see such as higher ionic strength, heating, a ligand with higher
also Ref. [159]). This method showed the overexpression binding affinity or pH changes [164].
of 5S rRNA in ovarian cancer cells compared to cervical
cancer cells [161].
4.2.1 Direct affinity separation with MNPs
4.1.3 Special CE microchip separations A classical direct approach uses complementary DNA.
RNA was purified via modified magnetic beads in one
Similar to CGE, RNA, DNA or proteins can be separated study, which presented a complementary DNA hairpin
on a microchip using the same sieving matrix. However, structure on the surface. RNA could be separated from
it is also possible to use different materials, such as an a DNA-/RNA-mixture, as the DNA–RNA-hybrid has a
SnO2 nanowire structure [162]. The same group also higher melting temperature than the DNA–DNA-hybrid,
published a follow-up study that now uses nanopillars and, therefore, could be eluted later. A hairpin was chosen
and nanoslits [163]. Both studies are so interesting that due to its higher selectivity towards the complementary
we discuss them in detail in the Supporting Information string. The fluorescence data from a self-made microgel-
section. based optical device (etalon) indicates a high purity
CE is a good option for further analysis of oligonu- [165].
cleotide samples. Currently, the focus is clearly on CGE Boronated nanoparticles are another possibility because
applications for separation by size. This is achieved boronate affinity chromatography is a technique to sep-
with HEC, as one main sieving matrix at the moment. arate molecules with a cis-diol-group under basic con-
Other compounds, such as BGE or the denaturants, for ditions, for example, glycopeptides, saccharides or RNA.
example, acetic acid, formaldehyde or formaldehyde/1,2,5- This is beneficial, as DNA is missing the 2′-hydroxyl group
thiadiazole mixtures, need to be considered. CGE is a good and cannot be captured. Under optimised conditions of
working horse for separation by size and allows fast and these non-MNPs, the RNA binding capacity was 172 mg/g.
automated runs. However, depending on the case, the clas- However, with an additional 100 mmol/L BaCl2 , 601 mg/g
sic gel electrophoresis may be acceptable for the desired could be achieved. This was explained by BaCl2 disturbing
application if a state-of-the-art protocol is used, which is the stacked conformation of the RNA bases, and therefore,
recommended. Besides this praise for CGE, one should not they are accessible to the boronated particles. Elution was
forget the other CE applications, such as iCIEF, which can performed with a 0.1 mol/L borate buffer (pH 8.5) with
separate and characterise RNA-loaded LNPs [148]. Such 0.5 mol/L NaCl. Particles were washed after each purifi-
applications are especially interesting for pharmaceutical cation cycle with 50 mmol/L NaCl and water. The authors
drug analysis. did not mention how the particles were separated from the
sample solution prior to elution, but as the particles are
not magnetic, it was most likely done via centrifugation.
4.2 Nanoparticle (NP) or magnetic The elution efficiency of the particles was determined to be
nanoparticle (MNP) separations 78%. It was also concluded that the particles are reusable, as
after 10 purification rounds, the binding capacity dropped
Magnetic or non-magnetic nanoparticle (MNP) separa- only slightly. As the samples were the same, no infor-
tions are considered a section here, even if they usually mation on cross-contamination is available if different
apply the previously mentioned separation principles, samples would be used [166].
especially affinity-based ones (see Sections 3.5.1 and 3.5.2).
Nanoparticles can be an additional and helpful option for
oligonucleotide separation. MNPs advantageously allow 4.2.2 Indirect affinity separation with MNPs
purification directly from the crude sample without much
equipment. For oligonucleotide purification, the mag- MNPs can also be used to purify the target indirectly.
netic particles are usually coated with polymers, which To investigate miRNA interactions with mRNA, the cor-
exhibit affinity to DNA and/or RNA, but complementary responding miRNA needs to be purified prior to this.
DNA/RNA sequences or DNA-binding proteins are also This was achieved by incubating the cell samples in
possible. The only important aspect is that even if the sur- formaldehyde with mRNAs, specifically cross linking
face has a high and specific affinity for the DNA/RNA, the miRNA and a protein [167]. After that, specifically
the binding can be released by an appropriate buffer sys- designed biotinylated DNA anti-sense oligonucleotides
tem without damaging the magnetic particles and the were added, which bind only to the specific mRNA.
DNA/RNA. The elution can be achieved via various means This mRNA:miRNA:DNA complex was then purified
2418 MINKNER et al.
using magnetic streptavidin beads. After elution of the 6 CONCLUDING REMARKS

mRNA:miRNA:DNA complex from the beads, DNase and
proteinase were used to remove DNA and proteins from The separation of oligonucleotides or, even more spe-
the complex before the cross linking was reversed. This cific, the separation of RNA is a broad topic. Particularly
purification strategy shows the benefit of using anti- mRNAs recently emerged, which are large and highly
sense oligonucleotides and magnetic beads for (affinity) charged molecules. Size and charge create options for
purification [167]. separation but can also lead to separation difficulties,
The last study is only mentioned on a side note, as this depending on the method. Precise, accurate and robust
is for a unique application. In translating ribosome affin- methods to analyse mRNA vaccines are urgently needed
ity purification (with magnetic beads), specific in situ cells to guarantee these pharmaceuticals’ high-quality stan-
are transfected to express a tag, for example, enhanced dards and be prepared for future oligonucleotides-based
GFP, on the large ribosomal subunit protein L10a. This developments.
way, the total RNA information of one specific cell type Even though mRNA has been a relevant topic for a long
can be investigated at one point in time. If the ribosomes time, experience from biological research, for example,
are purified, the RNA translated at this moment will be with different oligonucleotide species or proteins, cannot
co-purified, presupposed, the ribosome is maintained on be transferred without appropriate adjustments. The aims
the RNA. This is an indirect way for affinity purification, of the former and desired quality control methods are too
and for some applications, it may be a way to separate the different. General properties, sample pre-treatments, and
targeted oligonucleotides [168]. valuable equipment and reagents can still be derived from
We discussed that MNPs are a good option for small- the scientific literature. Some excellent methods for (AEC
scale (affinity) purification of oligonucleotides. They are a and IP RP) chromatography and electrophoresis can also
valuable tool for pre-treatment, after which further purifi- be adjusted for quality control.
cation steps are applied. The focus here was mostly on Therefore, we try to give some insight and highlight
specific separations and one optimisation, but their benefit interesting aspects (see Table 4). For example, established
became nicely visible because they were successful in these techniques such as precipitation, extractions and spin
complex tasks. Therefore, it can be confidently assumed columns have benefits, especially for laboratory scale and
that they can be successfully used for less challenging pre-treatment. Something similar can be said regarding gel
separations. electrophoresis, but this technique is more suitable as an
analytical tool than as a preparative one, even if the latter
is possible as well. For all these methods, it should be made
5 TOXICOLOGICAL CONCERNS OF clear that the protocols are the state of the art to ensure the
UTILISED REAGENTS best known outcome for the intended purpose.
Similarly, chromatography is the main focus, as it is a
Substances used for separations may be toxic or are at least broad topic. Especially here, it is beneficial to know as
not suitable for administration to (especially ill) humans much as possible about the investigated target to facilitate
or animals. Nevertheless, these reagents often cannot (yet) the choice of the separation conditions. Among the
be replaced in separation processes. Therefore, these sub- various principles, AEC is the first choice for separating
stances must be removed in further purification steps. This oligonucleotides of different lengths with N − x deletions.
is usually not necessary when separating for analysis, as If, on the other hand, oligonucleotides with a different
they are often destructive, and the consumed samples are base sequence need to be separated, the IP RP chromatog-
not processed further. The need and the way of removal raphy is the best choice. Moreover, it is very versatile
are case dependent. Cleaning steps can include all types of and can be used to solve other separation problems such
methods, not only those mentioned in this overview, such as duplexes, RNA/DNA mixtures and stereoisomeric
as SEC, desalting or AEC. oligonucleotides. If separation can be achieved solely
For example, cleaning steps are mandatory if the sub- relying on size, one should perform SEC or rather SXC, as
stance is used for pharmaceutical purposes. This results in the recovery is usually more favourable. If the separation
the need to evaluate the reagents used for toxicity and to is not achievable with these mentioned ones, the highly
check whether they are still present after the final intended specific affinity purification will most likely be able to
purification step. Evaluating all reagents used is beyond solve it, even though you may also face obstacles there. It
the scope of this review, but we selected several critical can be worthwhile to think outside of the box for affinity
and typical reagents and provided their classification by tags, not only for special applications but also in general.
the European Chemicals Agency (see Table 3). This aspect also includes the MNPs, which usually apply
MINKNER et al.
TA B L E 3 Toxicological assessment of selected substances that can be used for the separation of oligonucleotides
Globally harmonised
system and additional Derived no effect level
Substance For example used in classification Maximum exposure limit (DNEL)
Acetonitrile LC, IP RP (Section 3.3) Flammable, corrosive, health Lt. exposure limit: 70 mg/m3 Lt. (Inh): 70 mg/m3
hazard St. (Inh): 102 mg/m3
Lt. (oral): 400 µg/kg bw/day
St. (oral): 600 µg/kg bw/day
Acrylamide PAGE, CGE (Sections 2.2 Serious health hazard, acute Lt. exposure limit: 0.1 mg/m3 Lt. (Inh): 70 mg/m3
and 4.1.2) toxicity, carcinogenic, St. (Inh): 120 mg/m3
mutagenic, suspected to be
toxic to reproduction, skin
sensitising
Chloroform Extraction (Section 2.1) Serious health hazard, acute Lt. exposure limit: 10 mg/m3 Lt. (Inh): 2.5 mg/m3
toxicity, suspected to be Emission limit values; average St. (Inh): 5 mg/m3
carcinogenic and toxic to limit in water: 2.5 µg/L Lt. (oral): 0.33 mg/kg bw/day
reproduction
Dibutylammonium IP RP (Section 3.3) Health hazarda
acetate
Ethanol Precipitation, LC (Section 2.1) Flammable, serious health Lt. (Inh): 950 mg/m3
hazard Lt. (oral): 87 mg/kg bw/day
Formaldehyde PAGE, sample preparation Corrosive, serious health Lt. exposure limit: 0.37 mg/m3 Lt. (Inh): 3.2 mg/m3
(Sections 2.2 and 3.5.3) hazard, acute toxicity St. exposure limit: 0.74 mg/m3 Lt. (oral): 4.1 mg/kg bw/day
Guanidine Sample preparation (Section Health hazard Lt. (Inh): 3.5 mg/m3
hydrochloride 3.2) St. (Inh): 10.5 mg/m3
Hexafluoroisopropanol IP RP (Section 3.3) Corrosive, health hazard NOAEL (rat): 60 mg/kg
(HFIP) bw/day
LOAEL (rat): 300 mg/kg
bw/day
(Continues)
2419
2420
TA B L E 3 (Continued)
Globally harmonised
system and additional Derived no effect level
Substance For example used in classification Maximum exposure limit (DNEL)
Hexylammonium IP RP (Section 3.3) No data
acetate (HAA)
Lithium bromide Precipitation, LC (Section 2.1) Health hazard Lt. (oral): 1.09 mg/kg bw/day
St. (oral): 3.27 mg/kg bw/day
Lithium chloride Precipitation, LC (Section 2.1) Health hazard Lt. (oral): 7.32 mg/kg bw/day
St. (oral): 21.96 mg/kg bw/day
Methanol Precipitation, LC, CE Flammable, serious health Lt. exposure limit: 260 mg/m3 L/S (Inh): 130 mg/m3
(Sections 2.1 and 3) hazard, acute toxicity L/S (oral): 4 mg/kg bw/day
Phenol Extraction (Section 2.1) Corrosive, serious health Lt. exposure limit: 8 mg/m3 Lt. (Inh): 8 mg/kg bw/day
hazard, acute toxicity, St. exposure limit: 16 mg/m3 St. (Inh): 16 mg/kg bw/day
suspected to be mutagenic Lt. (oral): 500 µg/kg bw/day
Polyethylene glycol SXC (Section 3.4) Lt. (Inh): 40.2 mg/m3
(PEG) Lt. (oral): 40 µg/kg bw/day
Sodium perchlorate AEC (Section 3.2) Oxidising, health hazard Lt. (Inh): 280 µg/m3
Triethylamine (TEA) IP RP (Section 3.3) Flammable, corrosive, health Lt. exposure limit: 8 mg/m3 Lt. (Inh): 8.4 mg/m3
hazard St. exposure limit: 8.4 mg/m3 St. (Inh): 12.6 mg/m3
Triethylammonium IP RP (Section 3.3) Health hazard
bicarbonate (TEAB)
Triethylammonium IP RP (Section 3.3) Health hazard
acetate (TEAA)
Triton-X 100 Extraction (Section 2.1) Corrosive, health hazard,
hazardous to the
environment, endocrine
disrupting
Note: Within the EU, national regulations may differ, but at most, they may be set stricter, not weaker.
Abbreviations: AEC, anion exchange chromatography; CE, capillary electrophoresis; CGE, capillary gel electrophoresis; Inh., inhalation; IP RP, ion-pair reversed-phase; L/S, long/short term; LOAEL, lowest observed
adverse effect level; Lt., long term; NOAEL, no observed adverse effect level; St., short term; SXC, steric exclusion chromatography.
a
Data is derived from dibutylammonium chloride.
Source: Data are from the European Chemicals Agency: https://echa.europa.eu/de/home (as of 22.06.2022).
MINKNER et al.
MINKNER et al.
TA B L E 4 Schematic overview: For different separation purposes, different separation techniques are recommended, indicated by the plus marks
Small
Ion scale:
Gel RP pair Affinity Laboratory Spin LiCl
electrophoresis SEC/SXC MS CE AIEX LC RP (tag) protocols Kits columns precipitation
Investigated Integrity + +++ ++ ++
separation
purpose
Size +++ +++ +++ +++ +
Length (N − x ++* ++ ++ +++ ++
deletion)
Purity + ++ +++ ++ +++
Stereo isomeric +++ +++ +
isolation
Sequence + ++ +++
Isoelectric point
+++icEIF
DNA/RNA ++ +++ +++ ++ ++ ++ ++
separation
Note: This scheme is very general and simplified; not marked methods may be still applicable for an intended purpose but not the first choice based on our literature search. The amount of the plus marks represents a
suggested level of recommendation for the use of the methods. +++: strongly recommended; ++: recommended; +: suitable; ++*: gel electrophoresis is only suitable if there is a minimal length difference between the
oligonucleotides which can be resolved in the gel; +++icEIF : this applies only to icEIF.
Abbreviations: CE, capillary electrophoresis; MS, mass spectrometry; RP, reversed-phase; SEC, Size exclusion chromatography; SXC, steric exclusion chromatography.
2421
2422 MINKNER et al.
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175. Martins R, Queiroz JA, Sousa F. Histidine affinity

chromatography-based methodology for the simultaneous How to cite this article: Minkner R, Boonyakida
isolation of Escherichia coli small and ribosomal RNA. Biomed J, Park EY, Wätzig H. Oligonucleotide separation
Chromatogr. 2012;26:781–8. techniques for purification and analysis: What can
we learn for today’s tasks?. Electrophoresis.
S U P P O RT I N G I N F O R M AT I O N 2022;43:2402–2427.
Additional supporting information can be found online https://doi.org/10.1002/elps.202200079
in the Supporting Information section at the end of this
article.

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Received: 30 March 2022 Revised: 9 September 2022 Accepted: 23 September 2022

Oligonucleotide separation techniques for purification and

Robert Minkner1 Jirayu Boonyakida2,3 Enoch Y. Park2,3 Hermann Wätzig1

2402 www.electrophoresis-journal.com Electrophoresis 2022;43:2402–2427.

1 INTRODUCTION Engineered RNAs include ribozymes, riboswitches and

1.4.2 Enzymatic synthesis (IVT) of RNAs 2.1 Extraction, precipitation and

TA B L E 1 Example ion-pair reversed-phase (IP RP) chromatography methods

using magnetic streptavidin beads. After elution of the 6 CONCLUDING REMARKS

175. Martins R, Queiroz JA, Sousa F. Histidine affinity

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