Journal of Chromatography A, 1355 (2014) 1–14
`
`Contents lists available at ScienceDirect
`
`Journal of Chromatography A
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h r o m a
`
`Review
`Ribonucleic acid purification
`R. Martins, J.A. Queiroz, F. Sousa ∗
`
`CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 25 January 2014
`Received in revised form 23 May 2014
`Accepted 27 May 2014
`Available online 6 June 2014
`
`Keywords:
`Affinity chromatography
`Amino acids
`Isolation methods
`Purification
`RNA
`
`Research on RNA has led to many important biological discoveries and improvement of therapeutic
`technologies. From basic to applied research, many procedures employ pure and intact RNA molecules;
`however their isolation and purification are critical steps because of the easy degradability of RNA, which
`can impair chemical stability and biological functionality. The current techniques to isolate and purify
`RNA molecules still have several limitations and the requirement for new methods able to improve RNA
`quality to meet regulatory demands is growing. In fact, as basic research improves the understanding
`of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic
`tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the
`purification of biological compounds, requiring high purity degree. In addition, its application in bio-
`pharmaceutical manufacturing is well established. This paper discusses the importance and the progress
`of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In
`particular and in view of the high specificity, affinity chromatography has been recently applied to
`RNA purification processes. Accordingly, recent chromatographic investigations based on biorecogni-
`tion phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been
`used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose
`applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential
`contribution of these methods to overcome the challenges of RNA purification.
`© 2014 Elsevier B.V. All rights reserved.
`
`Contents
`
`1.
`2.
`3.
`
`4.
`
`3.2.
`
`Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
`RNA chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2
`RNA isolation and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
`3.1.
`RNA isolation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
`3.1.1.
`Chemical and solid-phase extractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4
`3.1.2.
`Polyacrylamide gel electrophoresis (PAGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
`3.1.3.
`Lithium chloride (LiCl) precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`6
`RNA purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
`3.2.1.
`Reverse phase (RP) and ion pairing (IP) RP chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
`3.2.2.
`Size exclusion chromatography (SEC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
`3.2.3.
`Anion exchange chromatography (AEX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`7
`RNA affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
`4.1.
`Oligo(dT) chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
`4.2.
`RNA affinity tags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
`4.3.
`Amino acid-based affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`9
`4.3.1. Histidine affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
`4.3.2.
`Arginine affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`11
`
`∗ Corresponding author. Tel.: +351 275 329 002; fax: +351 275 329 099.
`E-mail address: fani.sousa@fcsaude.ubi.pt (F. Sousa).
`
`http://dx.doi.org/10.1016/j.chroma.2014.05.075
`0021-9673/© 2014 Elsevier B.V. All rights reserved.
`
`(cid:38)(cid:56)(cid:53)(cid:40)(cid:57)(cid:36)(cid:38)(cid:3)(cid:40)(cid:59)(cid:21)(cid:19)(cid:19)3
`Page 1 of 14
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`

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`2
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`5.
`
`R. Martins et al. / J. Chromatogr. A 1355 (2014) 1–14
`
`Conclusion and future trends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
`Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`12
`Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`12
`
`1. Introduction
`
`Until recently, RNA was overlooked compared to DNA or pro-
`teins, consigned to a simple intermediate role in the flow of
`information from genes to functioning molecules in living cells.
`RNA is now known to play many more functional roles and to be
`responsible for a multitude of essential biological processes [1]. In
`the last 20 years RNA was the subject of four Nobel prizes win-
`ning discoveries – 1989 for catalytic RNA, 1993 for splicing, 2006
`for RNA interference (RNAi), and 2009 for the ribosomal struc-
`ture [2] and new roles for RNA in biology continue to emerge at a
`glance. All of these discoveries have revealed so far that RNA is truly
`a remarkable and multi-talented cellular component with funda-
`mental implication on biotic evolution and heredity. Furthermore,
`the widespread involvement of RNA in the regulation of numer-
`ous genes has highlighted its vast therapeutic potential [3]. These
`and similar breakthroughs have led to the emergence of numerous
`types of RNA-based therapeutics either using RNA as a therapeutic
`agent or a therapeutic target. Table 1 shows potential therapeu-
`tic approaches for RNA, specifying the involved RNA or RNA-based
`molecules and their mechanism of activity.
`The successful results of these novel therapeutic approaches are
`reinforcing the focus on RNA investigation and are rendering RNA
`molecules into new targets for pharmaceutical and biotechnolo-
`gical industries [4]. Due to the increasing number of structural,
`biophysical and biomedical studies that require large quantities of
`homogeneous good-quality RNA, a widespread need to improve
`production scale and RNA isolation and purification schemes has
`been recognized.
`As RNA emerges into the new class of biotherapeutic products,
`pharmaceutical-grade RNA, produced under the current good man-
`ufacturing practice (cGMP) is crucial. Thus, it will be essential that
`the bioproduct fulfil the requirements of regulatory authorities
`such as Food and Drug Administration (FDA), European Medicine
`Agency (EMA) and World Health Organization (WHO). However,
`non-consensus still exists for regulation of mRNA vaccination and
`RNA oligonucleotides-based therapies. In the European Union,
`mRNA-based therapies are based on the regulation for advanced
`therapy medicinal products – EC No 1394/2007 – [5] which refers to
`directive 2001/83/EC [6]. Here, a ‘Gene therapy medicinal product
`is an active substance which contains or consists of a recombi-
`nant nucleic acid used in or administered to human beings’. In
`the United States of America, in contrast, mRNA vaccines are not
`categorized as gene therapy [7]. On the other hand, RNA oligonu-
`cleotides products, such as siRNAs or aptamers, are regulated as
`drugs under FDA’s Centre for Drug Evaluation and Research and are
`not considered as an advanced therapy in the European Union (not
`classified as gene therapy) [8]. Currently, no regulatory authority
`has formal guidelines available for RNA oligonucleotide products
`or mRNA molecules. The guidelines established for human cell-
`based medicinal products [5] and DNA vaccines [9] are providing
`the guidance in the regulatory framework for RNA-based therapies
`[8,10]. Nevertheless, those guidelines do not focus on particular
`features of RNA, such that as shown in clinical trials, RNA-based
`therapies do not confer the risks of integration into the genome. The
`scientific community, in the form of volunteer members from the
`industry and regulatory agencies worldwide, are actively engaged
`in addressing topics such as quality specifications and impurities
`in RNA bioproducts [11].
`
`Consequently, economically feasible processes for RNA isolation
`and purification, as well as the implementation of methodologies
`able to control RNA quality suitable for industrial manufacturing,
`will be increasingly necessary, especially when the RNA products
`are finally released to the market.
`However, the isolation and purification of RNA are critical steps
`because of the easy degradability of RNA, consequence of the pecu-
`liar structural chemistry, which can impair chemical stability and
`biological functionality, and can limit the success of subsequent
`RNA investigations.
`Therefore, this paper focuses the challenging task of isolating
`and purifying RNA molecules. The current state of the isolation
`and purification methodologies used for RNA preparation will be
`discussed regarding the growing demands in RNA applicability.
`Moreover, promising affinity approaches based on chromato-
`graphic purification exploiting the biorecognition between amino
`acids ligands and RNA molecules will be introduced. These new
`strategies bring new insights into the way RNA can be purified,
`contributing to the future development of new and more robust
`bioseparation methods.
`
`2. RNA chemistry
`
`RNA has a number of unique chemical characteristics that have
`profound structural consequences with remarkable implications in
`cell biology and are a real challenge in research activities.
`RNA is a polymer organized in a long chain of ribonucleotide
`monophosphates, but it resembles DNA in many ways (Fig. 1). RNA
`shares the same chemical units as DNA, however there are two
`fundamental differences that distinguish DNA from RNA. RNA has
`the nucleotide uracil (U) instead of thymine (T) and the ribose
`2(cid:3)-OH group on each RNA nucleotide is absent in DNA. Conse-
`quently, the deoxyribose sugar in DNA is less reactive because
`of C-H bonds. This leads to a greater resistance of DNA to alka-
`line hydrolysis. Accordingly, RNA is less stable than DNA because
`its vicinal 2(cid:3)-OH group makes the 3(cid:3)-phosphodiester bond sus-
`ceptible to nucleophilic cleavage, and so it is readily hydrolyzed
`by hydroxide ions [12]. In addition, RNAs adopt dissimilar shapes
`when they are base-paired into a double helix. RNA takes on the
`geometry structure referred as an A-form helix while DNA takes
`on the B-form. DNA is commonly found in a double-stranded
`structure while RNA often comes as single-strand and is quite
`flexible [13]. However, single-stranded DNA can also occur as an
`intermediate in some biological processes. During these processes,
`single-stranded DNA assume specific folded structures to perform
`essential biological functions during DNA replication, recombina-
`tion, repair, and transcription [14]. In its turn, RNA can twist itself
`into a variety of complex structures. Its propensity to form sec-
`ondary structures facilitates RNA interactions with other molecules
`by covering some sequences and exposing others for recognition.
`Besides, RNA can assume tertiary structures that present surfaces
`for interactions and contain internal environments that create
`binding sites for metal ions, so that they can promote catalytic
`reactions [15].
`The high chemical reactivity of RNA provides more instability
`to the molecule, increasing the susceptibility to degradation. This
`instability is very important for cells, as they can change their pat-
`terns of protein synthesis very quickly in response to biological
`needs [16].
`
`Page 2 of 14
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`

`

`Table 1
`RNA molecules with therapeutic involvement.
`
`RNA type
`
`Cell function
`
`Therapeutic concept
`
`References
`
`R. Martins et al. / J. Chromatogr. A 1355 (2014) 1–14
`
`3
`
`Protein synthesis
`Messenger RNA (mRNA)
`Ribossomal RNA (rRNA)
`Transfer RNA (tRNA)
`
`Codes for protein
`Translation
`Translation
`Apoptosis regulation
`Post-transcriptional modification or DNA replication
`Splicing and other functions
`Small nuclear RNA (snRNA)
`
`Small nucleolar RNA (snoRNA)
`
`Nucleotide modification of RNAs
`
`Regulation
`Antisense RNA
`
`MicroRNA (miRNA)
`
`Small interfering RNA (siRNA)
`
`Ribozymes
`
`Riboswitch
`
`Aptamers
`
`Transcriptional attenuation
`mRNA stabilization or degradation
`Translation block
`mRNA cleavage and Translation
`repression
`mRNA cleavage and Translation
`repression
`RNA enzyme. Catalyze RNA cleavage
`and ligation reactions
`Regulate gene expression by binding to
`small metabolites
`Oligoribonucleotide part of a
`riboswitch that binds to a specific
`target molecule with high affinity
`
`These properties consign RNA versatility in cellular processes,
`namely in gene regulation, which open the possibility of exploring
`new therapeutic opportunities [17]. On the other hand, the pecu-
`liar three-dimensional compaction and structural instability of RNA
`are huge challenges in laboratory, as the biological activity and
`integrity can be easily compromised during extraction and purifi-
`cation procedures. Thus, improved methodologies for recovering
`RNA of high quality is a constant concern [18–20].
`
`3. RNA isolation and purification
`
`RNA methods differ from those used for DNA and proteins [2].
`The extraction, isolation, and analysis of RNA are routinely more
`difficult in comparison to that required for DNA. As raised before,
`RNA chemistry adds complexity to sample preparation because
`the ubiquitous presence of RNA-degrading enzymes (RNases) both
`in biological samples and in the laboratory environment easily
`degrade RNA, compromising the integrity and biological activity
`of RNA molecules [18]. Therefore, minimizing RNA degradation
`by protecting it against RNases requires that all glassware, plastic
`ware, instrument tubing and reagents be RNase free. Additionally,
`among the many challenges is the need for maximizing recovery
`
`Fig. 1. Structural characteristic of RNA. RNA differs from DNA in the nucleotide
`uracil, as a thymine exists in DNA, as well as the constituent sugar molecule that
`is a ribose in RNA and a desoxyribose in DNA. DNA molecules take a double helix
`structure, while RNA molecules are originally synthesized as single-strands.
`
`Vaccination
`Antibiotic target
`Understand many human
`diseases
`
`Understand many human
`diseases
`Understand many human
`diseases
`
`Kreiter et al. [7]
`Tenson and Mankin [118]
`van Raam and Salvesen [119], Belostotsky
`et al. [120]
`
`Matera et al. [121]
`
`Kiss [122]
`
`Inhibitor of mRNA translation
`
`Dias and Stein [123], Brantl [124]
`
`Gene silencing
`
`Gene silencing
`
`mRNA reprogramming and
`repair
`Regulate gene expression
`Antibacterial drug target
`Decoy mechanism that inhibits
`various target proteins
`
`Kusenda et al. [125], Lin et al. [126]
`
`Doench et al. [127], Ghildiyal and Zamore
`[128]
`Phylactou, et al. [129]
`
`Tucker and Breaker [130], Wittmann and
`Suess [25], Blount and Breaker [131]
`Mayer [132], Ni et al. [133]
`
`yield, while removing unwanted components, minimizing sam-
`ple transfers, and avoiding non-specific binding to containers [21].
`Therefore, the quality and quantity of RNA preparations are the
`main concerns of isolation procedures, since the lack of integrity,
`the presence of contaminants or the low RNA quantity may strongly
`constrain the success of several RNA based-procedures in basic and
`clinical research [22]. Additional challenges also emerge with the
`advance of clinical trials using RNA intended to be administered in
`humans.
`Presently, RNA molecules can be obtained by extraction from a
`biological matrix, such as cells or tissues, or they can be produced by
`chemical or enzymatic (in vitro transcription) synthesis. Chemical
`synthesis is normally used for the generation of short oligoribonu-
`cleotides (<50 nucleotides) while in vitro transcription can produce
`longer RNAs. Synthesized RNAs are being greatly employed in
`structural, biochemical and biophysical studies [23,24] as well as
`in the development of new therapeutic approaches by RNA inter-
`fering technology, RNA aptamers, ribozymes or mRNA vaccination
`[25–27]. In these cases, the final RNA product needs to be purified
`from impurities derived from the synthesis process. These impuri-
`ties are, besides enzymes, nucleotides, aberrant oligonucleotides,
`salts or buffer. Longer oligoribonucleotides are more contaminated
`with aberrant species than short ones. The failure products are
`prematurely halted as shorter oligonucleotides. Some are mis-
`match failure sequences where there are missing nucleotides in
`the middle of the sequence, rather than at the end. Other by-
`products of synthesis may have greater molecular weight than the
`target oligoribonucleotide (heterogeneous RNAs in length). This
`is a result of incomplete post-synthesis deprotection, or due to
`the branching of an oligonucleotide backbone during the synthesis
`[28].
`On the other hand, biological RNAs are preferably used in basic
`research for the study of cellular mechanisms, as they reflect intrin-
`sic cell features [29], in clinical investigations for pharmacokinetic
`and pharmacodynamics analysis [30] and in some strategies of
`mRNA vaccination using bulk tumour mRNA [31]. In RNA extrac-
`tion from a biological matrix, the main principle is the disruption
`of cells and subsequent elimination of host contaminants, such as
`genomic DNA (gDNA) and proteins, in order to obtain intact and
`pure RNA molecules [32].
`
`Page 3 of 14
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`4
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`R. Martins et al. / J. Chromatogr. A 1355 (2014) 1–14
`
`Table 2
`Isolation techniques used in RNA preparation (nt, nucleotides; PAGE, polyacrylamide gel electrophoresis; cGMP, current good manufacture practice).
`
`Method
`
`Principle
`
`Advantages
`
`Disadvantages
`
`References
`
`Acid guanidinium
`thiocyanate–phenol–chloroform
`
`Silica purification
`
`Preparative denaturing
`PAGE
`
`Lithium chloride
`precipitation
`
`Chaotrope helps cells lysis and
`inactivates RNases. DNA and
`Proteins are denatured and
`removed in the organic phase
`during acid phenol/chloroform
`extraction. RNA is precipitated
`with alcohol and salt.
`Chaotrope helps cells lysis and
`inactivates RNases. Polyanionic
`RNA and DNA bind to silica
`particles by hydrogen
`interaction in the presence of
`chaotrope. DNA is digested
`with DNase. Contaminants are
`washed away and RNA is
`eluted with low ionic strength.
`Separates molecules based on
`their electrical charge and
`hydrodynamic properties,
`which are a function of chain
`length. The desired RNA is
`eluted from the gel matrix,
`concentrated, equilibrated in
`buffer and refolded.
`Selective separation of long or
`short RNA sequences from
`impurities. Elevated
`concentration of LiCl is added
`to impure RNA preparations
`−20 ◦C
`follow by incubation at
`for several hours or overnight.
`Precipitates or supernatants
`are recovered according to
`required RNA type.
`
`Inexpensive
`Enhanced protection
`against RNases
`Good yields and purity
`
`Requirement of
`toxic chemicals
`Time-consuming
`Highly operator dependent
`Inhibit enzyme activity
`
`Chomczynski and Sacchi
`[32]
`
`Fast
`Does not require use of
`organic solvents or
`alcohol precipitation
`Amenable to
`automation
`
`Low binding capacities
`Purification based on
`adsorption/desorption
`mechanisms on solid surfaces.
`Does not discriminate between
`RNA or DNA
`Requires DNase treatment
`
`Wen et al. [38]
`
`Highly purified product
`
`Time-consuming
`Introduces contaminants
`Uses denaturing conditions
`Low yields
`
`Doudna [24], Hagen and
`Young [51]
`
`Separation of small
`RNAs from long RNAs
`Recovery of long RNAs
`from impurities of
`in vitro transcription
`synthesis.
`
`Inefficient precipitations
`Introduces lithium metal into
`preparations
`Employ phenol/chloroform
`extraction to improve isolation
`Time consuming
`
`Pascolo [55], Baker, et al.
`[56], Nilsen [57],
`Romanovskaya, et al. [60]
`
`Many methods have been developed in an attempt to circum-
`vent the several challenges of purifying RNA molecules and to
`achieve the goal of good-quality RNA [33–36]. Therefore, consid-
`ering the recent developments in RNA understanding and the
`growing demand on its purification, the next discussion intends
`to briefly introduce the isolation and purification methods used in
`RNA preparation. This description will draw particular attention
`to the main problems that can limit the success of RNA research
`or that can make the procedures not suitable to obtain RNA to
`be further applied in clinical investigation, also focusing on the
`cost-effectiveness for preparative-scale or large-scale industrial
`applications (Tables 2 and 3).
`
`3.1. RNA isolation methods
`
`3.1.1. Chemical and solid-phase extractions
`Traditionally two types of isolations are used in RNA prepara-
`tions, (1) chemical extraction using denaturing agents and organic
`solvent precipitation and (2) solid-phase extraction by immobi-
`lizing RNA on a glass support. These methods generally include a
`chaotropic agent, denaturant, or other chemical in the lysis step to
`inactivate RNases.
`Although the RNA extraction methods are similar to DNA pro-
`cedures, the main difference is the working pH. RNA is extracted
`under acidic conditions, while for DNA mild alkaline extractions
`are preferred [37]. This difference is related to the chemical sta-
`bility of each molecule, as previously discussed (Section 2). The
`acidic pH is the critical factor to ensure the separation of RNA
`from DNA and proteins [32,33]. Therefore, chemical extraction
`that involves acid phenol/chloroform extraction is currently the
`most employed method either performed with home-made solu-
`tions or commercial ready-to-use reagents, because it leads to high
`recovery yields and purity of total RNA [32]. Briefly, this technique
`
`allows RNA separation from DNA and proteins after extraction with
`an acidic solution consisting of guanidinium thiocyanate, sodium
`acetate, phenol, and chloroform that allow the formation of two
`phases. RNA remains in the upper aqueous phase of the whole mix-
`ture, while proteins and DNA remain in the interphase or lower
`organic phase. Recovery of total RNA is then achieved by precip-
`itation with isopropanol [33,37]. However, these extractions are
`extremely toxic and hazardous and highly operator dependent.
`Sometimes they can involve up to six or more steps and three
`sample transfers leading to time consuming and laborious RNA
`preparations. Although this method is almost always included in
`RNA purification schemes, the organic solvents such as alcohols and
`phenol/chloroform may interfere in the majority of routine molec-
`ular biology techniques because they can inhibit enzymes activity.
`Moreover, as those compounds convey health risks, this procedure
`is not tolerable for the welfare of the researcher and should not
`be an integral part of the process for a therapeutic formulation.
`However, RNAs that are being employed in clinical trials are often
`extracted using phenol/chloroform [31]. This should certainly be
`considered and advised by regulatory authorities.
`The other methods for RNA isolation are based in solid-phase
`extraction using silica membranes, as prefilled columns or as mag-
`netic beads, in combination with phenol/guanidine-based lysis of
`samples. This technology was developed to offer safer and sim-
`pler operations, as they are amenable to automation using liquid
`handling robotics [38]. In fact, these techniques significantly reduce
`sample preparation time, but can still involve multiple sample
`transfers and time-consuming evaporation steps. Low RNA yields
`are often obtained due to the low binding capacity of the car-
`tridges, which can be easily overloaded [38,39]. Moreover, these
`processes lack specificity as they are non-discriminatory for DNA
`or RNA. Silica matrices have no specific characteristics to dis-
`criminate between nucleic acids. Silica surface is composed of
`
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`
`5
`
`Table 3
`Purification methods used in RNA preparation (PAGE, polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; dsRNA, double-stranded RNA; SPE, solid
`phase extraction; DEAE, diethylaminoethyl).
`
`Chromatographic
`method
`
`Matrix functionalization
`
`Advantages
`
`Disadvantages
`
`References
`
`Reversed-
`phase
`
`Modified silica with hydrocarbon
`chains (normally C8 or C18)
`
`Ion-pairing
`reversed-phase
`
`Polystyrene-divinyl benzene beads
`
`Size exclusion
`
`Cross-linked dextran
`Cross-linked agarose gel
`
`Anion-
`exchange
`
`Polystyrene/divinyl benzene
`poly-(methyl-methacrylate)
`poly(glycidyl
`methacrylate-co-ethylene
`dimethacrylate) functionalized
`with strong quaternary amine
`
`DEAE Sepharose
`
`Latex coated monolith
`anion-exchange (Quaternary
`ammonium ion, diethyl methyl
`amine)
`
`Based on differences in hydrophobicity
`High resolution
`Efficient purification
`
`In combination with SEC improves the
`purification of biological non-coding RNAs
`Synthetic mRNA purification at preparative
`scale
`Non-denaturing conditions
`Use of simpler solvents
`Powerful as final polishing step in
`oligoribonucleotides purified by HPLC
`Effective alternative to preparative PAGE
`purification of in vitro transcribed RNA
`Non-denaturing conditions
`Purification of synthetic dsRNA, rather than
`separated single-strands
`Effective alternative to preparative PAGE
`purification of in vitro transcribed RNA
`Non-denaturing conditions
`Large scale preparation of dsRNA using
`monolithic columns
`Effective alternative to preparative PAGE
`purification of in vitro transcribed RNA
`Natively folded RNA
`Non-denaturing conditions
`Scaled up methods
`Separates short synthetic RNAs from isomers
`impurities
`Evaluation and characterization of therapeutic
`synthetic RNAs
`High efficiencies and capacities
`Lab-scale
`
`Limited to short RNA
`sequences (<50nt)
`Requirement of toxic chemicals
`Uses denaturing conditions
`Difficult to scale up
`Still use toxic solvents
`
`Time-consuming
`Highly operator dependent
`
`RNA folding can be
`compromised by
`chromatographic conditions
`
`Three columns in series to
`achieve purification
`Does not purify RNAs with
`homogeneous 3(cid:3)ends
`
`McGinnis, et al.
`[21]
`
`Dickman [66],
`Chionh et al. [68],
`Kariko et al. [69]
`
`Lukavsky and
`Puglisi [52], Kim
`et al. [53],
`McKenna et al.
`[64]
`Noll, et al. [75],
`Koubek, et al.
`[79],
`Romanovskaya,
`et al. [60]
`
`Easton, et al. [20]
`
`Difficult to scale up
`
`Thayer et al. [78]
`
`silanols and their presence is responsible for the adsorption prop-
`erties of silica gel [40]. Interaction with silica surface has been
`intensively studied to illustrate adsorption/desorption processes
`[41–44]. However, the mechanistic details are not fully understood.
`In the majority of reported protocols, nucleic acids extraction from
`biological samples is performed by inducing the binding onto a sil-
`ica surface in the presence of high salts concentration (e.g. 4 M or
`6 M guanidinium salts) [45,46]. The chaotropic agent guanidinium
`salt favours nucleic acids binding to silica because it destroys the
`hydration shell that is surrounding the nucleic acids. This allows
`positively charged ions to form a salt bridge between the neg-
`atively charged silica and the negatively charged DNA and RNA
`under high salt conditions. By using this high salt environment,
`the nucleic acids can be washed, in order to remove all other con-
`taminants [43]. It is also known that pH has a strong influence
`on the adsorption of nucleic acids on silica surfaces [41]. Several
`companies have tried to develop refined silica membranes in order
`to improve RNA binding over DNA. RNeasy® (Qiagen), PureLinkTM
`RNA (Invitrogen), NucleoSpin® RNA (MACHEREY-NAGEL) are some
`examples of new silica membranes-based approaches with suc-
`cessful results. These technologies combine the selective binding
`properties of silica-based membranes with the speed of micro spin
`technology. In general, samples are lysed and then homogenized
`in the presence of large amounts of a highly denaturing guani-
`dine salt-containing buffer, which protects RNA from endogenous
`RNases ensuring the intact purification of the molecules, and cre-
`ates appropriate binding conditions for the favoured adsorption
`of RNA to the silica membranes. After homogenization, ethanol
`is added to the samples to improve the binding. Impurities are
`removed by subsequent washing buffers. High-quality RNA is then
`eluted in RNase-free water or under low ionic strength condi-
`tions. Nevertheless, in most cases the isolation of pure RNA is
`
`achieved by a secondary enrichment, either through enzymatic
`removal of DNA (DNase treatment) or by a second step using
`specific columns [39]. These limitations can greatly diminish the
`success of several molecular biology investigations, in particular
`gene expression analysis. Sample contamination with DNA will
`provide false results in gene amplification by real time-P