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Nucleic Acid Structure and Function

"The importance of deoxyribonucleic acid (DNA) within living cells is undisputed" (Watson & Crick, 1953). This opening sentence of James Watson and Francis Crick's second major paper, published shortly after the announcement of their proposed structure for the genetic material, has proven to be an understatement. Today, it is readily apparent that Watson and Crick's breakthrough set off a firestorm of discovery and innovation that has continued for over 50 years. The material in this set of articles describes the science surrounding the structure and function of DNA. Here, you will find information on the chemical structure of DNA; details about the organization of DNA into chromosomes , genes , and gene families; and data regarding important categories of sequences within DNA , such as introns, exons, promoters, telomeres, and centromeres. As pointed out by Watson and Crick, the structure of DNA is central to its function, namely its duplication and its expression of the information contained in its nucleotide sequence. Thus, these articles explore both functions, taking a close look at the processes of DNA replication, transcription , and translation . Changes in the DNA sequence lead to most of the genetic disorders that affect humans and other organisms. Learning how these " mutations " cause disease allows investigators to more accurately diagnose and treat various disorders. Furthermore, researchers' ability to manipulate the genetic sequence has given rise to a new set of powerful technologies and industries that are collectively known as biotechnology . Such advances and techniques are also explored in depth throughout this set of articles.

Image: National Human Genome Research Institute.

Genetically Modified Organisms (GMOs): Transgenic Crops and Recombinant DNA Technology

Recombinant DNA Technology and Transgenic Animals

Restriction Enzymes

The Biotechnology Revolution: PCR and the Use of Reverse Transcriptase to Clone Expressed Genes

Copy Number Variation

Copy Number Variation and Genetic Disease

Copy Number Variation and Human Disease

DNA Deletion and Duplication and the Associated Genetic Disorders

Tandem Repeats and Morphological Variation

DNA Transcription

RNA Transcription by RNA Polymerase: Prokaryotes vs Eukaryotes

Translation: DNA to mRNA to Protein

What is a Gene? Colinearity and Transcription Units

Barbara McClintock and the Discovery of Jumping Genes (Transposons)

Discovery of DNA as the Hereditary Material using Streptococcus pneumoniae

Discovery of DNA Structure and Function: Watson and Crick

Isolating Hereditary Material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase

Functions and Utility of Alu Jumping Genes

Transposons, or Jumping Genes: Not Junk DNA?

Transposons: The Jumping Genes

DNA Damage & Repair: Mechanisms for Maintaining DNA Integrity

DNA Replication and Causes of Mutation

Genetic Mutation

Major Molecular Events of DNA Replication

Semi-Conservative DNA Replication: Meselson and Stahl

Chemical Structure of RNA

Eukaryotic Genome Complexity

Genome Packaging in Prokaryotes: the Circular Chromosome of E. coli

RNA Functions

RNA Splicing: Introns, Exons and Spliceosome

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Basic structure

Biosynthesis and degradation.

• Chemical structure
• Biological structures
• Denaturation
• Ultraviolet absorption
• Methylation
• Supercoiling
• Sequence determination
• Messenger RNA (mRNA)
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
• Antisense RNAs
• Viral genomes
• RNA editing
• Basic mechanisms
• Enzymes of replication
• General recombination
• Site-specific recombination
• Transcription
• Translation

What nitrogen-containing bases occur in nucleic acids?

• Who discovered the structure of DNA?

nucleic acid

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• National Center for Biotechnology Information - PubMed Central - Understanding biochemistry: structure and function of nucleic acids
• Nature - The current landscape of nucleic acid therapeutics
• Table Of Contents

What are nucleic acids?

Nucleic acids are naturally occurring chemical compounds that serve as the primary information-carrying molecules in cells. They play an especially important role in directing protein synthesis. The two main classes of nucleic acids are deoxyribonucleic acid ( DNA ) and ribonucleic acid ( RNA ).

What is the basic structure of a nucleic acid?

Nucleic acids are long chainlike molecules composed of a series of nearly identical building blocks called  nucleotides . Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar, which is in turn attached to a phosphate group.

Each nucleic acid contains four of five possible nitrogen-containing bases:  adenine  (A),  guanine  (G),  cytosine  (C),  thymine  (T), and  uracil  (U). A and G are categorized as purines, and C, T, and U are called pyrimidines. All nucleic acids contain the bases A, C, and G; T, however, is found only in DNA, while U is found in RNA.

When were nucleic acids discovered?

Nucleic acids were discovered in 1869 by Swiss biochemist Friedrich Miescher .

nucleic acid , naturally occurring chemical compound that serves as the main information-carrying molecule of the cell and that directs the process of protein synthesis, thereby determining the inherited characteristics of every living thing. Nucleic acids are further defined by their ability to be broken down to yield phosphoric acid , sugars , and a mixture of organic bases ( purines and pyrimidines ).

The two main classes of nucleic acids are deoxyribonucleic acid ( DNA ) and ribonucleic acid ( RNA ). DNA is the master blueprint for life and constitutes the genetic material in all free-living organisms and most viruses . RNA is the genetic material of certain viruses, but it is also found in all living cells, where it plays an important role in certain processes, such as the making of proteins.

Nucleotides : building blocks of nucleic acids

Nucleic acids are polynucleotides—that is, long chainlike molecules composed of a series of nearly identical building blocks called nucleotides . Each nucleotide consists of a nitrogen -containing aromatic base attached to a pentose (five- carbon ) sugar , which is in turn attached to a phosphate group.

Each nucleic acid contains four of five possible nitrogen-containing bases : adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). A and G are categorized as purines , and C, T, and U are collectively called pyrimidines . All nucleic acids contain the bases A, C, and G; T, however, is found only in DNA, while U is found in RNA.

The pentose sugar in DNA ( 2′-deoxyribose ) differs from the sugar in RNA (ribose) by the absence of a hydroxyl group (―OH) on the 2′ carbon of the sugar ring. Without an attached phosphate group, the sugar attached to one of the bases is known as a nucleoside . The phosphate group connects successive sugar residues by bridging the 5′-hydroxyl group on one sugar to the 3′-hydroxyl group of the next sugar in the chain. These nucleoside linkages are called phosphodiester bonds and are the same in RNA and DNA.

Nucleotides are synthesized from readily available precursors in the cell. The ribose phosphate portion of both purine and pyrimidine nucleotides is synthesized from glucose via the pentose phosphate pathway. The six- atom pyrimidine ring is synthesized first and subsequently attached to the ribose phosphate. The two rings in purines are synthesized while attached to the ribose phosphate during the assembly of adenine or guanine nucleosides. In both cases the end product is a nucleotide carrying a phosphate attached to the 5′ carbon on the sugar. Finally, a specialized enzyme called a kinase adds two phosphate groups using adenosine triphosphate (ATP) as the phosphate donor to form ribonucleoside triphosphate, the immediate precursor of RNA. For DNA, the 2′-hydroxyl group is removed from the ribonucleoside diphosphate to give deoxyribonucleoside diphosphate. An additional phosphate group from ATP is then added by another kinase to form a deoxyribonucleoside triphosphate, the immediate precursor of DNA.

During normal cell metabolism, RNA is constantly being made and broken down. The purine and pyrimidine residues are reused by several salvage pathways to make more genetic material. Purine is salvaged in the form of the corresponding nucleotide, whereas pyrimidine is salvaged as the nucleoside.

Module 3: Important Biological Macromolecules

Nucleic acids, discuss nucleic acids and the role they play in dna and rna.

DNA is the set of instructions for our cells. Our DNA determines who and what we are.

Learning Objectives

• Describe the basic structure of nucleic acids
• Compare and contrast the structure of DNA and RNA

Structure of Nucleic Acids

Nucleic acids are the most important macromolecules for the continuity of life. They carry the genetic blueprint of a cell and carry instructions for the functioning of the cell.

The two main types of nucleic acids are  deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) . DNA is the genetic material found in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is found in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope.

The entire genetic content of a cell is known as its genome, and the study of genomes is genomics. In eukaryotic cells but not in prokaryotes, DNA forms a complex with histone proteins to form chromatin, the substance of eukaryotic chromosomes. A chromosome may contain tens of thousands of genes. Many genes contain the information to make protein products; other genes code for RNA products. DNA controls all of the cellular activities by turning the genes “on” or “off.”

The other type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus but instead use an intermediary to communicate with the rest of the cell. This intermediary is the  messenger RNA (mRNA) . Other types of RNA—like rRNA, tRNA, and microRNA—are involved in protein synthesis and its regulation.

DNA and RNA are made up of monomers known as nucleotides. The nucleotides combine with each other to form a polynucleotide, DNA or RNA. Each nucleotide is made up of three components: a nitrogenous base, a pentose (five-carbon) sugar, and a phosphate group (Figure 1). Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to one or more phosphate groups.

Figure 1. A nucleotide is made up of three components: a nitrogenous base, a pentose sugar, and one or more phosphate groups. Carbon residues in the pentose are numbered 1′ through 5′ (the prime distinguishes these residues from those in the base, which are numbered without using a prime notation). The base is attached to the 1′ position of the ribose, and the phosphate is attached to the 5′ position. When a polynucleotide is formed, the 5′ phosphate of the incoming nucleotide attaches to the 3′ hydroxyl group at the end of the growing chain. Two types of pentose are found in nucleotides, deoxyribose (found in DNA) and ribose (found in RNA). Deoxyribose is similar in structure to ribose, but it has an H instead of an OH at the 2′ position. Bases can be divided into two categories: purines and pyrimidines. Purines have a double ring structure, and pyrimidines have a single ring.

The nitrogenous bases, important components of nucleotides, are organic molecules and are so named because they contain carbon and nitrogen. They are bases because they contain an amino group that has the potential of binding an extra hydrogen, and thus, decreases the hydrogen ion concentration in its environment, making it more basic. Each nucleotide in DNA contains one of four possible nitrogenous bases: adenine (A), guanine (G) cytosine (C), and thymine (T). RNA nucleotides also contain one of four possible bases: adenine, guanine, cytosine, and uracil (U) rather than thymine.

Adenine and guanine are classified as  purines . The primary structure of a purine is two carbon-nitrogen rings. Cytosine, thymine, and uracil are classified as pyrimidines which have a single carbon-nitrogen ring as their primary structure (Figure 1). Each of these basic carbon-nitrogen rings has different functional groups attached to it. In molecular biology shorthand, the nitrogenous bases are simply known by their symbols A, T, G, C, and U. DNA contains A, T, G, and C whereas RNA contains A, U, G, and C.

The pentose sugar in DNA is deoxyribose, and in RNA, the sugar is ribose (Figure 1). The difference between the sugars is the presence of the hydroxyl group on the second carbon of the ribose and hydrogen on the second carbon of the deoxyribose. The carbon atoms of the sugar molecule are numbered as 1′, 2′, 3′, 4′, and 5′ (1′ is read as “one prime”). The phosphate residue is attached to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms a 5′–3′  phosphodiester linkage. The phosphodiester linkage is not formed by simple dehydration reaction like the other linkages connecting monomers in macromolecules: its formation involves the removal of two phosphate groups. A polynucleotide may have thousands of such phosphodiester linkages.

DNA Double-Helix Structure

Figure 2. DNA is an antiparallel double helix. The phosphate backbone (the curvy lines) is on the outside, and the bases are on the inside. Each base interacts with a base from the opposing strand. (credit: Jerome Walker/Dennis Myts)

DNA has a double-helix structure (Figure 2). The sugar and phosphate lie on the outside of the helix, forming the backbone of the DNA. The nitrogenous bases are stacked in the interior, like the steps of a staircase, in pairs; the pairs are bound to each other by hydrogen bonds. Every base pair in the double helix is separated from the next base pair by 0.34 nm.

The two strands of the helix run in opposite directions, meaning that the 5′ carbon end of one strand will face the 3′ carbon end of its matching strand. (This is referred to as antiparallel orientation and is important to DNA replication and in many nucleic acid interactions.)

Only certain types of base pairing are allowed. For example, a certain purine can only pair with a certain pyrimidine. This means A can pair with T, and G can pair with C, as shown in Figure 3. This is known as the base complementary rule. In other words, the DNA strands are complementary to each other. If the sequence of one strand is AATTGGCC, the complementary strand would have the sequence TTAACCGG. During DNA replication, each strand is copied, resulting in a daughter DNA double helix containing one parental DNA strand and a newly synthesized strand.

Practice Question

Figure 3. In a double stranded DNA molecule, the two strands run antiparallel to one another so that one strand runs 5′ to 3′ and the other 3′ to 5′. The phosphate backbone is located on the outside, and the bases are in the middle. Adenine forms hydrogen bonds (or base pairs) with thymine, and guanine base pairs with cytosine.

A mutation occurs, and cytosine is replaced with adenine. What impact do you think this will have on the DNA structure?

Ribonucleic acid, or RNA, is mainly involved in the process of protein synthesis under the direction of DNA. RNA is usually single-stranded and is made of ribonucleotides that are linked by phosphodiester bonds. A ribonucleotide in the RNA chain contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and the phosphate group.

There are four major types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and microRNA (miRNA). The first, mRNA, carries the message from DNA, which controls all of the cellular activities in a cell. If a cell requires a certain protein to be synthesized, the gene for this product is turned “on” and the messenger RNA is synthesized in the nucleus. The RNA base sequence is complementary to the coding sequence of the DNA from which it has been copied. However, in RNA, the base T is absent and U is present instead. If the DNA strand has a sequence AATTGCGC, the sequence of the complementary RNA is UUAACGCG. In the cytoplasm, the mRNA interacts with ribosomes and other cellular machinery (Figure 4).

Figure 4. A ribosome has two parts: a large subunit and a small subunit. The mRNA sits in between the two subunits. A tRNA molecule recognizes a codon on the mRNA, binds to it by complementary base pairing, and adds the correct amino acid to the growing peptide chain.

The mRNA is read in sets of three bases known as codons. Each codon codes for a single amino acid. In this way, the mRNA is read and the protein product is made. Ribosomal RNA (rRNA) is a major constituent of ribosomes on which the mRNA binds. The rRNA ensures the proper alignment of the mRNA and the ribosomes; the rRNA of the ribosome also has an enzymatic activity (peptidyl transferase) and catalyzes the formation of the peptide bonds between two aligned amino acids. Transfer RNA (tRNA) is one of the smallest of the four types of RNA, usually 70–90 nucleotides long. It carries the correct amino acid to the site of protein synthesis. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide chain. microRNAs are the smallest RNA molecules and their role involves the regulation of gene expression by interfering with the expression of certain mRNA messages.

DNA versus RNA

While DNA and RNA are similar, they have very distinct differences. Table 1 summarizes features of DNA and RNA.

One other difference bears mention. There is only one type of DNA. DNA is the heritable information that is passed along to each generation of cells; its strands can be “unzipped” with small amount of energy when DNA needs to replicate, and DNA is transcribed into RNA. There are mutliple types of RNA: Messenger RNA is a temporary molecule that transports the information necessary to make a protein from the nucleus (where the DNA remains) to the cytoplasm, where the ribosomes are. Other kinds of RNA include ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and microRNA.

Even though the RNA is single stranded, most RNA types show extensive intramolecular base pairing between complementary sequences, creating a predictable three-dimensional structure essential for their function.

As you have learned, information flow in an organism takes place from DNA to RNA to protein. DNA dictates the structure of mRNA in a process known as transcription, and RNA dictates the structure of protein in a process known as translation. This is known as the Central Dogma of Life, which holds true for all organisms; however, exceptions to the rule occur in connection with viral infections.

In Summary: Nucleic Acids

Nucleic acids are molecules made up of nucleotides that direct cellular activities such as cell division and protein synthesis. Each nucleotide is made up of a pentose sugar, a nitrogenous base, and a phosphate group. There are two types of nucleic acids: DNA and RNA. DNA carries the genetic blueprint of the cell and is passed on from parents to offspring (in the form of chromosomes). It has a double-helical structure with the two strands running in opposite directions, connected by hydrogen bonds, and complementary to each other. RNA is single-stranded and is made of a pentose sugar (ribose), a nitrogenous base, and a phosphate group. RNA is involved in protein synthesis and its regulation. Messenger RNA (mRNA) is copied from the DNA, is exported from the nucleus to the cytoplasm, and contains information for the construction of proteins. Ribosomal RNA (rRNA) is a part of the ribosmes at the site of protein synthesis, whereas transfer RNA (tRNA) carries the amino acid to the site of protein synthesis. microRNA regulates the use of mRNA for protein synthesis.

Check Your Understanding

Answer the question(s) below to see how well you understand the topics covered in the previous section. This short quiz does  not  count toward your grade in the class, and you can retake it an unlimited number of times.

Use this quiz to check your understanding and decide whether to (1) study the previous section further or (2) move on to the next section.

• Introduction to Nucleic Acids. Authored by : Shelli Carter and Lumen Learning. Provided by : Lumen Learning. License : CC BY: Attribution
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Nucleic Acid

Reviewed by: BD Editors

A nucleic acid is a chain of nucleotides which stores genetic information in biological systems. It creates DNA and RNA, which store the information needed by cells to create proteins. This information is stored in multiple sets of three nucleotides, known as codons.

How Nucleic Acids Work

The name comes from the fact that these molecules are acids – that is, they are good at donating protons and accepting electron pairs in chemical reactions – and the fact that they were first discovered in the nuclei of our cells.

Typically, a nucleic acid is a large molecule made up of a string, or “polymer,” of units called “ nucleotides .” All life on Earth uses nucleic acids as their medium for recording hereditary information – that is nucleic acids are the hard drives containing the essential blueprint or “source code” for making cells.

For many years, scientists wondered how living things “knew” how to produce all the complex materials they need to grow and survive, and how they passed their traits down to their offspring.

Scientists eventually found the answer in the form of DNA – deoxyribonucleic acid – a molecule located in the nucleus of cells, which was passed down from parent cells to “daughter” cells.

When the DNA was damaged or passed on incorrectly, the scientists found that cells did not work properly. Damage to DNA would cause cells and organisms to develop incorrectly, or be so badly damaged that they simply died.

Later experiments revealed that another type of nucleic acid – RNA, or ribonucleic acid – acted as a “ messenger ” that could carry copies of the instructions found in DNA. Ribonucleic acid was also used to pass down instructions from generation to generation by some viruses.

Function of Nucleic Acids

Nucleic acids store information like computer code.

By far the most important function of nucleic acids for living things is their role as carriers of information.

Because nucleic acids can be created with four “bases,” and because “base pairing rules” allow information to be “copied” by using one strand of nucleic acids as a template to create another, these molecules are able to both contain and copy information.

To understand this process, it may be useful to compare the DNA code to the binary code used by computers. The two codes are very different in their specifics, but the principle is the same. Just as your computer can create entire virtual realities simply by reading strings of 1s and 0s, cells can create entire living organisms by reading strings of the four DNA base pairs.

As you might imagine, without binary code, you’d have no computer and no computer programs. In just the same way, living organisms need intact copies of their DNA “source code” to function.

The parallels between the genetic code and binary code have even led some scientists to propose the creation of “genetic computers,” which might be able to store information much more efficiently than silicon-based hard drives. However as our ability to record information on silicon has advanced, little attention has been given to research into “genetic computers.”

Protecting the Information

Because the DNA source code is just as vital to a cell as your operating system is to your computer, DNA must be protected from potential damage. To transport DNA’s instructions to other parts of the cell, copies of its information are made using another type of nucleic acid – RNA.

It’s these RNA copies of genetic information which are sent out of the nucleus and around the cell to be used as instructions by cellular machinery.

Cells also use nucleic acids for other purposes. Ribosomes – the cellular machines that make protein – and some enzymes are made out of RNA.

DNA uses RNA as a sort of protective mechanism, separating the DNA from the chaotic environment of the cytoplasm. Within the nucleus, the DNA is protected. Outside of the nucleus, movements of organelles, vesicles, and other cellular components could easily damage the long, complex DNA strands.

The fact that RNA can act both as hereditary material and an enzyme strengthens the case for the idea that the very first life might have been a self-replicating, self-catalyzing RNA molecule.

Examples of Nucleic Acids

The most common nucleic acids in nature are DNA and RNA. These molecules form the foundation for the majority of life on Earth, and they store the information necessary to create proteins which in turn complete the functions necessary for cells to survive and reproduce. However, DNA and RNA are not the only nucleic acids. However, artificial nucleic acids have also been created. These molecules function in the same way as natural nucleic acids, but they can serve a similar function. In fact, scientists are using these molecules to build the basis of an “artificial life form”, which could maintain the artificial nucleic acid and extract information from it to build new proteins and survive.

Generally speaking, nucleic acids themselves differ in every organism based on the sequence of nucleotides within the nucleic acid. This sequence is “read” by cellular machinery to connect amino acids in the correct sequence, building complex protein molecules with specific functions.

Nucleic Acids and Genetics

The genetic code.

Today, scientists know that the source code for cells is quite literally written in nucleic acids. Genetic engineering changes organisms’ traits by adding, removing, or rewriting parts of their DNA – and subsequently changing what “parts” the cells produce.

A sufficiently skilled genetic “programmer” can create the instructions for a living cell from scratch using the nucleic acid code. Scientists did exactly that in 2010, using an artificial DNA synthesizer to “write” a genome from scratch using bits of source code taken from other cells.

All living cells on Earth “read” and “write” their source codes in almost exactly the same “language” using nucleic acids. Sets of three nucleotides, called codons, can code for any given amino acid, or for the stop or start of protein production.

Other properties of nucleic acids may influence DNA expression in more subtle ways, such as by sticking together and making it harder for transcription enzymes to access the code they store.

The fact that all living cells on Earth “speak” almost the same genetic “language” supports the idea of a universal common ancestor – that is, the idea that all life on Earth today started with a single primordial cell whose descendants evolved to give rise to all modern living species.

From a chemical perspective, the nucleotides that are strung together to create nucleic acids consist of a five-carbon sugar, a phosphate group , and a nitrogen-containing base. The image below shows structural drawings of the four DNA and the four RNA nitrogenous bases used by living things on Earth in their nucleic acids.

It also shows how the sugar-phosphate “backbones” bond at an angle that creates a helix – or a double helix in the case of DNA – when multiple nucleic acids are strung together into a single molecule:

Nucleic Acids are Polymers of Nucleotides

DNA and RNA are both polymers made of individual nucleotides. The term “polymer” comes from “poly” for “many” and “mer” for parts, referring to the fact that each nucleic acid is made of many nucleotides.

Because nucleic acids can be made naturally by reacting inorganic ingredients together, and because they are arguably the most essential ingredient for life on Earth, some scientists believe that the very first “life” on Earth may have been a self-replicating sequence of amino acids that was created by natural chemical reactions.

Nucleic acids have been found in meteorites from space, proving that these complex molecules can be formed by natural causes even in environments where there is no life.

Some scientists have even suggested that such meteorites may have helped create the first self-replicating nucleic acid “life” on Earth. This seems possible, but there is no firm evidence to say whether it is true.

Nucleic Acid Structure

Because nucleic acids can form huge polymers which can take on many shapes, there are several ways to discuss the “structure of nucleic acid”. It can mean something as simple as the sequence of nucleotides in a piece of DNA, or something as complex as the way that DNA molecule folds and how it interacts with other molecules. Nucleic acids are formed mainly with the elements carbon, oxygen, hydrogen, nitrogen, and phosphorus.

Please refer to our Nucleic Acid Structure article for more information.

Monomer of Nucleic Acids

Nucleotides are the individual monomers of a nucleic acid. These molecules are fairly complex, consisting of a nitrogenous base plus a sugar-phosphate “backbone.” There are four basic types of nucleotide , adenine (A), guanine (G), cytosine (C), and thymine (T).

When our cells join nucleotides together to form the polymers called nucleic acids, it bonds them by replacing the oxygen molecule of the 3′ sugar of one nucleotide’s backbone with the oxygen molecule of another nucleotide’s 5′ sugar.

This is possible because the chemical properties of nucleotides allow 5′ carbons to bond to multiple phosphates. These phosphates are attractive bonding partners for the 3′ oxygen molecule of the other nucleotide’s 3′ oxygen, so that oxygen molecule pops right off to bond with the phosphates, and is replaced by the oxygen of the 5′ sugar. The two nucleotide monomers are then fully linked with a covalent bond through that oxygen molecule, turning them into a single molecule.

Nucleotides are the monomers of nucleic acids, but just as nucleic acids can serve purposes other than carrying information, nucleotides can too.

The vital energy-carrying molecules ATP and GTP are both made from nucleotides – the nucleotides “A” and “G,” as you might have guessed.

In addition to carrying energy, GTP also plays a vital role in G-protein cell signaling pathways. The term “G-protein” actually comes from the “G” in “GTP” – the same G that’s found in the genetic code.

G-proteins are a special type of protein that can cause signaling cascades with important and complex consequences within a cell. When GTP is phosphorylated, these G-proteins can be turned on or off.

1. Which of the following is NOT a reason why some scientists think the first life might have been made of RNA?

2. If there are only four base pairs of RNA and DNA, then why do we list five? (A, G, C, T, and U?)

3. Why might the “handedness” of our nucleic acids be important?

4. What is the difference between deoxyribonucleic acid (DNA) and ribonucleic adic (RNA)?

5. Which of the following is NOT a function of a nucleic acid?

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Article Contents

Editorial: critical reviews and perspectives in nucleic acids research.

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David R Corey, Helle D Ulrich, Editorial: Critical reviews and perspectives in Nucleic Acids Research , Nucleic Acids Research , Volume 50, Issue 13, 22 July 2022, Page 7201, https://doi.org/10.1093/nar/gkac598

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Articles that summarize knowledge are important contributions to science. Such articles should provide a single authoritative source of information relative to a particular scientific field or question. At Nucleic Acids Research , the editorial team realizes that rapid growth of potentially relevant papers increases the challenge of preparing review articles that will appeal to our broad readership.

Recognizing these challenges, we have recently revised our guidelines ( https://academic.oup.com/nar/pages/criteria_scope ) to make our expectations clear. First and foremost, we expect that authors demonstrate a passion for explaining their topic. Authors should see an important unmet need, a topic where researchers lack up-to-date syntheses of the best information on a subject. Authors should also have deep expertise. The scientific literature is vast. Not all papers are equal in quality. Authors should be able to identify the best, most rigorous studies. They should provide guidance to readers on the hallmarks of rigorous (or less than rigorous) research. Manuscripts should provide some guidance about challenges for future research.

We welcome expert opinion, and we hope that authors will enjoy sharing their hard-earned authority to judge with the community. We note, however, that we continue to welcome more traditional review articles that summarize key findings in a field. Implicit in such papers, however, is the fact that authors possess the expertise to identify the most reliable data and set it into context. We are looking for papers that weave a field together, not present it as a series of unconnected findings. Critical Review and Perspective articles can be commissioned by the editorial team, but we also welcome suggestions from authors. We are happy to provide feedback to authors if we feel that a potential submission is not ready but might have the potential to be improved.

In this issue, we present the first commissioned Critical Review and Perspective. This Perspective is not a typical NAR review, because rather than focus on a particular scientific question, it focuses on the scientific process itself. In 1989, Dr Stanley Crooke left his position as head of Research and Development at Smith Kline Beckman to start Ionis Pharmaceuticals, a company dedicated to a daring new concept—turning synthetic oligonucleotides into drugs. There were many scientific challenges. How can a large negatively charged synthetic molecule enter cells? How can it be made economically on large scale? How can it be chemically modified for biological activity? In 1989, the basic science underlying the field was primitive—the first automated small scale synthesizers had begun to enter laboratories just a few years previously. Nothing was known about the pharmacological properties of oligonucleotides. With so little scientific background, identification of appropriate disease targets was speculative.

Dr Crooke dedicated over thirty years to tackling these problems at Ionis. The teams he has helped supervise have created several approved drugs. One drug, Spinraza, has had a major impact on the treatment of spinal muscular atrophy, providing hope to patients and their families and demonstrating that nucleic acids drugs can have a major impact. While pursuing drug development, Dr Crooke has led his own research group and encouraged Ionis employees in general to publish their work and support basic science through collaborations.

The question of how to address difficult scientific problems is one that is often faced by the authors submitting work to Nucleic Acids Research . The question of rigor is one that NAR editors and reviewers face when evaluating thousands of submissions each year. We hope that Dr Crooke's perspective will be a useful guide for those seeking to understand how to balance rigorous basic science with the practical demands of drug development. We expect that this Perspective will not only inspire scientists in industry, but also in academia.

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Nucleic Acids - Structure and Function

What You Need to Know About DNA and RNA

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The nucleic acids are vital biopolymers found in all living things, where they function to encode, transfer, and express genes . These large molecules are called nucleic acids because they were first identified inside the nucleus of cells , however, they are also found in mitochondria and chloroplasts as well as bacteria and viruses. The two principal nucleic acids are deoxyribonucleic acid ( DNA ) and ribonucleic acid ( RNA ).

DNA and RNA in Cells

DNA is a double-stranded molecule organized into chromosome found in the nucleus of cells, where it encodes the genetic information of an organism. When a cell divides, a copy of this genetic code is passed to the new cell. The copying of the genetic code is called replication .

RNA is a single-stranded molecule that can complement or "match up" to DNA. A type of RNA called messenger RNA or mRNA reads DNA and makes a copy of it, through a process called transcription . mRNA carries this copy from the nucleus to ribosomes in the cytoplasm, where transfer RNA or tRNA helps to match amino acids to the code, ultimately forming proteins through a process called translation .

Nucleotides of Nucleic Acids

Both DNA and RNA are polymers made up of monomers called nucleotides. Each nucleotide consists of three parts:

• a nitrogenous base
• a five-carbon sugar (pentose sugar)
• a phosphate group (PO 4 3- )

The bases and the sugar are different for DNA and RNA, but all nucleotides link together using the same mechanism. The primary or first carbon of the sugar links to the base. The number 5 carbon of the sugar bonds to the phosphate group. When nucleotides bond to each other to form DNA or RNA, the phosphate of one of the nucleotides attaches to the 3-carbon of the sugar of the other nucleotide, forming what is called the sugar-phosphate backbone of the nucleic acid. The link between the nucleotides is called a phosphodiester bond.

DNA Structure

Both DNA and RNA are made using bases, a pentose sugar, and phosphate groups, but the nitrogenous bases and the sugar are not the same in the two macromolecules.

DNA is made using the bases adenine, thymine, guanine, and cytosine. The bases bond to each other in a very specific way. Adenine and thymine bond (A-T), while cytosine and guanine bond (G-C). The pentose sugar is 2'-deoxyribose.

RNA is made using the bases adenine, uracil, guanine, and cytosine. Base pairs form the same way, except adenine joins to uracil (A-U), with guanine bonding with cytosine (G-C). The sugar is ribose. One easy way to remember which bases pair with each other is to look at the shape of the letters. C and G are both curved letters of the alphabet. A and T are both letters made of intersecting straight lines. You can remember that U corresponds to T if you recall U follow T when you recite the alphabet.

Adenine, guanine, and thymine are called the purine bases. They are bicyclic molecules, which means they consist of two rings. Cytosine and thymine are called the pyrimidine bases. A pyrimidine bases consists of a single ring or heterocyclic amine.

Nomenclature and History

Considerable research in the 19th and 20th centuries led to the understanding of the nature and composition of the nucleic acids.

• In 1869, Friedrick Miescher discovered nuclein in eukaryotic cells. Nuclein is the material found in the nucleus, consisting mainly of nucleic acids, protein, and phosphoric acid.
• In 1889, Richard Altmann investigated the chemical properties of nuclein. He found it behaved as an acid, so the material was renamed nucleic acid . Nucleic acid refers to both DNA and RNA.
• In 1938, the first x-ray diffraction pattern of DNA was published by Astbury and Bell.
• In 1953, Watson and Crick described the structure of DNA.

While discovered in eukaryotes, over time scientists realized a cell need not have a nucleus to possess nucleic acids. All true cells (e.g., from plants, animals, fungi) contain both DNA and RNA. The exceptions are some mature cells, such as human red blood cells. A virus has either DNA or RNA, but rarely both molecules. While most DNA is double-stranded and most RNA is single-stranded, there are exceptions. Single-stranded DNA and double-stranded RNA exist in viruses. Even nucleic acids with three and four strands have been found!

• The Difference Between Purines and Pyrimidines
• The Elemental Composition of the Human Body
• Thymine Definition, Facts, and Functions
• Nucleic Acid Facts
• 10 RNA Facts
• Nitrogenous Bases - Definition and Structures
• What Is a Peptide? Definition and Examples
• Enzyme Biochemistry - What Enzymes Are and How They Work
• What You Need To Know About Adenosine Triphosphate or ATP
• The Differences Between DNA and RNA
• Types of Chemical Bonds in Proteins
• Protein and Polypeptide Structure
• Major Lipids and Their Properties
• What Is Fixed Nitrogen or Nitrogen Fixation?
• Amino Acids
• What Are Amphipathic Molecules? Definition, Properties, and Functions

Syllabus Edition

First teaching 2023

First exams 2025

Nucleic Acid Structure & Function ( HL IB Biology )

Revision note.

DNA & RNA: Comparison

Differences between dna and rna.

• Unlike DNA , RNA nucleotides never contain the nitrogenous base thymine (T) – in place of this they contain the nitrogenous base uracil (U)
• Unlike DNA , RNA nucleotides contain the pentose sugar ribose (instead of deoxyribose)

Comparing DNA and RNA nucleotides diagram

An RNA nucleotide compared with a DNA nucleotide

• Unlike DNA , RNA molecules are only made up of one polynucleotide strand (they are single-stranded )
• Unlike DNA, RNA polynucleotide chains are relatively short compared to DNA

RNA structure

mRNA as an example of the structure of an RNA molecule

Nucleotide Structure Summary Table

You need to know the difference between DNA and RNA molecules (base composition, number of strands, pentose sugar present). You also need to be able to sketch the difference between ribose and deoxyribose.

Complementary Base Pairing

The role of complementary base pairing.

• Adenine (A) will pair up with Thymine (T)
• Cytosine (C) will pair up with Guanine (G)
• Two hydrogen bonds form between A and T
• Three hydrogen bonds form between C and G
• We say that one strand acts as a template of the other
• This allows DNA to be copied very precisely during DNA replication which in turn ensures that the genetic code is accurately copied and expressed in newly formed cells

Complementary base pairs and hydrogen bonding diagram

A section of DNA showing nucleotide bonding and complementary base pair bonding

DNA: Information Storage Molecule

Diversity of dna base sequences.

• Despite the genetic code only containing four bases (A, T, C, G), they can combine to form a very diverse range of DNA base sequences in DNA molecules of different lengths
• This means that DNA has an almost limitless capacity for storing genetic information in living organisms
• One way in which this storage capacity can be measured is by the number of genes contained within the DNA of an organism
• Even the most simplistic forms of life may contain several thousand genes within their DNA

Comparing the Number of Genes between Different Organisms Table

• The storage capacity of DNA can also be measured in the number of base pairs contained within the genome of an organism
• That is about 10 9 DNA base pairs
• Given the fact that a nucleus is microscopic in size, is an indication of how incredibly well packaged this amount of genetic information is
• This gives DNA an enormous capacity for storing genetic 'data' with great economy

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Author: Marlene

Marlene graduated from Stellenbosch University, South Africa, in 2002 with a degree in Biodiversity and Ecology. After completing a PGCE (Postgraduate certificate in education) in 2003 she taught high school Biology for over 10 years at various schools across South Africa before returning to Stellenbosch University in 2014 to obtain an Honours degree in Biological Sciences. With over 16 years of teaching experience, of which the past 3 years were spent teaching IGCSE and A level Biology, Marlene is passionate about Biology and making it more approachable to her students.

What is Nucleic Acid?

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Nucleic acids are essential for all forms of life, and it is found in all cells. Nucleic acids come in two natural forms called deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Image Credit: Christoph Burgstedt/Shutterstock.com

Nucleic acids are made of biopolymers, which are naturally-occurring, repeated sets of monomers (making polymers) that then create nucleotides, which form nucleic acids.

To understand the structure of nucleic acid, it is important to understand the structure of the nucleotides that make up nucleic acid.

The structure of nucleic acid

A nucleotide is made up of three parts that are attached by bonds. The three parts are a phosphate group, a 5-carbon sugar, and a nitrogen base.

Phosphate group

The phosphate group is made up of a phosphorus atom with four negatively charged oxygen atoms attached to it.

5-carbon sugar

The 5-carbon sugar (known as a pentose) includes ribose and deoxyribose, which are present in nucleic acid. Both ribose and deoxyribose have five carbon atoms and one oxygen atom. Attached to the carbon atoms are hydrogen atoms and hydroxyl groups.

In ribose sugar, there are hydroxyl groups attached to the second and third carbon atoms. In deoxyribose sugar, there is a hydroxyl group attached to the third carbon atom, but only a hydrogen atom is attached to the second carbon atom.

Nitrogen base

The nitrogen molecule acts as a base in nucleic acid because it can give electrons to other molecules and create new molecules through this process. It can bind to carbon, hydrogen, and oxygen molecules to create ring structures.

Ring structures come in single rings (pyrimidines) and double rings (purines). Pyrimidines include thymine, cytosine, and uracil. Purines include adenine and guanine. Purines are larger than pyrimidines, and their size differences help to determine their pairings in DNA strands.

Nucleic acid bonds

The bonds that hold together the phosphorus, sugar, and nitrogen molecules are called glycosidic bonds and ester bonds.

Glycosidic bonds are made between the first carbon atom in a 5-carbon sugar and the ninth nitrogen atom in a nitrogenous base.

Ester bonds are made between the fifth carbon atom in a 5-carbon sugar and the phosphate group.

These bonds not only hold together a single nucleotide, but they also hold together chains of nucleotides that create polynucleotides that form deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

To create these chains, the phosphate group that is bound to the fifth carbon atom in a 5-carbon sugar will bind to the third carbon atom in the next 5-carbon sugar. This will repeat to create a chain held together by a sugar-phosphate backbone.

If the sugar in this chain is a ribose sugar, a strand of RNA will be created.

To create DNA, the RNA strand bonds to a polynucleotide that has a similar but anti-parallel structure with bonds called hydrogen bonds. These hydrogen bonds link the pyrimidines and purines in the nitrogen bases together. In a process called complementary base pairing, guanine bonds to cytosine, and adenine bonds to thymine. This enhances the energy efficiency of the base pairings, and they will always be found in this pattern.

Image Credit: Billion Photos/Shutterstock.com

The function of nucleic acid

Each type of nucleic acid carries out a different function in the cells of all living things.

DNA is responsible for storing and coding genetic information in the body. The structure of DNA allows for genetic information to be inherited by children from their parents.

As the nucleotides adenine, thymine, guanine, and cytosine in DNA will only pair in a certain sequence (adenine with thymine, and guanine with cytosine), every time a cell duplicates the strand of DNA can specify the sequence in which the nucleotides should be copied. As such, accurate copies of DNA can be made and passed down from generation to generation.

Inside DNA, instructions for all the proteins an organism will make are stored.

RNA plays an important role in protein synthesis and regulates the expression of the information stored in DNA to make these proteins. It is also how genetic information is carried in certain viruses.

• The various functions of RNA include:
• Creating new cells in the body
• Translating DNA into proteins
• Acting as a messenger between DNA and ribosomes
• Helps ribosomes choose the correct amino acids to create new proteins in the body.

These functions are carried out by RNA with different names. These names include:

• Transfer RNA (tRNA)
• Ribosomal RNA (rRNA)
• Messenger RNA (mRNA).

However, not all nucleic acids are involved in processing the information stored in cells. The nucleic acid adenosine triphosphate (ATP), made up of an adenine nitrogenous base, a 5-carbon ribose sugar, and three phosphate groups, is involved in generating energy for cellular processes.

The bonds between the three phosphate groups are high energy bonds, and supply the cell with energy. All living cells use ATP for energy to allow them to carry out their functions.

To supply energy, the last phosphate group in the chain is removed, which releases energy. This process changes ATP to adenosine diphosphate (ADP). Removing two phosphate groups from ATP generates the energy needed to create adenosine monophosphate (AMP).

ATP can be created again through a recycling process in mitochondria that recharges the phosphate groups and adds them back onto the chain.

ATP is involved in the transportation of proteins and lipids in and out of cells, known as endocytosis and exocytosis respectively. ATP is also important in maintaining the overall structure of a cell as it helps to build the cytoskeletal properties of the cell.

In terms of specific bodily functions, ATP is important in muscle contraction. This includes the contractions made by the heart as it beats, as well as movements made by larger muscle groups.

Nucleic acid is an essential part of all living things and is the building block for both DNA and RNA. It is found in all cells and also in some viruses. Nucleic acids have a very diverse set of functions, such as cell creation, the storage and processing of genetic information, protein building, and the generation of energy cells.

Although their functions may differ, the structures of DNA and RNA are very similar, with only a few fundamental differences in their molecular make-up differentiating them.

• Alberts, B. et al. Molecular biology of the cell. (2002). https://www.ncbi.nlm.nih.gov/books/NBK26821/
• Bergman, J. ATP: the perfect energy currency for the cell. (2002). https://www.trueorigin.org/atp.php
• BYJU (n.d.). https://byjus.com/biology/structure-of-rna/
• MEDSimplified. (2017). https://www.youtube.com/watch?v=0lZRAShqft0

Last Updated: Mar 5, 2021

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Nucleic Acids Analytical Methods for Viral Infection Diagnosis: State-of-the-Art and Future Perspectives

Emanuele luigi sciuto.

1 Azienda Ospedaliero, Universitaria Policlinico “G. Rodolico-San Marco”, Via Santa Sofia 78, 95123 Catania, Italy

Antonio Alessio Leonardi

2 CNR-IPCF, Istituto per i Processi Chimico-Fisici, Viale F. Stagno D’Alcontres 37, 98158 Messina, Italy; [email protected] (A.A.L.); [email protected] (A.I.)

3 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d’Alcontres 5, 98166 Messina, Italy; [email protected] (G.C.); [email protected] (G.D.L.)

Giovanna Calabrese

Giovanna de luca, maria anna coniglio.

4 Department of Medical and Surgical Sciences and Advanced Technologies “G.F. Ingrassia”, University of Catania, Via Sofia 87, 95123 Catania, Italy; [email protected]

Alessia Irrera

Sabrina conoci.

5 Istituto per la Microelettronica e Microsistemi, Consiglio Nazionale delle Ricerche (CNR-IMM), Ottava Strada n.5, 95121 Catania, Italy

The analysis of viral nucleic acids (NA), DNA or RNA, is a crucial issue in the diagnosis of infections and the treatment and prevention of related human diseases. Conventional nucleic acid tests (NATs) require multistep approaches starting from the purification of the pathogen genetic material in biological samples to the end of its detection, basically performed by the consolidated polymerase chain reaction (PCR), by the use of specialized instruments and dedicated laboratories. However, since the current NATs are too constraining and time and cost consuming, the research is evolving towards more integrated, decentralized, user-friendly, and low-cost methods. These will allow the implementation of massive diagnoses addressing the growing demand of fast and accurate viral analysis facing such global alerts as the pandemic of coronavirus disease of the recent period. Silicon-based technology and microfluidics, in this sense, brought an important step up, leading to the introduction of the genetic point-of-care (PoC) systems. This review goes through the evolution of the analytical methods for the viral NA diagnosis of infection diseases, highlighting both advantages and drawbacks of the innovative emerging technologies versus the conventional approaches.

1. Introduction

The Coronavirus pandemic has recently focused the attention of researchers worldwide on infectious diseases, thus requiring the best screening strategy for safeguarding public health.

Infectious diseases are disorders provoked by a wide group of microorganisms including viruses, bacteria, fungi, etc., that under certain conditions become dangerous for human safety and capable of rapid transmission among humans or animals. This leads to a series of signs and symptoms that are mostly dependent on the type of microorganism causing the infection and the human district involved.

Viruses are submicroscopic microorganisms composed of two core elements: the nucleic acid genome, either double- (ds) or single-stranded (ss) DNA or RNA, and a protein-based shell called “capsid”. They exist in different habitats but are obliged intracellular parasites, thus they need to infect a living organism as a host for their growth and replication [ 1 ]. The infectious diseases caused by viruses usually respond to rest and home remedies but, for more severe cases, they need hospitalization and specific therapies. However, some types of infections have no valid treatments yet due to the microorganism drug resistance or its structural variability, as in the case of the human immunodeficiency virus syndrome, hemorrhagic fever caused by Ebola virus, and severe acute respiratory syndrome (SARS) caused by variants of the coronaviruses [ 2 ].

Therefore, in order to face the invasive transmission of some types of viruses, early detection and isolation of infected people is crucial to provide a strategy able to control the infection sources and appropriate treatment of the infecting agent.

In this review, a complete overview of the approaches developed for virus detection and infections diagnosis will be given, reporting the state-of-the-art of both the conventional methods and the advancements brought by the emerging new detection technologies.

2. Conventional Methods for Virus Detection

Virus detection for the diagnosis of infections is generally performed by an indirect or direct identification.

Methods based on indirect detection involve virus isolation through its introduction and proliferation into suitable host cells via traditional culture [ 3 ] or faster centrifuge-enhanced techniques [ 4 , 5 ]. Once proliferated, virus can be detected by the evaluation of morphological alterations and other cytopathic effects (CPEs) expressed by the infected cells or before cell damage by using intracellular staining of viral functional proteins (pre-CPE analysis).

Methods based on direct identification instead consist of straight virus detection from its biological source without pre-proliferation and propagation and are generally performed by immunological or molecular approaches.

The immunological identification uses antibodies as probes for the virus direct detection within a sample. Antibodies are available in polyclonal, monoclonal, and recombinant formats [ 6 , 7 , 8 ] and are used to interact specifically with a series of antigens exposed by the viral structural and functional proteins. The immune interactions can be achieved by different strategies, such as the blotting techniques for membrane-mediated interactions or the Enzyme-Linked Immunosorbent Assay (ELISA) for sandwich-type interactions [ 9 ]. These methods are generally not cost effective, and, above all, they must be carried out by specialized personnel in a qualified laboratory environment. Additionally, immunoassays do not achieve the same sensitivity as molecular detection methods [ 10 ].

Molecular methods for virus identification are based on the nucleic acid tests (NATs). These are multistep procedures where the pathogen NA is first extracted and purified from a biological sample (blood, urine, saliva, etc.), using different sample preparation kits, and then detected by PCR. This reaction allows amplification of a specific genetic sequence, unique to an individual organism, through the catalytic action of polymerase enzymes and thermal cycling. So far, the PCR reaction has been extensively studied and optimized, introducing the advancement of quantitative real time PCR (or qPCR), which allows quantitative evaluation of the amplified genetic target through fluorescent labels [ 11 , 12 ].

Although NAT has reached the greatest maturity in infectious disease diagnosis, conventional approaches are mostly still time-, cost- and labor-consuming, dependent on expensive machines (since the PCR reaction requires large and expensive thermal cyclers), and not suitable for miniaturized and decentralized analysis in the point-of-care format, since they are constrained to dedicated laboratories, thus becoming inadequate for the extensive use required during a pandemic outbreak of an infectious disease [ 13 ]. Figure 1 schematizes the main steps of the NAT analytical process.

NAT analytical steps based on PCR: ( a ) sample collection; ( b ) NA extraction; ( c ) PCR amplification; ( d ) real-time PCR quantification; ( e ) sequence detection results.

Due to the above limitations, NATs are further evolving towards faster, portable, and integrated technologies called genetic point-of-care (PoC).

3. NATs Improvements towards PoC Applications

The term point-of-care (PoC) refers to all diagnostic tests that can be performed as close as possible to the patient, providing analytical results in a very short period for an immediate diagnosis and/or therapeutic decision [ 14 ]. The genetic PoC, in particular, must be able to manage, integrate, and merge the fundamental steps required by a complete molecular analysis (NA extraction, amplification, and detection) of a sample for a unique, portable, and user-friendly solution to be used outside a laboratory by unspecialized personnel. Therefore, in order to reach the right level of miniaturization, integration, and automation, evolution of conventional NATs for virus detection was mandatory.

Most of the improvements concerned the NA extraction step, focusing on the enhancement of the purification and isolation step, and the qPCR reaction, focusing on both the chemistry of probes used for the amplification/detection and the thermal cycling.

3.1. NATs Evolution: NA Extraction

NA extraction is a crucial step in molecular analysis as the efficiency of the entire NAT procedure depends on the quality of the genetic material isolated from the biological sample. Tissue biopsy, blood, urine, or saliva samples contain an amount of viral genome that is very low compared to the co-present human genetic material. Therefore, the purity of the extracted material must be very high, and an improvement of the purification and isolation steps is mandatory.

Over the last 20 years, solid phase extraction (SPE) has become the most common method for viral NA extraction due to some advantageous features, such as the minimal need for hazardous chemicals, reduced and easier manipulation, automation capability, and increased throughput [ 15 ]. SPE is based on the capture of a target genetic material from the lysed cell debris by binding/absorption on a solid surface (purification step) and the subsequent elution of purified NA using buffers with specific chemical properties (isolation step) [ 16 ].

SiO 2 is one of the most used solid materials for SPE extraction since it is well known that, under proper ionic strength conditions, it specifically captures DNA. Actually, most of the current commercial extraction kits use silica in the form of (a) core-shell beads featuring a magnetic core covered by an SiO 2 shell that are moved by external magnets, as in the case of the Magazorb kits [ 17 ] ( Figure 2 a); or (b) micro-filter mounted in a plastic column, such as the spin-column of the Qiagen kits [ 18 ] ( Figure 2 b).

DNA absorption and isolation by solid phase extraction based on SiO 2 -coated magnetic beads and separation device ( a ) and with silica filters and spin columns through centrifugation ( b ).

Silicon is also a widely used material for SPE. It is very appealing for PoC system integration due to its optimal physical properties, including a low heat capacity, good thermal conductivity, and the possibility to incorporate electrode and microelectronics circuitry that imprint the so-called intelligence on board.

A drawback of the conventional SPE protocol, however, is that the chemical agents (i.e., alcohols and chaotropic salts) used during the extraction procedure can inhibit the subsequent qPCR reaction [ 19 ]. This implies several washing steps to remove all chemical traces and requires complex and expensive architectures for the fluid’s management.

Therefore, new technologies for SPE have been developed that focus on the silicon derivation, such as functionalization based on amino groups, chitosan, and graphene oxides [ 20 , 21 , 22 , 23 , 24 ], and patterned structures that increase the surface–area ratio (SVR), in order to enlarge the exchange areas used for the target NA binding/transport and allowing an excellent quality of separation and purification of the genetic material from the cell debris. Most of these advanced silicon-based technologies have been used in combination with microfluidics, which brings the advantages of volume downscaling, a reduction of the reagent and sample consumption, and a high degree of miniaturization and integration, suitable for PoC system development [ 25 ].

As an example, in 2017, Takano et al. reported an advanced and simplified SPE method based on low-cost polycationic silica particles for the enhanced extraction of free DNA from human urine [ 26 ]. The positively charged coated particles, prepared by mixing silica gel with polycationic polymer poly-Lys, were able to capture up to 1.3 μg of cell-free DNA with a negatively charged backbone from 50 mL of urine without the need for high concentrations of chaotropic agents, thus increasing the purity of the extracted DNA.

Birch et al. instead developed a dual porous silica (DPS) structure for SPE composed of two monolithic disks, synthesized from tetramethyl orthosilicate and potassium silicate, incorporated into a microfluidic device [ 27 ]. With this structure, the DPS was able to rapidly extract a large quantity of DNA from human urine samples (less than 35 min) with high integrity for downstream analysis, allowing SPE incorporation into the microfluidic device without leakage of the sample or reagents.

In 2017, Petralia et al. developed another example of a patterned silica structure in a microfluidic device for miniaturized SPE purification and isolation of viral NA [ 28 ]. Using the hepatitis B virus (HBV) genome as the analytical sample, a downsized (few centimeters) biofilter made of silicon micropillars, opportunely designed to increase the SVR and the amount of bound target NA, was fabricated and characterized. The pillars–NA interaction was mediated by a charge-based reversible adsorption occurring between the pillars’ superficial epoxydic groups and the negatively charged oxygens exposed by the NA backbone. The NA was first selectively linked to the pillars, washing away all the unbounded molecules including cells debris and other chemicals, and then eluted by deionized water. Thanks to the synergy of the absorption chemistry and the designed layout, the silicon pillars showed an extraction efficiency about 16% higher than that measured with commercial kits within a low-cost miniaturized device, providing an extraction strategy for genetic fully integrated PoC systems.

3.2. NATs Evolution: Redox PCR Probes

Once extracted and purified, the NA material is analyzed by molecular methods, such as qPCR.

A wide series of probes for qPCR are used to allow real-time detection of the genetic target during its amplification, such as oligonucleotide probes and intercalating agents. These agents, in particular, are able to enter the two paired bases of a double-stranded nucleic acid (dsNA) and interact with the minor groove [ 29 ]. Conventional and commercial intercalating probes, such as ethidium bromide, SYBR Gold, Evagreen, and SYBR Green, have a transduction active center that emits fluorescence once intercalated in the amplified product, producing the optical signal for the target sequence detection. This type of transduction, however, implies some limitations for integration into PoC systems since the conventional optical modules required for detection are not easily miniaturizable, are expensive and constrained to cooling supports, and the fluorescent probes are not stable enough to be used as reagents on-board [ 30 ].

To overcome the above reported limitation, redox-active compounds have been proposed as alternative intercalating probes for qPCR allowing electronic detection. Some of these are complex, composed of metals (such as osmium, ruthenium, etc.) that have been properly engineered to have the metallic center covalently bound to a planar intercalating ligand, such as phenanthroline, bypiridine derivates (BPY), or dipyridophenazine (DPPZ) [ 31 , 32 , 33 , 34 ]. As reported in Figure 3 , the redox activity of these probes allows electrochemical detection in the NAT method that brings the advantages of a high level of miniaturizability and good sensitivity and robustness, which are ideal for integration into PoC systems.

Generic scheme of nucleic acid analysis through intercalative redox-active compounds: detail of the electrochemical stimulus generation, by target–probe NA interaction and redox compound intercalation inside the dsNA, and the electrochemical signal detection.

In 2011, Limoges et al. used a series of metallic redox-intercalating compounds as probes for qPCR combined with electrochemical detection by Square Wave voltammetry and reported the osmium complex Os[(bpy) 2 DPPZ] 2+ as the most performant, since it is chemically stable under qPCR thermal cycling, a strong intercalating agent towards the amplified dsNA, and able to enhance the sensitivity of the NAT method at about 10 3 copies/reaction [ 35 ].

Another application of the redox probe for qPCR was presented in Petralia et al. in 2015 [ 36 ]. Again, they used the Os[(bpy) 2 DPPZ] 2+ probe and developed a PCR-based electrochemical method for the detection of hepatitis-B virus (HBV) in a miniaturized silicon device, where 5’-thiolated oligonucleotides (used as primers) were chemically immobilized on the surface of a micro working electrode (WE) made of platinum with a density of about 4.0 × 10 12 molecules per cm 2 [ 37 ]. Thanks to the interaction between the viral DNA target and the anchored oligos and the subsequent intercalation of the osmium redox probe inside the amplified double-strand target, it was possible to detect the HBV DNA in solid state after 10, 20, 30, and 40 cycles of amplification with a huge increase of sensitivity with respect to the traditional qPCR systems. Moreover, this approach offered a high degree of versatility, since the technology can be employed to detect various double helix DNA targets by changing the specificity of the anchored oligos, and the possibility of miniaturization.

3.3. NATs Evolution: Isothermal PCR

Another improvement of the qPCR concerns the reduction of the thermal complexity by isothermal approaches.

Isothermal PCR is an alternative amplification method that uses a single temperature to rapidly and efficiently accumulate NA target sequences without the constraint of the thermal cycling [ 38 , 39 ]. One of the main features of this technology is the denaturation step of the dsNA target. Thanks to specific reagents (such as polymerase having strand-displacement ability, recombinase, and helicase), the double helix of the target is, in fact, enzymatically denatured, thus avoiding the use of high temperatures. Once denatured, the target is amplified by a combination of primers and extension phases that are set to be performed at the same temperature, thus excluding the need of a thermal cycling. This makes isothermal methods like Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), and Helicase-Dependent Amplification (HDA) faster, as they do not need to reach a high number of thermal cycles but use continuous amplification, which provides detectable amplicons within a few minutes, which is more sensitive than qPCR, since they involve the synergy of multiple specific primers for the annealing step [ 40 , 41 , 42 ]. Schemes of the above-mentioned isothermal approaches are reported in Figure 4 .

Isothermal approaches for NATs. ( a ) Loop-Mediated Isothermal Amplification. Reproduced with permission from [ 43 ]. Copyright 2021, Elsevier. ( b ) Recombinase Polymerase Amplification. Adapted with permission from [ 44 ]. Copyright 2021, Elsevier. ( c ) Helicase-Dependent Amplification. Reproduced with permission from [ 45 ]. Copyright 2021, American Society for Microbiology—Journals.

The isothermal amplification simplifies the architectural setup of the PCR-based method. In this case, in fact, since the thermal cycling is excluded, the temperature management does not need additional components (such as Peltier cells, resistors, capacitors, etc.) and high-power demand for the amplification process, thus reducing costs and architecture complexity. Moreover, such isothermal methods as LAMP and HAD can be coupled with an electrochemical detection that, as reported before, is perfectly suitable for integration into PoC systems [ 46 ].

4. Genetic PoC Systems for Viral Infection Diagnosis

4.1. pcr-based genetic poc.

All the above reported NAT improvements were preliminary to the introduction of new functional modules for integration into genetic PoC systems, giving a full and accurate analysis of the viral NA in a sample-in-answer-out format to face the need for massive, fast, and decentralized diagnosis of infections. The development of integrated systems combining molecular detection with advanced extraction technologies for selective viral NA isolation has provided high-quality diagnosis of infectious disease. In fact, the possibility to specifically separate and extract few copies of viral genomes among more abundant copies of human or animal nucleic acid in a typical biological sample (blood, urine, saliva) has increased the amount of template available for the subsequent amplification and detection steps, providing an enhancement of the analytical resolution.

Examples of emerging PoC systems are reported in Figure 5 . The synergy between microfluidics and silicon technology played a key role in the development of most of these systems, bringing a series of advantages in terms of reagent consumption optimization by volume reduction, incorporation of microelectronic circuits, and robustness of the material used. This led to the development of a series of genetic PoC systems for complete analysis of viral pathogens in infections [ 47 ]. A first product was the commercial GeneXpert (Cepheid) system, endorsed by the World Health Organization (WHO) in 2010, for the early detection of HIV-associated tuberculosis, reported in Figure 5 a. The microfluidic device integrated a mechanical SPE extraction step, based on lysis by sonication and surface affinity, and PCR amplification and detection of the purified material, completing the analysis within 2 h [ 48 ]. A GeneXpert system was also developed for the severe and highly contagious Ebola virus disease (EVD), providing detection in 94 min with an LoD of 0.13 plaque-forming unit (PFU)/mL of Ebola Zaire virus [ 49 ]. This product was followed by others PoCs systems, such as the FilmArray (BioMerieux), combining NA extraction, based on SPE using silica-coated magnetic beads, and detection, by multiplex PCR, in a plastic cartridge of some tens of cm [ 50 ]. However, although performing all the main steps for a complete NA analysis, most of these PoC systems were just relocations of conventional NAT steps and required expensive and bulky instruments, thus leaving the integration and automation issue still unsolved.

Schemes of the genetic PoC systems. ( a ) Fully integrated microfluidic PoC system for SARS-CoV-2 RNA analysis. Adapted with permission from [ 51 ] Copyright 2021, Elsevier. ( b ) Lab-on-disk for viral NA analysis. Adapted with permission from [ 52 ]. Copyright 2021, John Wiley & Sons—Books. ( c ) Sp-SlipChip microfluidic device and PI dPCR system for BKV DNA analysis in urine samples: detail of sample processing, droplet formation, and viral DNA detection by dPCR. Adapted with permission from [ 53 ]. Copyright 2021, Elsevier. ( d ) Paper-based integrated PoC for tropical virus diagnostics. Adapted with permission from [ 54 ]. Copyright 2021, Elsevier.

Over the last years, many design and fabrication efforts have focused on improving the microfluidics of genetic PoC towards more integrated, automated, sensitive, and rapid devices. As an example, Sun et al. developed a genetic PoC system based on a portable silicon microfluidic chip for the live detection of virus in nasal swab samples [ 55 ]. Inside the chip, 10 microchannels with a deposited set of primers allowed an LAMP amplification of specific sequences of some equine viruses, such as equine herpesvirus 1 (EHV1) and equine influenza virus (EIV), and then optically detected by fluorescence imaging operated by a customized smartphone, revealing down to 5.5 × 10 4 copies/mL of viral DNA. These performances make the system an example of genetic PoC for multiplex low-cost and portable analysis enabling remote diagnosis of viral coinfections for efficient epidemic surveillance. Rodriguez-Mateos et al. presented a fully integrated microfluidic PoC system for genomic SARS-CoV-2 RNAs analysis ( Figure 5 a), combining an NA extraction step based on immiscible filtration and surface tension (IFAST) with a detection via colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) [ 51 ]. Within a downsized architecture, using a simplified detection of color change by the naked eye, the platform was capable of detecting up to 470 copies/mL of virus in saliva samples in about 1 h, with a 100% specificity, leading to a potential increase of the COVID-19 screening speed and an early detection prior to the viral transmission.

The shape of the analytical system represented an important issue in the microfluidics evolution, inspiring a lot of commercial products, such as the LIAT analyzer (IQuum), proposing a lab-in-a-tube technology for HIV detection [ 56 ], or the GenePoC system, reporting a downsized microfluidic cartridge for Influenza A and B virus detection [ 57 ]. A very appealing solution was introduced by the lab-on-disk technology. The disk shape allowed an improvement of PoC automation thanks to the possibility of rotation by centrifugal forces inducing fluid movements and other operations, such as liquid mixing, aliquot, switching, valving, and storage. In 2019, Zhou et al. developed a microfluidic-RT-LAMP chip system for the simultaneous detection of three types of coronaviruses (porcine epidemic diarrhea, porcine deltacoronavirus, and swine acute diarrhea syndrome-coronavirus) [ 58 ]. The system consisted in a polymethyl methacrylate disk containing a series of chambers (devolved to the sample storage, reaction, waste collection, etc.) for viral NA amplification by RT-LAMP and a detector integrating temperature control, high-speed centrifugation, and fluorescence reading and analysis, provided by iGeneTech (Ningbo, China). With this composition, the system was able to detect and quantify the NA of all three viral targets within 1.5 h, with a limit of detection (LoD) of about 1 × 10 2 copies/mL, showing potential for rapid, sensitive, and high-throughput PoC diagnosis. In 2020, Sciuto et al. developed and characterized a lab-on-disk PoC technology for fully integrated viral NA analysis ( Figure 5 b) [ 52 ]. It was a hybrid structure composed of a plastic microfluidic disk containing all chambers required for the viral NA extraction and purification through SPE using magnetic beads (lysis, binding, washing, and elution), and a silicon module for NA amplification and detection by qPCR. The movements of beads, sample, and reagents were induced by a disk reader equipped with magnets and actuating all the centrifugal forces, thermal control, and optical fluorescence imaging required for NA analysis. Using the hepatitis B virus (HBV) DNA genome as the viral target, it was shown that the system was able to purify the viral NA from the starting sample and detect down to eight copies/reaction of the target, bringing an improvement in terms of automation and sensitivity with respect to the conventional qPCR platforms.

Microfluidics evolution paved the basis for the emerging droplet manipulation technology [ 59 , 60 ]. This strategy simplified the integration of microfluidic components in PoC systems by using droplets with embedded superparamagnetic particles and reagents/samples. By merging and splitting with each other through magnetic actuators, the droplets are able to achieve all the fluidic functions and operations required by the NA analysis, substituting the conventional virtual pumps, valves, mixer, SPE substrates, and PCR reactors. This was the case of PoC systems, such as the droplet-based platform developed by Pipper et al., which allowed the detection of the avian influenza virus H5N1 from a throat swab within 30 min [ 61 ].

Recently, the literature reported some evidence of PoC systems based on droplet manipulation combined with digital nucleic acid analysis, such as digital PCR (dPCR) and digital isothermal amplification. This method provides sensitive detection and precise quantification of target nucleic acids by the partitioning of the sample into droplets, each containing either zero or one (or, at the most, a few) template molecules. The single partition goes through an individual PCR reaction and, thanks to the fluorescence detection, is considered positive (1, fluorescent) if it contains target and negative (0) if not. The combination of droplets that tested negative or positive with the Poisson statistics allows estimation of the exact number of the NA target copies in each partition and calculation of the absolute template concentration in the original sample [ 62 ].The utility of the digital NA analysis has been demonstrated in many diagnostic applications. As an example, in 2021, Yu et al. proposed a self-partitioning SlipChip (sp-SlipChip) microfluidic device for the slip-induced generation of droplets to detect human papilloma virus (HPV) DNA by digital LAMP [ 63 ]. The chip was composed of top and bottom plates that can slip past each other, generating aqueous droplets used in the final fluorescent digital amplification, being capable of quantifying the viral DNA strain within a concentration range of 7.0 × 10 2 –1.4 × 10 7 copies/mL. Recently, Xu et al. proposed an advanced spSlipChip microfluidic device combined with a portable integrated (PI) dPCR system, reported in Figure 5 c, for quantitative analysis of the BK virus (BKV) genome in urine samples [ 53 ]. Through to a simplified slip-induced self-partitioning mechanism, enhanced by a series of microchannels and bridges, the SlipChip device was able to operate the formation of droplets, containing urine lysate sample and PCR mix, without a precise alignment of the contacting plates. A dedicated reader for both thermal control and fluorescence imaging, then, performed dPCR quantification of the BKV DNA with a dynamic range of 3.0 × 10 4 to 1.5 × 10 8 copies/mL within 2 h.

The microfluidics evolution in fully integrated genetic PoC proceeded in parallel with the improvement of the structuring material used. The literature reports some PoC systems substituting silicon with other materials that have received particular attention due to their simplified, low-cost, and robust properties [ 64 , 65 , 66 ]. In 2020, Seok et al. proposed a paper-based PoC system reporting a fully integrated platform for the molecular diagnostics of three kinds of tropical viruses (Zika, dengue, and Chikungunya virus) in human serum, described in Figure 5 d [ 54 ]. The system consisted of a chip made of paper containing a series of pads and dried chemicals for both the automatic fluidic flow-based extraction and the LAMP detection of viral RNAs. With this structure, the paper-based system was able to detect the target viruses in the range of 5–5000 copies/mL simultaneously and with extreme accuracy. Some evidence has also reported graphene as an alternative material for viral analysis, due to its unique optical and electrical properties, as from Liu et al., who developed a graphene conductive film performing highly sensitive detection of rotavirus [ 67 ], or Chen et al., who fabricated a graphene field-effect transistor (GFET)-based portable system for the detection of human influenza virus H1N1 [ 68 , 69 ].

4.2. PCR-Free Genetic PoC

So far, virus detection in genetic PoC systems has mostly been based on NA amplification by PCR technologies. Despite its maturity, this approach implies several challenges, such as the selection of reagents with the right stability for an on-board usage (up to 1 year), the simplification of sample preparation and PCR chemistry to reduce the number of steps involved, and the integration of several modules for the thermal and opto-electrical control requiring certain energy consumption. Moreover, PCR may fail to amplify the target NA due to the high genetic variability of some viruses [ 70 ]. Since NA amplification was quite constraining, alternative PCR-free virus detection strategies, reported in Figure 6 , have been proposed in genetic PoC.

PCR-free virus detection strategies in genetic PoC systems. ( a ) Gold nanoparticle-based genomic microarray for the specific identification of avian influenza virus. Reproduced with permission from [ 71 ]. Creative Commons CC BY, Springer Nature. ( b ) Miniaturized electrochemical device for the PCR-free detection of HBV. Adapted from [ 72 ] ( c ) Fully integrated PoC system for PCR-free detection of HCV in blood. Adapted from [ 73 ] with permission from the Royal Society of Chemistry.

Zhao et al., for example, developed a PCR-free system for the rapid detection of avian influenza virus H5N1 ( Figure 6 a) [ 71 ]. The system was a modified microarray platform with immobilized oligos used to directly and specifically hybridize the H5N1 RNA and, then, allow another poly-A-tailed intermediate oligos to form a sandwich complex with the target. This complex was bound by stained gold nanoparticles, giving a light scattering signal that was detected and quantified by a dedicated optical reader. The assay identified the H5N1 viral RNA with extreme accuracy, discriminating from other influenza virus subtyping (H1N1, H3N2), and velocity, since no NA amplification was required.

In 2017, Sciuto et al. proposed a technology for portable and rapid electrochemical detection of HBV without NA amplification ( Figure 6 b) [ 72 ]. The device consisted of a silicon miniaturized chip containing three microelectrodes and the chemical strategy employed was based on the hybridization between the target viral genome and two specific oligonucleotide probes grafted on top of the WE of the chip. Once formed, the hybrid complex was exposed to the redox-active compound Os(bpy) 2 DPPZ (see Section 3.2 ), releasing a current signal processed by a portable electronic board for Square-Wave voltammetry. With this structure, the system was able to detect and quantify the HBV genome without any preliminary amplification, reaching a low LoD of 20 copies of DNA analyzed.

Similarly, Liu et al. developed a fully integrated and user-friendly PoC chip for the PCR-free detection of hepatitis C virus (HCV) in blood ( Figure 6 c) [ 73 ]. The chip used a passive fluidic method capable of first extracting the target RNA from blood and then detecting it by label-free charge-based electrochemical assay. Using specific PNA sequences anchored on top of nanostructured microelectrodes and the combined action of two redox-active probes (Ru(NH 3 ) 6 3+ and Fe(CN) 6 3− ), the chip was able to electrochemically detect the target RNA within 30 min without the need for amplification.

5. Conclusions

The growing demand for massive viral infections diagnosis, together with the low resource settings of certain infrastructures, has resulted in the need to develop increasingly more integrated, low-cost, and portable platforms for rapid and decentralized analysis. This also suggests the need to achieve early detection of viruses, improving the level of diagnostics.

Virus detection has evolved over time, moving from conventional indirect identification by cultural evaluations of viral cytological effects in host cells to direct immunological detection of viral antigens and direct molecular analysis of the viral genome by NAT. This molecular approach provided the most accurate and complete analysis by viral NA extraction and its PCR detection. However, although it has reached the greatest maturity and the highest level of affordability, NAT is still constrained to dedicated laboratories and long and complex procedures so that considerable efforts, bringing new SPE and isothermal PCR methods for NA extraction and detection, have focused on performing NATs in more integrated and automated systems called genetic PoC.

In the last decade, PoC approaches have allowed complete viral molecular analysis in the sample-in-answer-out format to be achieved, reducing the cost, time, and complexity of conventional methods. The literature reported a series of genetic PoCs that, thanks to the synergy between the silicon technology and microfluidics and the incorporation of reagents and electronic circuits on board, that have evolved towards fully integrated, automated, and portable systems, as reported by the droplet manipulations-based technologies and lab-on-disk platforms. Moreover, new PCR-free PoC strategies have been proposed in order to improve NA detection in terms of time and cost consumption by excluding the amplification step.

Altogether, this evidence proves that the molecular analysis achieves the best level of accuracy and portability, providing new measures to face infection outbreaks and global pandemics.

Author Contributions

Conceptualization, E.L.S. and S.C.; writing—original draft preparation, E.L.S.; writing—review and editing, G.D.L., G.C., A.A.L., A.I., M.A.C. and S.C.; supervision, M.A.C. and S.C. All authors have read and agreed to the published version of the manuscript.

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

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Programmable Framework Nucleic Acid-Modified Nanomagnetic Beads for Efficient Isolation of Exosomes and Exosomal Proteomics Analysis

Affiliations.

• 1 Technology Innovation Center of Mass Spectrometry for State Market Regulation, Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, PR China.
• 2 College of Life Sciences, Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, China Jiliang University, Hangzhou 310018, PR China.
• PMID: 39161057
• DOI: 10.1021/acs.analchem.4c01193

Exosomes are increasingly being regarded as emerging and promising biomarkers for cancer screening, diagnosis, and therapy. The downstream molecular analyses of exosomes were greatly affected by the isolation efficiency from biosamples. Among the current exosome isolation strategies, affinity nanomaterials performed comparably better with selectivity and specificity. However, these techniques did not take the structure and size of exosomes into account, which may lead to a loss of isolation efficiency. In this article, a framework nucleic acid was employed to prepare a well-designed nanosized bead Fe 3 O 4 @pGMA@DNA TET@Ti 4+ for enrichment of exosomes. The abundant phosphate groups in the framework nucleic acid provide binding sites to immobilize Ti 4+ , and its rigid three-dimensional skeleton makes them act as roadblocks to barricade exosomes and provide affinity interactions on a three-dimensional scale, resulting in the improvement of isolation efficiency. The model exosomes can be effectively isolated with 92% recovery in 5 min. From 100 μL of HeLa cell culture supernatant, 34 proteins out of the top 100 commonly identified exosomal proteins were identified from the isolated exosomes by the novel beads, which is obviously more than that by TiO 2 (19 proteins), indicating higher isolation efficiency and exosome purity by Fe 3 O 4 @pGMA@DNA TET@Ti 4+ beads. The nanobeads were finally applied for comparing exosomal proteomics analysis from real clinical serum samples. Twenty-five upregulated and 10 downregulated proteins were identified in the lung cancer patients group compared to the health donors group, indicating that the novel nanobeads have great potential in isolation of exosomes for exosomal proteomics analysis in cancer screening and diagnosis.

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