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I'm struggling with a question I've been asked.
"Why is double stranded genetic material more 'durable' than single stranded one?"
I know that double stranded genetic material is more stable due to multople strands. The single stranded isn't as stable as the double stranded since it lacks the second strand and therefore the bases are open.
Stable isn't the same as durable, and I've never heard in class that one is more durable than the other one. Did I miss something or is this just a mistake?
I'm interpreting "durability" as the DNA's resistance to physical stress, such as shearing. The Bustamante lab at UC Berkeley does a lot of very cool single-molecule biophysics looking at forces involved in protein-protein interactions and protein-DNA interactions. This Bustamante et al. review paper, Single-molecule studies of DNA mechanics includes a look at the force required to break ssDNA and dsDNA:
Single molecules of dsDNA were broken with a receding water meniscus  at forces estimated to be 960 pN (correcting Young's modulus doubles the published scission force of 480 pN). Short dsDNA molecules pulled with an AFM tip  sustained forces over 1700 pN.
pN = picoNewton
AFM = atomic force microscopy
dsDNA is more resistant to stretching/shearing forces because the double helix arrangement is "springy". I recommend giving the paper a read for more info. Very cool stuff.
Innate recognition of non-self nucleic acids
The immune system has evolved a plethora of innate receptors that detect microbial DNA and RNA, including Toll-like receptors in the endosomal compartment and RIG-I-like receptors and Nod-like receptors in the cytosol. Here we discuss the recognition of and responses to non-self nucleic acids via these receptors as well as their involvement in autoimmune diseases.
The function of the immune system is to protect the organism from invading pathogens. To avoid collateral damage to the body's own tissues, it must be able to distinguish infectious non-self entities from self tissues. Antigen-specific lymphocytes - T cells and B cells - recognize pathogens through T-cell receptors and immunoglobulins, respectively, which are generated by somatic gene rearrangement. But although these antigen-specific receptors allow the recognition of a vast number of different molecules, they have no intrinsic ability to distinguish non-self from self. Instead, it is believed that signals delivered through the so-called pattern recognition receptors of the innate immune system are fundamental in recognizing infectious non-self entities, thus preparing the body for the initiation of a full antigen-specific immune response that targets invading pathogens but not self tissues . The receptors utilized by the innate immune system recognize microbial components, known as pathogen-associated molecular patterns, that are essential for the survival of the microorganism and are therefore difficult for it to alter. Different receptors interact with different pathogen molecules, and show distinct expression patterns, activate specific signaling pathways and lead to distinct anti-pathogen responses [2, 3]. The molecules recognized include, for example, components of bacterial and fungal cell walls, flagellar proteins and viral surface proteins - molecules that are unique to the pathogen and not found in the host. Another major group of pathogen molecules specifically recognized by innate immune receptors comprises microbial DNA and RNA. Because nucleic acids are present in all organisms, the host has evolved specialized mechanisms for recognizing non-self nucleic acids while maintaining tolerance (non-responsiveness) to self nucleic acids. In this article, we will review several systems of pattern recognition receptors involved in the recognition of non-self nucleic acids, including the Toll-like receptors (TLRs), the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and the Nod-like receptors (NLRs) (Table 1). Mechanisms for recognizing non-self nucleic acids are not fail-safe, however, and under abnormal conditions recognition of self DNA and RNA occurs, leading to the development of autoimmunity. This is discussed in the last section of this review.
Solid-state nanopores offer a promising method for rapidly probing the structural properties of biopolymers such as DNA and RNA. We have for the first time translocated RNA molecules through solid-state nanopores, comparing the signatures of translocating double-stranded RNA molecules and of single-stranded homopolymers poly(A), poly(U), poly(C). On the basis of their differential blockade currents, we can rapidly discriminate between both single- and double-stranded nucleic-acid molecules, as well as separate purine-based homopolymers from pyrimidine-based homopolymers. Molecule identification is facilitated through the application of high voltages (∼600 mV), which contribute to the entropic stretching of these highly flexible molecules. This striking sensitivity to relatively small differences in the underlying polymer structure greatly improves the prospects for using nanopore-based devices for DNA or RNA mapping.
USING OLIGOCALC TO CALCULATE THE PROPERTIES OF OLIGONUCLEOTIDES
OligoCalc has a familiar ‘calculator’ interface and the basic properties can be calculated by pasting or entering the sequence followed by one of the following actions: clicking out of the sequence box, entering a ‘tab’, hitting ‘return’ or clicking ‘Calculate’. OligoCalc will use the currently entered sequence, selected options and entered conditions to calculate the length, molecular weight, estimated absorbance at 260 nm, the micromolar concentration and micrograms of oligonucleotide present in a 1 ml solution with an absorbance of one for the sequence entered. The calculator is available at the URL http://basic.northwestern.edu/biotools/OligoCalc.html and loading that URL in a browser will display the interface shown in Figure 1.
Entry and main calculation screen for OligoCalc.
Entry and main calculation screen for OligoCalc.
Once the user has entered a sequence, several additional options can be selected or entered. The absorbance at 260 nm (A260) can be entered for the oligonucleotide, and will result in the calculation of the micromolar concentration of oligonucleotide as well as the micrograms of the oligonucleotide present in a 1 ml solution with that absorbance. The millimolar concentration of salt [Na + ] can be entered and will adjust the salt adjusted and nearest neighbor melting temperature calculations. The default value is 50 mM. The nanomolar concentration of primer in the hybridization solution can also be entered and will adjust the nearest neighbor melting temperature. The composition of the oligonucleotide (DNA or RNA, single-stranded or double-stranded) can be selected and will change many of the calculations, although the absorption coefficients are only accurate for single-stranded oligonucleotides. There are a number of 5′ and 3′ modifications that can be selected, and will change the molecular weight and in some cases the absorbance coefficient for the oligonucleotide. The entry of IUPAC codes are also supported (for instance W for A or T) and results in a range of values being reported for melting temperature, %GC content, molecular weight, concentration and micrograms present in a 1 ml solution with a A260 of 1, with the range representing the highest and lowest values possible for the set of possible oligonucleotides.
Clicking the ‘Swap Strands’ button swaps the entered strand for its reverse complement, and updates the properties of the oligonucleotide based on the new strand sequence. Note that if the ‘dsDNA’ or ‘dsRNA’ molecule type has been selected, swapping strands has no effect on the overall properties, since both strands are already taken into account by the calculations.
Clicking the ‘mfold button results in a new window that posts to the mfold web server ( 1, 2) with likely hairpin and self-complementary areas highlighted.
A final option available is ‘BLAST’, which opens a new window and posts the sequence entered to the NCBI BLAST page and starts a blastn analysis of the entered sequence against the current set of non-redundant sequences (nr) available at the NCBI ( 3), with filtering for low-complexity sequences enabled.
There is also considerable documentation available, including a page of chemical modifications, including the chemical names for the common synonyms and links to the structures of the modifications, when available. This page is available at http://www.basic.northwestern.edu/biotools/OligoCalcModifications.html.
Other 5´/3´ end modifications
Several other modifications, such as the inverted deoxythymidine bases and dideoxynucleotides (Figure 2) have been reported to suppress serum nuclease activity when appended to the end of synthetic oligonucleotides (27). Many other modifications may be attached through &ldquolinkers&rdquo at either the 5´ or 3´ end, including ﬂuorescent tags, biotin or other affinity labels, or reactive groups for attachment to beads or surfaces. These linkers are typically connected to the 5´ or 3´ end via a phosphodiester. What is the interaction of these modified ends with exonucleases?
We have surveyed a range of these modifications under typical in vitro exonuclease assays. In general, while many provide modest inhibition as compared to a 5´-phosphate, all exonucleases tested could cleave all modifications connected through phosphodiester bonds. Interestingly, this poor inhibition held true for the inverted dT modifications, which have been reported to grant extra stability versus degradation by serum exonucleases for aptamers and other modified oligonucleotides. In our hands, 3´-inverted dT blocked only the relatively weak 3´&rarr 5´ exonuclease activity of DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210) and Exonuclease T (Exo T, NEB #M0265), but did not block more active exonucleases such as in T7 DNA Polymerase (NEB #M0274), Exo I or Exo III. Similarly, 5´-inverted dT partially inhibited only Lambda Exo activity, which is known to require a 5´-phosphate for efficient initiation. Other 5´&rarr 3´ exonucleases were not significantly inhibited by this modification, showing complete digestion after a one-hour incubation under the recommended usage conditions.
We do not recommend 5´/3´ end modification as a good strategy for producing nucleotides resistant to exonuclease degradation in vitro. Researchers should be aware that these modifications will be cleaved by the majority of exonucleases, potentially leading to the loss of ﬂuorescent labels and affinity tags. If a modification stable to exonuclease activity is needed, a better strategy is to use internal labels connected to the 5-methyl position of dT (e.g., Fluorescein dT, Figure 2). If these modified dT bases are used near the end of an oligo, they can be protected with surrounding pt bonds (Figure 4). The linkage to the base is not susceptible to enzymatic cleavage, and the pt bonds will protect the backbone from digestion, as described above.
Figure 4: Designing oligonucleotides with nuclease-resistant modifications
(A) End ﬂuorescein (FAM) labeled-DNA is rapidly degraded by exonucleases. (B) pt bonds between nucleotides prevent the DNA strand from being degraded, but the end label can still be cleaved. (C) An internal FAMdT surrounded by pt bonds will prevent the exonuclease from removing the label.
Summary of Differences Between DNA and RNA
- Pentose sugar in the nucleotide of DNA is deoxyribose whereas in the nucleotide of RNA it is ribose.
- DNA is copied via self-replication while RNA is copied by using DNA as a blueprint.
- DNA uses thymine as a nitrogen base while RNA uses uracil. The difference between thymine and uracil is that thymine has an extra methyl group on the fifth carbon.
- The adenine base in DNA pairs with thymine while the adenine base in RNA pairs with uracil.
- DNA cannot catalyze its synthesis while RNA can catalyze its synthesis.
- The secondary structure of DNA consists of mainly B-form double helix while the secondary structure of RNA consists of short regions of A-form of a double helix.
- Non Watson-Crick base pairing (where guanine pairs with uracil) is allowed in RNA but not in DNA.
- A DNA molecule in a cell can be as long as several hundred million nucleotides whereas the cellular RNAs range in length from less than one hundred to many thousands of nucleotides.
- DNA is chemically much more stable than RNA.
- The thermal stability of DNA is less compared to RNA.
- DNA is susceptible to ultraviolet damage while RNA is relatively resistant to it.
- DNA is present in the nucleus or mitochondria while RNA is present in the cytoplasm.
DNA and RNA are made of long chains of repeating nucleotides.
Each nucleotide is made of 3 parts:
A pentose sugar
A phosphate group
One of the four types of nitrogen base
To form a strand, nucleotides are linked into chains, with the phosphate and sugar groups alternating.
In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae
Background: The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.
Results: A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.
Conclusion: In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.
Recent Advances in Chemical Modification of Peptide Nucleic Acids
Peptide nucleic acid (PNA) has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification.
Peptide nucleic acid (PNA) is a DNA analogue that has the entire sugar-phosphodiester backbone replaced by a pseudopeptide linkage built of 2-aminoethylglycine residues (Figure 1) . PNA is highly stable chemically and, because of the unnatural backbone, highly resistant to enzymatic degradation, which makes it an excellent candidate for in vivo applications as an oligonucleotide analogue. The neutral pseudopeptide backbone eliminates electrostatic repulsion (a factor that negatively affects oligonucleotide binding) and PNA binds to DNA and RNA with excellent affinity. PNA binds to double helical DNA via two competing binding modes, triple helix (PNA : DNA, 1 : 1), and strand invasion, where PNA displaces one of the DNA strands, typically followed by a triplex formation (PNA : DNA, 2 : 1) . PNA also forms exceptionally strong and sequence-specific Watson-Crick duplexes with single-stranded DNA and RNA . Interestingly, the sequence specificity of duplexes involving PNA is substantially higher than that of unmodified nucleic acids. Because of these superior qualities, PNA has become a powerful tool in chemical biology and biotechnology [3–5]. The main applications of PNA are as hybridization probes and molecular diagnostics of high affinity and selectivity for single-stranded DNA and RNA. PNA also holds a promise of becoming a novel gene therapy agent for targeting specific RNA molecules [3, 4].
Although PNA binds single-stranded DNA and RNA with superior affinity and selectivity, there are other properties of PNA that can be further improved. Most importantly, in vivo applications of unmodified PNA are hindered by poor cellular uptake and endosomal entrapment . Current methods to enhance the cellular uptake of PNA, such as conjugation with cell penetrating peptides (CPP) [7, 8], are complicated and require high PNA-peptide concentrations that may cause off-target binding and toxicity in vivo. Another problem is the limited sequence scope of double-stranded nucleic acids that can be recognized by PNA. While PNA can bind any sequence of single-stranded DNA and RNA with high affinity and selectivity, recognition of double helical DNA has been limited to polypurine tracts and binding to double helical RNA has been little explored. The present paper focuses on most recent developments in chemical modification of PNA to enhance cellular uptake and recognition of double helical nucleic acids. Several comprehensive reviews have recently discussed modification of PNA backbone [9, 10] and nucleobases  in a broader context.
2. Conjugation of PNA with Cationic Peptides to Improve the Cellular Uptake
Inefficient crossing of cellular membrane of mammalian cells by unmodified PNA has been a major problem for practical in vivo applications of PNA. Because of the neutral backbone, PNA does not associate with delivery vehicles based on cationic lipids. To use such standard oligonucleotide transfectants as Lipofectamine, PNA needs to be hybridized to complementary oligodeoxynucleotide (ODN) that aids the electrostatic complexation with the positively charged lipids . Recently, a new approach to PNA delivery was developed by Wooley, Taylor and coworkers  who used cationic shell-cross-linked knedel-like nanoparticles (cSCKs) to deliver either PNA-ODN hybrid or PNA covalently attached to cSCKs nanoparticles through a biodegradable disulfide linkage. cSCKs nanoparticles have a hydrophobic core and a positively charged cross-linked shell. The latter is highly functionalizable and mediates the cellular delivery through, most likely, an endocytotic mechanism. An elegant extension of this technology is reported in this special issue by Taylor and coworkers .
Perhaps, the most popular approach to enhance cellular delivery has been conjugation of PNA with cell penetrating peptides that deliver the conjugate through the endocytosis pathway [7, 8]. However, the low ability of PNA-CPP conjugates to escape from endosomes has been the bottleneck of this approach. Various endosomolytic compounds have been explored unfortunately, most are too toxic for in vivo applications . Conjugates with arginine-rich peptides have shown promising activity in HeLa cells in the absence of endosomolytic agents . However, even in the most promising cases large amount of conjugates remained in endosomes, leaving plenty of room for further improvement . The relatively high concentrations of PNA-CCP, which are required for efficient delivery, may cause off-target binding and toxicity in vivo. Moreover, CPPs are relatively large peptides, which complicate the preparation and use of PNA-CPP conjugates. Recently, several groups have demonstrated that relatively simple cationic modifications in PNA can substantially improve their cellular uptake and produce effect similar to that of longer and more complex CPPs.
The groups of Corey [16, 17] and Gait [15, 18, 19] showed that conjugation of PNA with short oligolysine (Figure 2, 1 and 2, resp.) enabled efficient delivery in fibroblast and various cancer cell lines (T47D, MCF-7, Huh7, and HeLa). As few as four lysine residues achieved similar efficiency as R6-Penetratin, a CPP previously optimized for cellular delivery of PNA . Using short oligolysine instead of longer CPP significantly reduced the complexity and effort required for PNA use in cell culture. Lysine conjugates have also been used to deliver PNA in mice [20, 21]. Most recently, Gait and coworkers showed that introduction of a terminal Cys residue further increased the cellular uptake of Cys-Lys-PNA-Lys3 conjugate . While some studies showed that conjugates built of the unnatural D-lysine were more effective , presumably due to higher biostability, other studies found little difference between the L and D series . In a similar study, Fabbri et al.  demonstrated that PNA conjugated at the carboxyl terminus with octaarginine was efficiently taken up in human leukemic K562 cells and inhibited activity of the target microRNA-210.
Nielsen and coworkers have recently reported on conjugates of PNA with cationic ligands that showed improved cellular delivery and activity [24, 25]. In one study, addition of a lipid domain to the cationic peptides increased the activity of PNA conjugate by two orders of magnitude . The lypophilic fatty acid contributed by promoting both endosomal uptake and endosomal escape of PNA. In another study, conjugation of PNA with polyethylenimine showed significantly higher antisense activity than PNA-octaarginine conjugates . Polyethylenimine conjugates had lower toxicity than PNA-octaarginine conjugates. The polyethylenimine conjugate activity did not depend on the presence of lysosomolytic agents, which suggested that these conjugates are able to escape endosomes efficiently. These studies suggest that chemical approaches can be used to tailor cationic modifications that will improve cellular uptake and avoid the problem of endosomal entrapment.
Conjugation of PNA with a lipophilic triphenylphosphonium cation has been shown to increase the cellular delivery [26, 27]. In this special issue, Pandey, Patino and coworkers  report on cyclic and hairpin PNAs conjugated to the triphenylphosphonium cation via a disulfide linkage. The conjugates inhibit HIV replication by targeting the HIV-1 TAR RNA loop. Most recently, Shiraishi and Nielsen  reported on cellular uptake and antisense activity of PNA conjugated with cholesterol and cholic acid in HeLa pLuc705 cells. Although the conjugates alone were inactive, the delivery was dramatically improved by addition of Lipofectamine leading to nanomolar antisense activity.
As the numerous recent studies reviewed above suggest, design and optimization of CPP and other cationic ligands for cellular delivery of PNA is still a vigorous and important area of research. The focus has shifted to addressing endosomal escape, improving the end point activity and potential in vivo applications.
3. Cationic Backbone Modifications to Improve the Cellular Uptake of PNA
An alternative approach to conjugation of PNA has been direct modification of PNA’s backbone. Several groups have explored cationic modifications of PNA [30–32]. Ly and coworkers introduced guanidine groups at
-positions  of PNA’s backbone by custom synthesis of monomers starting from diaminoethane and L or D arginine instead of glycine (Figure 3, L series shown). The
-guanidine-modified PNA (GPNA) derived from the unnatural D-arginine had higher affinity for complementary DNA  and RNA  good sequence selectivity was maintained. GPNA was readily taken up by several cell lines (HCT116, human ES, and HeLa), which was attributed to the cationic guanidine modifications. GPNA was less toxic to cells than a PNA-polyarginine conjugate and induced potent antisense inhibition of E-cadherin in A549 cells . Our laboratory recently studied the triple helix formation between double helical RNA and α-GPNA. We found that the α-guanidine modification decreased RNA binding affinity and sequence selectivity of α-GPNA compared to unmodified PNA .
The γ-guanidine-modified PNA had higher affinity for complementary DNA and RNA than α-guanidine-modified PNA, presumably due to favorable preorganization of the γ-modified backbone into a right-handed helix . In contrast to α-modified PNA, Englund and Appella found that the S-isomer of γ-modified PNA (derived from the natural L-lysine) had higher affinity for complementary DNA than the R-isomer . Most recently, Manicardi et al.  used both α- and γ-modified GPNA 15-mers to inhibit microRNA-210 in K562 cells. Both isomers showed promising though not complete inhibition with the PNAs having eight consecutive γ-modification at the carboxyl terminus performing slightly better than other modification patterns .
Mitra and Ganesh reported similar results on DNA binding and cellular uptake of α- and γ-aminomethylene PNA (am-PNA, Figure 3) [38, 39]. The aminomethylene modification increased PNA binding to DNA, with γ-(S)am-PNA being significantly better than α-(R)am-PNA, which, in turn, was better than α-(S)am-PNA . The cellular uptake was enhanced by these modifications in roughly the same order, with γ-(S)am-PNA giving the most promising results.
4. PNA Modifications to Expand the Recognition of Double-stranded Nucleic Acids
Recognition of single-stranded DNA and RNA following the Watson-Crick base pairing rules is relatively straightforward. Recognition of double-stranded nucleic acids is substantially more challenging because the Watson-Crick faces of nucleobases are already engaged in hydrogen bonding. PNA, as well as other oligonucleotide analogues, can recognize double-stranded nucleic acids by forming either a parallel triple helix (Figure 4(a), the amino end of PNA aligned with the 5′ end of DNA) or a strand-invasion complex, where PNA displaces one of the DNA strands. The strand-invasion is typically a competing mode for triplex (PNA : DNA, 1 : 1) and usually results in a strand-displacement triplex (PNA : DNA, 2 : 1). The PNA strand that is replacing the DNA strand aligns antiparallel with the DNA strand (Figure 4(b), the carboxyl end of PNA aligned with the 5′ end of DNA). Both binding modes are limited to nucleic acid duplexes featuring so-called polypurine tracts where one strand is built of purines, while the other strand consists of pyrimidines. This is because the standard Hoogsteen triplets (U*A-U and C+*G-C) recognize only purine bases (Figure 5(a)).
4.4: Nucleic Acids
There are two types of nucleic acids in biology: DNA and RNA. DNA carries the heritable genetic information of the cell and is composed of two antiparallel strands of nucleotides arranged in a helical structure. Each nucleotide subunit is composed of a pentose sugar (deoxyribose), a nitrogenous base, and a phosphate group. The two strands associate via hydrogen bonds between chemically complementary nitrogenous bases. Interactions known as "base stacking" interactions also help stabilize the double helix. By contrast to DNA, RNA can be either be single stranded, or double stranded. It too is composed of a pentose sugar (ribose), a nitrogenous base, and a phosphate group. RNA is a molecule of may tricks. It is involved in protein synthesis as a messenger, regulator, and catalyst of the process. RNA is also involved in various other cellular regulatory processes and helps to catalyze some key reactions (more on this later). With respect to RNA, in this course we are primarily interested in (a) knowing the basic molecular structure of RNA and what distinguishes it from DNA, (b) understanding the basic chemistry of RNA synthesis that occurs during a process called transcription, (c) appreciating the various roles that RNA can have in the cell, and (d) learning the major types of RNA that you will encounter most frequently (i.e. mRNA, rRNA, tRNA, miRNA etc.) and associating them with the processes they are involved with. In this module we focus primarily on the chemical structures of DNA and RNA and how they can be distinguished from one another.
The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA and RNA are made up of monomers known as nucleotides. Individual nucleotides condense with one another to form a nucleic acid polymer. Each nucleotide is made up of three components: a nitrogenous base (for which there are five different types), a pentose sugar, and a phosphate group. These are depicted below. The main difference between these two types of nucleic acids is the presence or absence of a hydroxyl group at the C2 position, also called the 2' position (read "two prime"), of the pentose (see Figure 1 legend and section on the pentose sugar for more on carbon numbering). RNA has a hydroxyl functional group at that 2' position of the pentose sugar the sugar is called ribose, hence the name ribonucleic acid. By contrast, DNA lacks the hydroxyl group at that position, hence the name, "deoxy" ribonucleic acid. DNA has a hydrogen atom at the 2' position.
The nitrogenous base
The nitrogenous bases 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 acting as a base by decreasing the hydrogen ion concentration in the local environment. Each nucleotide in DNA contains one of four possible nitrogenous bases: adenine (A), guanine (G), cytosine (C), and thymine (T). By contrast, RNA contains adenine (A), guanine (G) cytosine (C), and uracil (U) instead of thymine (T).
Adenine and guanine are classified as purines. The primary distinguishing structural feature of a purine is double carbon-nitrogen ring. Cytosine, thymine, and uracil are classified as pyrimidines. These are structurally distinguished by a single carbon-nitrogen ring. You will be expected to recognize that each of these ring structures is decorated by functional groups that may be involved in a variety of chemistries and interactions.
Take a moment to review the nitrogenous bases in Figure 1. Identify functional groups as described in class. For each functional group identified, describe what type of chemistry you expect it to be involved in. Try to identify whether the functional group can act as either a hydrogen bond donor, acceptor, or both?
The pentose sugar
The pentose sugar contains five carbon atoms. Each carbon atom of the sugar molecule are numbered as 1&prime, 2&prime, 3&prime, 4&prime, and 5&prime (1&prime is read as &ldquoone prime&rdquo). The two main functional groups that are attached to the sugar are often named in reference to the carbon to whch they are bound. For example, the phosphate residue is attached to the 5&prime carbon of the sugar and the hydroxyl group is attached to the 3&prime carbon of the sugar. We will often use the carbon number to refer to functional groups on nucleotides so be very familiar with the structure of the pentose sugar.
The pentose sugar in DNA is called deoxyribose, and in RNA, the sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the 2' carbon of the ribose and its absence on the 2' carbon of the deoxyribose. You can, therefore, determine if you are looking at a DNA or RNA nucleotide by the presence or absence of the hydroxyl group on the 2' carbon atom&mdashyou will likely be asked to do so on numerous occasions, including exams.
The phosphate group
There can be anywhere between one and three phosphate groups bound to the 5' carbon of the sugar. When one phosphate is bound, the nucleotide is referred to as a Nucleotide MonoPhosphate (NMP). If two phosphates are bound the nucleotide is referred to as Nucleotide DiPhosphate (NDP). When three phosphates are bound to the nucleotide it is referred to as a Nucleotide TriPhosphate (NTP). The phosphoanhydride bonds between that link the phosphate groups to each other have specific chemical properties that make them good for various biological functions. The hydrolysis of the bonds between the phosphate groups is thermodynamically exergonic in biological conditions nature has evolved numerous mechanisms to couple this negative change in free energy to help drive many reactions in the cell. Figure 2 shows the structure of the nucleotide triphosphate Adenosine Triphosphate, ATP, that we will discuss in greater detail in other chapters.
The term "high-energy bond" is used A LOT in biology. This term is, however, a verbal shortcuts that can cause some confusion. The term refers to the amount of negative free energy associated with the hydrolysis of the bond in question. The water (or other equivalent reaction partner) is an important contributor to the energy calculus. In ATP, for instance, simply "breaking" a phosphoanhydride bond - say with imaginary molecular tweezers - by pulling off a phosphate would not be energetically favorable. We must, therefore, be careful not to say that breaking bonds in ATP is energetically favorable or that it "releases energy". Rather, we should be more specific, noting that they hydrolysis of the bond is energetically favorable. Some of this common misconception is tied to, in our opinion, the use of the term "high energy bonds". While in Bis2a we have tried to minimize the use of the vernacular "high energy" when referring to bonds, trying instead to describe biochemical reactions by using more specific terms, as students of biology you will no doubt encounter the potentially misleading - though admittedly useful - short cut "high energy bond" as you continue in your studies. So, keep the above in mind when you are reading or listening to various discussions in biology. Heck, use the term yourself. Just make sure that you really understand what it refers to.
Double helix structure of DNA
DNA has a double helix structure (shown below) created by two strands of covalently linked nucleotide subunits. The sugar and phosphate groups of each strand of nucleotides are positioned on the outside of the helix, forming the backbone of the DNA (highlighted by the orange ribbons in Figure 3). The two strands of the helix run in opposite directions, meaning that the 5&prime carbon end of one strand will face the 3&prime carbon end of its matching strand (See Figures 4 and 5). We referred to this orientation of the two strands as antiparallel. Note too that phosphate groups are depicted in Figure 3 as orange and red "sticks" protruding from the ribbon. The phosphates are negatively charged at physiological pHs and therefore give the backbone of the DNA a strong local negatively charged character. By contrast, the nitrogenous bases are stacked in the interior of the helix (these are depicted as green, blue, red, and white sticks in Figure 3). Pairs of nucleotides interact with one another through specific hydrogen bonds (shown in Figure 5). Each pair of separated from the next base pair in the ladder by 0.34 nm and this close stacking and planar orientation gives rise to energetically favorable base-stacking interactions. The specific chemistry associated with these interactions is beyond the content of Bis2a but is described in more detail here for the curious or more advanced students. We do expect, however, that students are aware that the stacking of the nitrogenous bases contributes to the stability of the double helix and defer to your upper-division genetics and organic chemistry instructors to fill in the chemical details.
Figure 3. Native DNA is an antiparallel double helix. The phosphate backbone (indicated by the curvy lines) is on the outside, and the bases are on the inside. Each base from one strand interacts via hydrogen bonding with a base from the opposing strand. Attribution: Marc T. Facciotti (original work)
In a double helix, certain combinations of base pairing are chemically more favored than others based on the types and locations of functional groups on the nitrogenous bases of each nucleotide. In biology we find that:
Adenine (A) is chemically complementary with thymidine (T) (A pairs with T)
Guanine (G) is chemically complementary with cytosine (C) (G pairs with C).
We often refer to this pattern as "base complementarity" and say that the antiparallel strands are complementary to each other. For example, if the sequence of one strand is of DNA is 5'-AATTGGCC-3', the complementary strand would have the sequence 5'-GGCCAATT-3'.
We sometimes choose to represent complementary double-helical structures in text by stacking the complementary strands on top of on another as follows:
5' - GGCCAATTCCATACTAGGT - 3'
3' - CCGGTTAAGGTATGATCCA - 5'
Note that each strand has its 5' and 3' ends labeled and that if one were to walk along each strand starting from the 5' end to the 3' end that the direction of travel would be opposite the other for each strand the strands are antiparallel. We commonly say things like "running 5-prime to 3-prime" or "synthesized 5-prime to 3-prime" to refer to the direction we are reading a sequence or the direction of synthesis. Start getting yourself accustomed to this nomenclature.
Functions and roles of nucleotides and nucleic acids to look out for in Bis2a
In addition to their structural roles in DNA and RNA, nucleotides such as ATP and GTP also serve as mobile energy carriers for the cell. Some students are surprised when they learn to appreciate that the ATP and GTP molecules we discuss in the context of bioenergetics are the same as those involved in the formation of nucleic acids. We will cover this in more detail when we discuss DNA and RNA synthesis reactions. Nucleotides also play important roles as co-factors in many enzymatically catalyzed reactions.
Nucleic acids, RNA in particular, play a variety of roles in in cellular process besides being information storage molecules. Some of the roles that you should keep an eye out for as we progress through the course include: (a) Riboprotein complexes - RNA-Protein complexes in which the RNA serves both catalytic and structural roles. Examples of such complexes include, ribosomes (rRNA), RNases, splicesosome complexes, and telomerase. (b) Information storage and transfer roles. These roles include molecules like DNA, messenger RNA (mRNA), transfer RNA (tRNA). (c) Regulatory roles. Examples of these include various non-coding (ncRNA). Wikipedia has a comprehensive summary of the different types of known RNA molecules that we recommend browsing to get a better sense of the great functional diversity of these molecules.
General characteristics of the viruses
- The term ‘virus’ is derived from Latin which means “slimy poison fluid” or “venom”.
- The virus is an ultramicroscopic, infectious agent that is metabolically inert so require a living host or cell to multiply. are obligate intracellular parasites.
- Viruses cannot make energy or proteins on their own so these are dependent on their host cell. multiply inside the living cells using host cell machinery.
- The virus has different strains or types.
- The virus has its own genetic material either DNA or RNA which may be single or double-stranded.
- A virus can undergo Mutation.
- They can be destroyed by Ultraviolet Rays.
- These are not composed of cells. They lack cellular structures such as plasma membrane, nucleus, organelles, etc.
- They do not respire or perform a gaseous exchange.
- They do not move, grow in size but can reproduce by using the metabolism of their hosts.
- They can be crystallized and stored in bottles like chemicals.
- They lack the enzyme system and do not have the metabolic activity of their own. are not able to survive without a host cell, so active viruses reside inside a host body. They are present either in a bacterial cell, animal cell or plant cell.
- Size: Viruses are much smaller than Bacteria. Their Size ranges from 20 – 1400nm. The poliovirus is 30nm. Giant Mimi viruses are up to 800 nm.
- Different shapes of Viruses: Viruses are of different shapes. They are rod-shaped, bullet-shaped, filament shaped, icosahedral in shape and tadpole-shaped.
Viral Structure: Virus consists of nucleic acid and a protein. Genome or the nucleic acid is covered by a protein coat called the capsid. Some viruses have an envelope outside the capsid. A virus without the envelope is called the Naked virus.