Genetic information is encoded in a sequence of nucleobases which are bonded to a sugar–phosphate ba — Analytical Chemistry Chemistry Question
Nucleic acids
Genetic information is encoded in a sequence of nucleobases which are bonded to a sugar–phosphate backbone. Deoxyribonucleic acid (DNA) contains adenine (A), cytosine (C), guanine (G), and thymine (T), whereas ribonucleic acid (RNA) contains uracil (U) instead of thymine.
The most common structures of nucleobases are shown in Figure 1, but these are not the only possible ones. Since nucleobases contain a number of double bonds, they may occur in several different tautomeric forms. Note that even zwitterionic tautomers are possible in principle, but the tasks below deal only with uncharged molecular structures.
[VISUAL]
Figure 1. Structural formulae of nucleobases A, C, G, T, and U bonded to sugar-phosphate backbone (R).
Draw the structural formulae of all non-charged tautomers of cytosine. Assume the nucleobase is bonded to the sugar–phosphate backbone. Consider any pair of imino E/Z isomers as two different tautomers.
Model Answer
[VISUAL]
Draw the structural formulae of the non-standard pairs T–G*, T*–G, A–C* and A*–C, where any minor uncharged tautomer is marked with an asterisk. Keep the relative orientation of the sugar–phosphate backbone the same as in the standard pairs and maximize the number of hydrogen bonds between the nucleobases.
Model Answer
[VISUAL]
At 260 nm, sample 1 of a nucleic acid with an unknown concentration of adenine transmits 11% UV light. A standard solution in which the concentration of adenine amounts to 27 μmol dm−3, absorbs 57% UV light at the same wavelength.
Calculate the unknown concentration of adenine in sample 1. Neglect any absorption at 260 nm by the other nucleobases and assume that both measurements were performed under identical experimental conditions (cuvette length, buffer composition, temperature, etc.).
Model Answer
Unknown sample (1) transmittance: T1 = 0.11
Known sample (2) transmittance: T2 = 1 − 57 = 0.43
Using Lambert–Beer law: −log10 T = ε l c
(−log10 T2) / c2 = (−log10 T1) / c1
c1 = c2 × (−log10 T1) / (−log10 T2) = 27 [μmol dm−3] (−log10 0.11) / (−log10 0.43) = 70.6 μmol dm−3
Spectrophotometry in the near-UV region is a useful tool to monitor the hybridization of DNA as the temperature changes. Melting temperature Tm is defined as the temperature at which 50% of the original amount of DNA double helices are dissociated into separated strands. Nucleobases within dsDNA absorb less strongly than those in ssDNA, thus the dissociation of dsDNA manifests itself by an increase of absorbance. The plot below shows the absorbance at 260 nm as a function of temperature for two different DNA species (DNA1 and DNA2). Assume that both DNA species have equal molar absorption coefficients and that all the measurements were performed under otherwise identical conditions using identical equipment (initial concentrations, buffers, cuvette, etc.).
[VISUAL]
Considering the plot shown above, decide whether the following statements are true or false or whether that cannot be answered based only on the plot.
a) At 320 K, the concentration of dsDNA1 is lower than the concentration of dsDNA2.
True False Cannot be answered
b) The melting temperature Tm of DNA1 is higher than the melting temperature of DNA2.
True False Cannot be answered
c) dsDNA of the species DNA1 is more thermodynamically stable than that of the DNA2 with respect to their single-stranded forms.
True False Cannot be answered
d) dsDNA1 is composed of a larger number of nucleobase pairs than dsDNA2
True False Cannot be answered
Model Answer
True. According to the Lambert–Beer law, absorbance is directly proportional to concentration (as long as the cuvette length and the molar absorption coefficients are assumed equal). The higher absorbance of DNA1 actually means that the concentration of dsDNA1, which absorbs less radiation than ssDNA1, is lower.
a) False. Thermodynamic stability is described in terms of Tm, which can be read as the inflexion point of the sigmoidal curve; here Tm(DNA1) ~ 315 K and Tm(DNA2) ~ 340 K.
b) False. Since Tm(DNA1) ~ 315 K and Tm(DNA2) ~ 340 K, dsDNA2 is more stable than dsDNA1 with respect to their single-stranded forms.
c) Cannot be answered. The thermodynamic stability of a DNA double helix depends on both its length (i.e. the number of nucleobase pairs) and its sequence (roughly, the content of G–C nucleobase pairs). Since no information about the G–C pairs content is given, no conclusions about the DNA lengths can be drawn.
d) Cannot be answered. The thermodynamic stability of a DNA double helix depends on both its length (i.e. the number of nucleobase pairs) and its sequence (roughly, the content of G–C nucleobase pairs). Since no information about the G–C pairs content is given, no conclusions about the DNA lengths can be drawn.
The Rous sarcoma virus is a retrovirus. Its genetic information is stored in a single strand of RNA rather than in dsDNA; recall that RNA contains uracil instead of thymine (Figure 1). The virus uses an enzyme, reverse transcriptase, to synthesize its complementary DNA (cDNA) strand, which is then transcribed to messenger RNA (mRNA). Finally, the mRNA is translated to a polypeptide strand in the ribosome of the infected cell.
The following fragment of 8 nucleotides was identified in the RNA of the virus: 5′-CCCCAGGU-3′.
Write the sequences of cDNA and mRNA corresponding to the octanucleotide. Mind the orientation of the molecule, and identify the 5′- and 3′-termini.
Model Answer
cDNA: 5′-ACCTGGGG-3′, mRNA: 5′-CCCCAGGU-3′
How many possible single-stranded RNA octanucleotides do exist?
Model Answer
Each position of the 8-nucleobase sequence can be occupied by one of the four nucleobases (A, C, G, U). Hence, there are 4^8 = 65 536 theoretically possible single-stranded octanucleotides.