Question

The practical limit for the number of different RNA sequences that can be screened in a SELEX experiment is \(10^{15}\). a. Suppose you are working with oligonucleotides that are 36 nucleotides long. How many sequences exist in a randomized pool containing every sequence possible? b. What percentage of these can a SELEX experiment screen? c. Suppose you wish to select an RNA molecule that catalyzes the hydrolysis of a particular ester. From what you know about catalysis, propose a SELEX strategy that might allow you to select the appropriate catalyst.

Short answer

a) Total sequences: \(6.87 \times 10^{21}\). b) SELEX screens \(0.0000146\%\). c) Use SELEX rounds with ester substrates to enrich catalysts.

Step-by-step solution

Determine Total RNA Sequences

Each nucleotide can be one of four types: A, U, G, or C. Therefore, for a sequence of 36 nucleotides, the total number of possible sequences is given by the expression \(4^{36}\). Using a calculator, compute \(4^{36}\).

Calculate Total Possible RNA Sequences

First, compute \(4^3 = 64\).Compute again: \((4^3)^2 = 64^2 = 4096\).Now, compute: \(4^6 = 4096^6 = 16777216\).Finally, compute: \(4096 \cdot 16777216 = 6.87 \times 10^{21}\).The total possible sequences is approximately \(6.87 \times 10^{21}\).

SELEX Screening Fraction Calculation

Given: SELEX can screen \(10^{15}\) sequences, find the fraction of total sequences screened by SELEX.Calculate: \(\frac{10^{15}}{6.87 \times 10^{21}}\). Using a calculator: the fraction is approximately \(1.46 \times 10^{-7}\).

Convert Screening Fraction to Percentage

Convert the fraction from Step 3 to percentage: \(1.46 \times 10^{-7} \times 100\% = 0.0000146\%\). Therefore, about \(0.0000146\%\) of the possible sequences can be screened in a SELEX experiment.

Propose a SELEX Strategy

To search for an RNA molecule that catalyzes ester hydrolysis, start by preparing a pool of 36 nucleotide RNA sequences. Apply repeated rounds of SELEX by binding the RNA to a substrate carrying the ester bond and washing non-binding sequences. Amplify the bound sequences and introduce mutations to increase diversity. Continue the rounds of selection until you enrich the pool for sequences showing desired catalytic activity.

Key concepts

RNA sequences

RNA sequences are composed of chains of nucleotides, each being a molecule that includes a ribose sugar, a phosphate group, and a nitrogenous base.
In the context of RNA, the possible nitrogenous bases are adenine (A), uracil (U), guanine (G), and cytosine (C). When you string these nucleotides together, you form the backbone of RNA sequences. These sequences play a pivotal role in the genetic coding, decoding, regulation, and expression processes within biological systems. In our exercise, we're particularly interested in understanding the range of possible combinations for RNA sequences of a specific length, in this case, 36 nucleotides long.

To determine the diversity of RNA sequences available, you use the mathematical expression for permutations of bases:

• Each of the 36 positions in the RNA sequence can be filled by one of the four bases: A, U, G, or C.
• Thus, you have a total of \(4^{36}\) possible sequences.

This results in an astronomical number, showing that RNA sequences can potentially encode a vast variety of functions and characteristics.

oligonucleotides

Oligonucleotides are short sequences of nucleotides, usually ranging from 2 to 50 bases.
They can be synthesized to match a specific sequence and are integral tools in genetic research and biotechnology.
The synthesis of oligonucleotides allows researchers to investigate gene functions, manipulate genetic material, or discover RNA molecules with unique properties in experiments like SELEX (Systematic Evolution of Ligands by Exponential Enrichment).

Oligonucleotides are essential in these experiments because they form the initial randomized pool from which specific sequences with desirable traits are selected. For example, when working with oligonucleotides that are 36 nucleotides long, researchers create a pool that potentially contains all possible nucleotide sequences of that length.

• These molecules can be labeled, modified, and used as primers or probes in various applications.
• In a SELEX experiment, the goal is to find the oligonucleotides that bind effectively to a target molecule or demonstrate catalytic activity, such as the hydrolysis of ester bonds.

By iteratively enriching the pool for binding efficacy or activity, scientists can isolate the most effective oligonucleotides for their study.

catalysis

Catalysis refers to the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst.
Catalysts are not consumed in the reaction and can be used repeatedly.
In biological systems, RNA molecules are capable of catalyzing chemical reactions; these are known as ribozymes. When selecting an RNA molecule for catalysis, the focus is on enhancing the efficiency and specificity of these RNA enzymes toward the desired reaction.

The challenge lies in identifying RNA sequences that can effectively catalyze the reaction of interest, such as ester hydrolysis.

• The SELEX process aids by creating a large library of RNA sequences, employing selection rounds to highlight those sequences that demonstrate catalytic functions.
• Researchers repeatedly expose these sequences to conditions that favor catalysis, enabling the isolation of the most effective molecules.

This iterative process ensures that only the RNA sequences with the highest catalytic capabilities are selected, making them prime candidates for further examination and application.

ester hydrolysis

Ester hydrolysis is a reaction where an ester bond is broken down into an alcohol and an acid, usually in the presence of water.
This reaction is fundamental in both natural and synthetic chemistry, playing a critical role in metabolism and the breakdown of complex molecules.
In SELEX experiments, researchers may aim to discover RNA molecules capable of catalyzing this type of reaction, potentially leading to novel biocatalysts.

The goal is to identify RNA sequences (ribozymes) that can accelerate ester hydrolysis under specific conditions.

• SELEX starts by screening large libraries of RNA for those capable of binding to ester substrates.
• Subsequent rounds focus on retaining sequences that show catalytic activity, thereby finding RNA molecules that can effectively expedite the reaction.

This selection method not only advances our understanding of enzymatic activity but also helps in crafting biomolecules that mimic natural enzymes for various applications.